U.S. patent application number 13/063156 was filed with the patent office on 2012-02-16 for mirna targets.
This patent application is currently assigned to IMMUNE DISEASE INSTITUTE, INC.. Invention is credited to Ashish Lal, Judy Lieberman.
Application Number | 20120040851 13/063156 |
Document ID | / |
Family ID | 42040163 |
Filed Date | 2012-02-16 |
United States Patent
Application |
20120040851 |
Kind Code |
A1 |
Lieberman; Judy ; et
al. |
February 16, 2012 |
miRNA TARGETS
Abstract
The present invention provides systems for identifying,
isolating, and/or characterizing targets of micro RNAs.
Inventors: |
Lieberman; Judy; (Brookline,
MA) ; Lal; Ashish; (Brookline, MA) |
Assignee: |
IMMUNE DISEASE INSTITUTE,
INC.
Boston
MA
|
Family ID: |
42040163 |
Appl. No.: |
13/063156 |
Filed: |
September 18, 2009 |
PCT Filed: |
September 18, 2009 |
PCT NO: |
PCT/US2009/057498 |
371 Date: |
October 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61098707 |
Sep 19, 2008 |
|
|
|
61098696 |
Sep 19, 2008 |
|
|
|
Current U.S.
Class: |
506/9 ; 435/6.1;
435/6.12; 536/23.1 |
Current CPC
Class: |
A61P 37/00 20180101;
A61P 35/00 20180101; C12Q 1/6806 20130101; C12N 2320/11 20130101;
C12N 15/113 20130101; C12Q 1/6809 20130101; C12N 2310/141 20130101;
C12N 15/111 20130101; A61P 43/00 20180101; A61P 37/06 20180101 |
Class at
Publication: |
506/9 ; 435/6.1;
435/6.12; 536/23.1 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C07H 21/02 20060101 C07H021/02; C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method comprising steps of: providing a cell that contains an
miRNA of interest; and identifying one or more RNAs that interact
with the miRNA in the cell.
2. The method of claim 1, wherein the step of identifying comprises
isolating miRNA-RNA complexes.
3. The method of claim 1, wherein the miRNA is miR-24.
4. The method of claim 1, wherein the step of identifying comprises
determining that the one or more interacting RNAs is enriched in
miRNA-RNA complexes as compared with its cellular expression
level.
5. The method of claim 1, further comprising a step of: analyzing
the one or more interacting RNAs by a technique selected from the
group consisting of: reverse transcription (RT), polymerase chain
reaction (PCR), sequence analysis, expression level analysis,
network analysis, and combinations thereof.
6. A target RNA identified according to the method of claim 1.
7. A kit comprising components for performing the method of claim
1.
8. The kit of claim 6, comprising one or more components selected
from the group consisting of: nucleic acid standards, reagents for
labeling miRNAs, reagents for quantifying degree of target RNA
enrichment relative to cellular expression levels, and combinations
thereof.
9. The kit of claim 6, which kit further contains one or more
components selected from the group consisting of: nucleic acid
polymerases, nucleotides, nucleotide analogs, buffers, antibodies,
labels, and combinations thereof.
10. The kit of claim 6, which kit further contains one or more
components that regulate the concentration or the downstream
effects of the one or more interacting RNAs.
Description
RELATED APPLICATIONS
[0001] The present application is related to U.S. Ser. No.
61/098,696, filed on Sep. 19, 2008, entitled "miRNA Targets", and
U.S. Ser. No. 61/098,707, filed on Sep. 19, 2008, entitled
"Therapeutic and Diagnostic Strategies," the entire contents of
which are incorporated herein by reference.
BACKGROUND
[0002] microRNAs (miRNAs) regulate key steps of cell
differentiation and development (1-3) by suppressing gene
expression in a sequence-specific manner (4). In mammals, the
active strand miRNA sequence (typically--22 base pairs) is
partially complementary to binding sites in the 3'UTR of genes,
often with full complementarity to 7 or 8 nucleotides in the "seed
region" (residues 2-9) of the miRNA. Gene suppression in mammals is
thought to occur primarily by inhibiting translation (5). However,
miRNAs in mammals also cause mRNA decay (6, 7).
[0003] Current approaches to identify miRNA targets fall short of
the task. The major tools that have been used are (1) bioinformatic
algorithms that predict potential target genes that contain
conserved complementary sequences in their 3'UTR to a seed region
at the 5'-end of the miRNA active strand (12, 13), and (2) analysis
of mRNAs that are down-regulated when a miRNA is over-expressed
(14, 15).
[0004] The bioinformatic approach is hampered by the fact that the
existing algorithms have a high margin of error (the majority of
predicted genes are not real targets and some of the key targets,
such as RAS for let-7, are not predicted). For many miRNAs, current
algorithms predict hundreds or even thousands of potential targets,
making it difficult to identify the most important targets.
[0005] Gene expression array analysis does not readily distinguish
direct mRNA targets from mRNAs down-regulated through secondary
effects and misses most target genes that are regulated by blocking
translation rather than by mRNA degradation. Moreover, even when
mRNA degradation occurs, changes in mRNA levels may be small (often
less than 2-fold) and may be difficult to distinguish from
background fluctuations, especially in genomewide surveys.
[0006] Even combining these 2 approaches still is not helpful in
many situations. Recently mRNA targets of miRNAs have been
identified by their enrichment in co-immunoprecipitates with tagged
Argonaute proteins in Drosophila and human cell lines
overexpressing the miRNA of interest (16-19). However these studies
have not been shown to identify new miRNA targets. Argonaute
over-expression globally increases miRNA levels, perhaps obscuring
the effect of an individual over-expressed miRNA (20).
SUMMARY
[0007] The present invention provides an approach for identifying
miRNA targets. In some embodiments, such target identification
involves isolating mRNAs that bind to an miRNA active strand. In
some embodiments, the miRNA active strand is labeled (e.g.
biotinylated) to facilitate isolation of miRNA-target RNA
complexes. This approach is exemplified by application to miR-24
(see Examples, which describe identification of genes regulated by
miR-24). Combining additional analytical techniques (e.g.
microarray analysis, bioinformatics analysis, etc.) has enabled us
to overcome the hurdle of identifying miRNA-regulated genes.
[0008] Through its exemplary application to miR-24, the present
invention, among other things, demonstrates that miR-24 regulates a
network of genes that control cell cycle progression through G1, S
and G2/M as well as key DNA repair genes. Over-expressing miR-24
increases the G1 population, reduces DNA replication and increases
sensitivity to DNA damage (see U.S. Ser. No. 61/098,707, entitled
"Therapeutic and Diagnostic Strategies" and filed on Sep. 19,
2008), while antagonizing miR-24 increases cell proliferation.
DESCRIPTION OF THE DRAWING
[0009] FIG. 1. miR-24 is up-regulated during hematopoietic cell
differentiation into multiple lineages (B) miR-24 expression,
measured using qRT-PCR relative to untreated cells, increases in
K562 cells differentiated to megakaryocytes or erythrocytes and
HL60 cells differentiated to macrophages, monocytes and
granulocytes. Differentiation in all experiments was verified by
cell surface phenotype. Both the primary transcript corresponding
to the chromosome 19 miR-24 cluster (C, K562; D, HL60) and mature
miR-24 (E, K562; F, HL60) increase rapidly and remain elevated when
cells are differentiated with TPA. Mature miR-24 levels were
determined by qRT-PCR and normalized to U6 whereas GAPDH was used
as an internal control to measure changes in levels of pri-miR-24.
Error bars in (B-F) represent standard deviation from 3 independent
experiments (*, p<0.05; **, p<0.01; #, p<0.005; ##,
p<0.001; ***, p<0.0001).
[0010] FIG. 2. Identification of mRNAs down-regulated by miR-24
over-expression (A) HepG2 cells express low levels of miR-24,
assayed by qRT-PCR analysis normalized to U6, compared to HeLa,
WI-38, HL60 and K562 cells. (B) Effective increase in miR-24 RNA in
HepG2 cells 48 hr after transfection with miR-24 mimic compared to
control cells transfected with cel-miR-67. #, p<0.005. (C) Venn
diagram of genes down-regulated by miR-24 in HepG2 cells and genes
predicted to be regulated by miR-24 using TargetScan 4.2. In the
diagram, the 100 predicted genes, whose mRNA is also significantly
down-regulated, have either conserved (20) or nonconserved (80)
predicted miR-24 recognition sites. TargetScan 4.2 predicts 349
conserved miR-24 targets and many more that are not conserved. (D)
Genes identified by microarray as downregulated by miR-24
overexpression were confirmed to be downregulated by qRT-PCR
normalized to GAPDH. UBC is a housekeeping gene. Cells were
transfected with cel-miR-67 (black) or miR-24 mimic (white).
**p<0.01, #p<0.005, and ##p<0.001. The downregulated genes
are graphed in order of their downregulation on the microarray; the
Z ratio of the microarray analysis is shown below. Error bars
represent SD from three independent experiments (A, B, and C). (E)
Top 15 over-represented cellular processes for the 100 overlapping
genes in (C). Histogram displays over-represented processes sorted
by Score (-log [p-value]). A highly positive Score suggests that
the subnetwork is highly saturated with genes identified
experimentally and doesn't have many nodes not identified in the
experiment. The complete list of 100 genes in the overlap and
further statistical analysis is provided in Suppl. Table 3. The
dotted line represents the statistically significant limit. (F)
Direct interaction network of over-represented subnetworks (G)
Sites complementary to the miR-24 seed are enriched in the 3'UTR of
downregulated transcripts. The table shows the frequency of perfect
hexamer (positions 2-7), heptamer (positions 2-8), and octamer
(positions 2-9) miR-24 3'UTR seeds in the downregulated genes.
[0011] FIG. 3. Isolation of miRNA-bound target mRNAs (A) Biotin
pull-down assay. (B) Activity of biotinylated miR-24 (Bi-miR-24) is
similar to non-biotinylated miR-24 mimic. HepG2 cells were
transfected with control miRNA or miR-24 mimics or 3' biotin-miR-24
(Bi-miR-24). miRNA-transfected cells were cotransfected 48 hr later
with pGL3-RL and pMIR-REPORT.TM. plasmid (black) or pMIR-REPORT.TM.
containing a perfectly complementary target site for miR-24 (white)
and assayed for luciferase expression after 24 hr. The firefly
luciferase signal from pMIR-REPORT.TM. was normalized to the
Renilla luciferase from pGL3-RL. #, p<0.005. (C) HepG2 cells
were transfected with 30 nM Bi-miR-24 or Bi-cel-miR-67 miRNA, and
RNA isolated from the streptavidin pull-down was analyzed by
qRT-PCR for miR-24 and miR-16 (a control miRNA) after normalization
to U6. miR-24 was -500-fold higher in miR-24 pull-down as compared
to control pull-down (left panel). ##, p<0.001. miR-16 levels
were similar in each pull-down (right panel). (D) H2AX and CDK6
mRNAs (encoding 2 and 3, 3'UTR miR-24 binding sites, respectively),
assayed by qRT-PCR (normalized to GAPDH), are significantly
enriched in miR-24 (white), compared to control (black) pull-downs
from K562 cells. Enrichment was maximal when pull-down was done 24
hr after transfection. (E, F) Similar enrichment for miR-24 (E) and
let-7 (F) regulated mRNAs was observed in HepG2 cells 24 hr after
Bi-miR-24 or Bi-let-7 mimic transfection. The specific miRNA
pull-down (white) is compared to cel-miR-67 pull-down (black). The
housekeeping gene UBC is a negative control. Error bars represent
standard deviation from 3 independent experiments (B, C, E, F). *,
p<0.05 and *, p<0.01.
[0012] FIG. 4. Bioinformatic analysis of miR-24 pull-down genes
suggests that miR-24 regulates cell cycle progression (A) Venn
diagram of miR-24 target genes predicted by TargetScan 4.2 or
experimentally identified by pull-down or by miR-24-dependent
down-regulation in HepG2 cells. There is not much overlap between
the genes identified by these methods. (B) Over-represented
cellular processes of miR-24 pull-down genes. A histogram of the
top 15 over-represented processes in the 269 gene pull-down set is
ranked by Score (-log [p-value]). Dotted line indicates
statistically significant score. More details are provided in
Suppl. Table 6. (C) Direct interaction network of over-represented
subnetworks, with non-connected nodes removed. Genes annotated as
involved in any aspect of cell cycle regulation are indicated by a
blue symbol; other genes are represented in gray (symbols as in
FIG. 2F) Genes whose primary function is associated with a specific
phase of the cell cycle are indicated [G1/S transition (G1/S), DNA
replication and S phase (S), G2/M transition (G2/M) and mitosis
(M)].
[0013] FIG. 5. miR-24 regulates MYC expression (A) MYC mRNA is
selectively pulled-down from HepG2 cells with Bi-miR-24 (white)
compared to control Bi-cel-miR-67 (black). For each condition,
pulled down RNA was first normalized to GAPDH mRNA in the sample
and then to relative input cellular RNA (#, p<0.001). The
housekeeping gene UBC mRNA was not enriched in the pull-downs. (B)
Predicted binding sites in the MYC 3'UTR for miR-24 (MRE1-6) by
rna22. The miR-24 binding site (447-468) is in the 3'UTR of MYC
mRNA. miR-24 over-expression in HepG2 (C) or K562 cells (D)
decreases MYC mRNA, analyzed by qRTPCR and normalized to GAPDH
(black, cel-miR-67; white, miR-24). (E) MYC protein is decreased in
K562 cells upon overexpression of miR-24. Densitometry was used to
quantify protein levels; a,-tubulin served as loading control. (F)
miR-24 targets the MYC 3'UTR in a luciferase reporter assay. HepG2
cells were transfected with control miRNA (black) or miR-24 (white)
mimic for 48 hr and then with MYC 3'UTR-luciferase reporter (MYC)
or vector (V) for 24 hr. Mean.+-.SD, normalized to vector control,
of 3 independent experiments is shown (**, p<0.01). (G) (G-I)
miR-24 regulates MRE3 and MRE6 by luciferase reporter assay. HepG2
cells were cotransfected with cel-miR-67 (black) or miR-24 (white)
mimics and luciferase reporters containing the wild-type (wt)
MRE1-6 in (B) and (H) or mutated (mt) MRE3 and MRE6 in (G) and (I)
or vector (V). Luciferase activity was measured 48 hr after
transfection. In (G), red letters denote point mutations that
disrupt base pairing. Mean.+-.SD, normalized to vector control, of
three independent experiments is shown. *p<0.05,
**p<0.01.
[0014] FIG. 6. miR-24 inhibits cell proliferation (A) Bi-miR-24
binds to mRNAs encoding E2F2, H2AX, PCNA, AURKB, CCNA2, BRCA1 and
CHEK1, cell cycle and DNA repair genes identified in the pull-down
microarray analysis, but not to E2F1, E2F3 or SDHA (a housekeeping
gene), genes not enriched in the microarray. Streptavidin pull-down
was performed in HepG2 cells transfected with Bi-miR-24 (white) or
control Bi-miRNA (black). Data were normalized to GAPDH. The
Z-ratios refer to the enrichment in the pull-down microarray
analysis. (B) miR-24 silences the expression of luciferase genes
engineered with the 3'UTR of E2F2, but not with E2F1 or E2F3
3'UTRs, suggesting that E2F2 is a direct miR-24 target but E2F1 and
E2F3 are downregulated indirectly. Luciferase assays were performed
in HepG2 cells overexpressing miR-24 (white) or control mimics
(black). miR-24 targets the 3'UTR of E2F-regulated genes (AURKB,
BRCA1, CCNA2, CDC2, and FEN1) and CDK4, a MYC-regulated gene. CHEK1
and PCNA 3'UTRs are not regulated by miR-24. HepG2 cells were
cotransfected with a luciferase reporter containing the 3'UTR of
the indicated gene and control miRNA (black) or synthetic miR-24
(white) for 48 hr. Expression of the unmodified luciferase vector
(V) is unchanged by miR-24. (C) miR-24 significantly reduces mRNA
levels of E2F1, E2F2, and E2F3 and of some E2F target genes (RRM2,
CHEK1, CCNA2, FEN1, MCM4, MCM10, CDC2, and AURKB), but not BRCA1
and PCNA. CDK4, a key MYC target gene, is also downregulated. HepG2
cells were transfected with miR-24 (white) or control mimics
(black) for 48 hr, and E2F and their target mRNAs were measured by
qRT-PCR. Data normalized to GAPDH are expressed relative to control
mimic-transfected cells. UBC is a control housekeeping gene. The Z
ratios refer to the significantly downregulated mRNAs in the mRNA
microarray in miR-24-overexpressing cells. (D) Protein expression
of miR-24 target genes is substantially reduced in miR-24
mimic-transfected K562 cells 72 hr after transfection, relative to
control miRNAtransfected cells. Densitometry was used to quantify
protein relative to a-tubulin. HuR and a-tubulin are loading
controls. (E) miR-24 knockdown in K562 cells specifically decreases
miR-24 levels, assayed by qRT-PCR in cells transfected with miR-24
ASO (white) relative to control ASO (black). Expression relative to
U6 snRNA is depicted normalized to control cells. (F) miR-24
knockdown with ASO increases K562 cell proliferation measured by
thymidine uptake, both in the presence and absence of TPA. The
decline in proliferation with TPA is completely restored by
antagonizing miR-24. (G) miR-24 over-expression increases the G1
compartment in HepG2 cells. HepG2 cells transfected with miR-24 or
control mimic for 48 hr were stained with propidium iodide and
analyzed by flow cytometry. Representative analysis of three
independent experiments is shown. Error bars represent standard
deviation from 3 independent experiments (A-C, E, F). *, p<0.05;
**, p<0.01; #, p<0.005.
[0015] FIG. 7. Endogenous miR-24 and Bi-miR-24 sediment with
polysomes in K562 cell extracts. K562 cells were transfected with
Bi-miR-24 (pink) or Bi-cel-miR-67 (blue) for 24 hr, fixed with
formaldehyde and lysed by sonication. The lysates were fractionated
on 10-50% sucrose gradients and RNA was isolated. The relative
distribution of miR-24 (both endogenous and exogenous) in each
fraction was determined by qRT-PCR analysis. The profiles of
Bi-miR-24 and endogenous miR-24 in the control cells were similar
with most miR-24 associated with the more dense polysome-containing
fractions (6-10).
[0016] FIG. 8. Crosslinking or formaldehyde fixation enhances the
enrichment for let-7 target HRAS and CDK6 mRNAs in the streptavidin
pull-down of Bi-let-7. (A) let-7 RNA was selectively captured from
HeLa cells transfected with Bi-let-7 compared to cells transfected
with control miRNA. Enrichment was enhanced by crosslinking or by
isolating polysomes. (B) let-7 target mRNAs (assayed by qRT-PCR)
were also enriched by capturing let-7 vs control miRNA. Target mRNA
capture was enhanced by crosslinking or polysome purification.
[0017] FIG. 9. miR-24 target mRNAs do not bind to exogenous miR-24
added at the time of K562 cell lysis. Streptavidin pull-downs were
performed in the presence of 300 nM Bi-miR-24 or Bi-cel-miR-67. RNA
bound to the beads was isolated and analyzed by qRT-PCR. miR-24
target mRNAs were not enriched in either Bi-cel-miR-67 (black) or
Bi-miR-24 pull-down (white). In the manuscript we had already shown
that the pull-down increases with time after transfection,
presumably reflecting the time required for RISC incorporation of
the exogenous Bi-miRNA.
[0018] FIG. 10. (B) miR-24 knockdown in K562 cells specifically
decreases miR-24, assayed by qRT-PCR in cells transfected with
miR-24 ASO (white) relative to control ASO (black). Expression
relative to U6 snRNA is normalized to control cells. (C) miR-24
knockdown with ASO increases K562 cell proliferation measured by
thymidine uptake, both in the presence and absence of TPA. The
decline in proliferation with TPA is completely restored by
antagonizing miR-24. (D and E) Knocking down miR-24 in WI-38 or
IMR-90 cells also significantly increases proliferation as measured
by thymidine incorporation 48 hr posttransfection. miR-24 knockdown
by ASO measured by qRT-PCR is normalized to U6 snRNA. Error bars in
(B-E) represent SD from three experiments. **p<0.01,
##p<0.001. (F) miR-24 overexpression increases the G1
compartment in HepG2 cells. HepG2 cells transfected with miR-24 or
control mimic for 48 hr were stained with propidium iodide and
analyzed by flow cytometry. Representative analysis of three
independent experiments is shown. (G) K562 cells, synchronized in
G2/M by nocodazole and then released, were analyzed by flow
cytometry. A representative experiment is shown in the top panel,
and the mean (.+-.SD) percentage of cells in each phase of the cell
cycle (from three independent experiments) is shown below (light
gray, G1; dark gray, S; black, G2/M). (H) qRT-PCR analysis of
miR-24 (normalized to U6 snRNA) from the partially synchronized
K562 cells in (G) shows that miR-24 is most highly expressed in G1
and declines as cells progress to S and G2/M phase.
#p<0.005.
[0019] FIG. 11. Bioinformatic Analysis of miR-24-Downregulated
Genes Suggests that miR-24 Regulates Cell-Cycle Progression and DNA
Repair. Major direct interaction network of the 248 genes
significantly downregulated after transfection of HepG2 cells with
miR-24 mimics. Nodes with at least five interactions (including
autoregulation) are highlighted.
[0020] FIG. 12. (A) miR-24 levels significantly increased when K562
cells were transfected with 10 or 50 nM miR-24 mimics as measured
by qRT-PCR analysis. Expression relative to U6 snRNA is depicted
normalized to control cells. (B) A 4-fold increase in miR-24,
obtained by transfecting 10 nM miR-24 mimic, reduces target
proteins. Cell lysates of K562 cells, obtained 72 hr after
transfection with indicated concentrations of control or miR-24
mimics, were analyzed by immunoblot. a-tubulin and HuR are loading
controls. Error bars represent mean.+-.SD from three independent
experiments. *p<0.05, **p<0.01, and #p<0.005.
[0021] FIG. 13. (A) miR-24 downregulates luciferase activity of a
reporter gene containing wild-type (wt) E2F2 MRE1. Mutations in the
miR-24 pairing residues (mt) rescue luciferase expression
(sequences and luciferase assays for candidate E2F2 WT 3'UTR MREs
are shown in Figure S3). (B) Predicted binding sites in the 3'UTR
of genes whose 3'UTR was repressed by miR-24 in (C) and binding
site mutations tested (indicated in red). (C) Expression of
reporter genes containing wild-type (wt) AURKB MRE1, BRCA1 MRE5,
CDC2 MRE1, CDK4 MRE1, and FEN1 MRE1 is significantly reduced upon
cotransfection of HepG2 cells with miR-24 mimics (white) and not
the control mimic (black). Mutations in the miR-24 pairing residues
(mt) rescue luciferase expression (sequences and luciferase assays
for all tested wt MREs for these genes are shown in Figures S6 and
S7). The CCNA2 MRE1 is not regulated. (D) However, miR-24 regulates
a 181 nt region containing the CCNA2 MRE1 in the luciferase vector.
Mutations in the binding residues of CCNA2 MRE1 within the extended
sequence restore luciferase activity.
[0022] FIG. 14. E2F2 is a key miR-24 target gene. (A) Increased
cell proliferation from antagonizing miR-24 in K562 cells is
blocked by siRNA-mediated knockdown of E2F2, but not MYC. Knockdown
is shown by immunoblot (Figure S5). K562 cells were cotransfected
with or without miR-24 ASO plus control siRNA or siRNAs targeting
E2F2 and/or MYC. The rate of cellular proliferation was determined
72 hr later by thymidine incorporation. Error bars represent
mean.+-.SD from three independent experiments. (B and C)
Downregulation of E2F2 mRNA (B) and protein (C) during TPA-mediated
differentiation of K562 cells to megakaryocytes is mediated by
miR-24 and can be completely inhibited by antagonizing miR-24. K562
cells were transfected with miR-24 or a control (CTL) ASO for 72 hr
and then treated with TPA for 6 hr. mRNA was assessed by qRT-PCR
normalized to GAPDH and normalized to control cells transfected
with CTL ASO. E2F2 protein was quantified by densitometry and
normalized to a-tubulin. (D and E) Transfection of K562 cells with
a miR-24 mimic reduces cell proliferation, which can be rescued by
expressing miR-24-insensitive E2F2 lacking the 3'UTR. K562 cells
were cotransfected with a vector expressing HA-tagged E2F2 or GFP
and miR-24 or cel-miR-67 (CTL) mimics for 72 hr before measuring
thymidine uptake. (E) Immunoblot probed for HA tag. Error bars
represent mean.+-.SD from three independent experiments.
*p<0.05, **p<0.01, and #p<0.005. (F) Model of the miR-24,
miR-17.sub.--92, MYC, and E2F network of cell-cycle regulators.
Here, we show that miR-24 directly suppresses expression of MYC and
E2F2 (and indirectly suppresses E2F1 and E2F3) and thereby
regulates the G1/S transition. Expression of the opposing miRNAs
encoded by the miR-17.sub.--92 and miR-106b.sub.--25 clusters that
promote cell proliferation is transcriptionally activated by the
same transcription factors that miR-24 suppresses (O'Donnell et
al., 2005; Petrocca et al., 2008). Therefore, miR-24 would be
predicted to reduce expression of the proliferation-promoting miRNA
clusters indirectly. These miRNAs also knock down the E2F genes but
probably to fine-tune their proliferative effect. MYC may also
suppress miR-24 transcription (Gao et al., 2009).
[0023] FIG. 15. (A) Direct interaction network of over-represented
subnetworks built from the 100 genes down-regulated by miR-24 and
also predicted to be miR-24 targets by TargetScan 4.2. (B) Direct
gene interaction small subnetworks built from the 248 mRNAs that
are down-regulated in miR-24 over-expressing cells.
[0024] FIG. 16. (A) Candidate miR-24 microRNA recognition elements
(MRE) in the 3'UTR of E2F2 mRNA predicted by rna22. Numbers in
parenthesis represent the location in the E2F2 3'UTR. (B) Only E2F2
MRE1 was found to be repressed by miR-24 by luciferase assay.
Inserting the other E2F2 MREs (MRE2-5) in the 3'UTR of a luciferase
gene had no significant effect on luciferase expression in HepG2
cells after over-expressing miR-24. Data are an average of two
independent experiments. ** p<0.01
[0025] FIG. 17. miR-24 down-regulates luciferase expression from a
reporter. gene containing the MYC and E2F2 MREs, before its effect
on cell proliferation can be detected. (A) 24 hr after ectopic
introduction of miR-24 mimic into HepG2 cells there is no
significant change in cellular proliferation compared to control
mimic transduced cells as measured by thymidine uptake. (B)
Luciferase assays performed 24 hr post transfection of HepG2 with
miR-24 (white) or cel-miR-67 (black) show Significant decreases in
reporter expression from reporters encoding E2F2 MRE 1 or MYC MRE3
or MRE6 in miR-24 overexpressing cells (white). Luciferase activity
was normalized. The mean and S.D. of 3 independent experiments is
shown.
[0026] FIG. 18. miR-24 down-regulates target mRNAs when transfected
at physiological levels. K562 cells were transfected with miR-24 or
cel-miR-67 (CTL) mimics at 2 nM, 10 nM and 50 nM. miR-24 levels
significantly increased when cells were transfected with 10 nM or
50 nM miR-24 (FIG. 5C). Target gene mRNA was assessed by qRT-PCR 72
hr later. E2F2, CCNA2, MYC, BRCA1 and H2AX mRNAs were
down-regulated in cells transfected with 10 nM or 50 nM miR-24
mimic. miR-24 over-expression had no effect on PCNA mRNA levels as
shown before (FIG. 5A). Light grey, dark grey and black bars
correspond to miRNA concentrations of 2 nM, 10 nM and 50 nM,
respectively. Expression is normalized to mRNA level in cells
transfected with 2 nM control miRNA. Representative experiments are
shown; each experiment was done twice with similar results.
[0027] FIG. 19. Candidate miR-24 microRNA recognition elements
(MRE) in the 3'UTR of target mRNAs (AURKB, BRCA1, CCNA2, CDK4, CDC2
and FEN1) predicted by rna22 or PITA. Only BRCA1 MRE5 is predicted
by TargetScan 4.2. Numbers in parenthesis represent the location in
the 3'UTR of the target gene. The effect of inserting these MREs on
luciferase expression is shown in FIG. 20.
[0028] FIG. 20. Inserting the miR-24 recognition elements present
in the 3'UTR of AURKB (MRE1), BRCA1 (MRE5), CDK4 (MRE1), CDC2
(MRE1) and FEN1 (MRE1) in a luciferase gene significantly reduces
luciferase expression in HepG2 cells transfected with miR-24 mimics
(white) and not the control mimics (black). Sequences of these MREs
are provided in Suppl. FIG. 6. Data are an average of 3
experiments. Error bars represent standard deviation.
*p<0.05.
[0029] FIG. 21. siRNAs knockdown MYC and E2F2 expression. K562
cells were transfected with siRNAs targeting E2F2 or MYC or GFP
(Ct1) for 48 hr before immunoblot analysis for E2F2 (A) or (B) MYC.
a-Tubulin was probed as a loading control.
[0030] FIG. 22. (A) Inhibiting miR-24 partially rescues MYC mRNA
expression in K562 cells treated with TPA. K562 cells were
transfected with miR-24 ASO or a control (CTL) ASO for 72 hr and
then treated with TPA for 6 hr. MYC mRNA levels were measured by
qRTPCR analysis after normalizing to GAPDH mRNA. (B) Immunoblot
analysis shows that antagonizing miR-24 in K562 cells increase MYC
protein levels in untreated cells. However, MYC protein expression
is still down-regulated after TPA treatment in cells transfected
with miR-24 ASO (miR-24).
[0031] FIG. 23. Activity of Bi-miR-24 mimics is similar to
non-biotinylated miR-24 mimics. (A) Effective increase in miR-24
(normalized to U6 SnRNA) in K562 cells 24 hr after transfection
with 100 nM 3'-Biotinylated miR-24 compared to 3'-Biotinylated
cel-miR-67 mimic transfected cells. (B) Activity of biotinylated
miR-24 (Bi-miR-24) is similar to non-biotinylated miR-24 mimic.
K562 cells were transfected with 100 nM control miRNA or miR-24
mimics or Bi-miR-24 for 24 hr and then cotransfected with 100 ng
psiCHECK2 plasmid (black) or psiCHECK2 containing a perfectly
complementary target site for miR-24 (white) and assayed for
luciferase expression after 24 hr. The Renilla luciferase signal
was normalized to the Firefly luciferase.
[0032] FIG. 24. Endogenous and Biotinylated miR-24 associate with
RISC proteins. Both endogenous and Biotinylated-miR-24 specifically
associate with RISC complex proteins such as HA-Ago1 and HA-Ago2 in
K562 cells. K562 cells were co-transfected with 100 nM Bi-miR-24 or
Bi-cel-miR-67 and 2 .mu.g plasmids expressing HA-Ago1 or HA-Ago2 or
Vector alone for 48 hr following which Streptavidin pull-downs were
performed from cytoplasmic extracts for 16 hr. qRT-PCR analysis
(normalized to U6 SnRNA) show that HA-Ago1 or HA-Ago2 pull-down
endogenous or transfected Bi-miR-24.
[0033] FIG. 25. Endogenous miR-24 and Bi-miR-24 co-sediment with
polysomes in K562 cell extracts. K562 cells were transfected with
100 nM Bi-cel-miR-67 (A) or Bi-miR-24 (B) for 24 hr and left
untreated (.tangle-solidup.) or treated with 200 uM puromycin
(.box-solid.) before fixation with formaldehyde and lysis by
sonication. The lysates were fractionated on 10-50% sucrose
gradients and the relative distribution of miR-24 (both endogenous
and exogenous) in each fraction was determined by qRT-PCR analysis.
In the absence of puromycin, the profiles of Bi-miR-24 and
endogenous miR-24 were similar with most miR-24 associated with the
more dense polysome-containing fractions (7-9). Treatment with
puromycin resulted in a decrease in the abundance of miR-24 or
Bi-miR-24 in the heaviest fractions (8,9) with a subsequent
increase in the less dense fractions (2,6).
[0034] FIG. 26. (A) K562 cells were transfected with 100 nM
Bi-miR-24 or Bi-cel-miR-67 miRNA for 6, 12, 24, 48 and 60 hr. RNA
was isolated from the Streptavidin pull-downs and analyzed by
qRT-PCR for miR-24 after normalization to U6 SnRNA. miR-24 was
specifically enriched in miR-24 pull-down and not the control
pull-down. The enrichment of miR-24 in miR-24 pull-downs increased
with time and was highest at 24 hr and later. (B) H2AX and E2F2
mRNAs (encoding 2 and 1,3'-UTR miR-24 binding sites, respectively),
assayed by qRT-PCR (normalized to GAPDH), are significantly
enriched in miR-24 and not the control pull-downs from K562 cells.
Enrichment was maximal when pull-downs were performed 24 hr after
transfection.
[0035] FIG. 27. (A) K562 cells were transfected with 100 nM
Bi-miR-24 or Bi-cel-miR-67 mimics for 24 hr following which
Streptavidin pull-downs were performed and the abundance of miR-24
target mRNAs was determined by qRT-PCR analysis normalized to GAPDH
mRNA. H2AX, E2F2, MYC and AURKB mRNAs (containing 2, 1, 2 and 1
binding sites for miR-24) are specifically enriched in miR-24
pull-downs. The house-keeping mRNA UBC was not enriched. (B) mRNA
targets of miR-34a (CDK4, CDK6 and MYB) are enriched specifically
in Bi-miR-34a pull-downs performed from K562 cells transfected with
100 nM Biotinylated miR-34a mimics.
[0036] FIG. 28. Presents Supplementary Table 1. Down-regulated
genes in over-expressing miR-24 cells with accession numbers,
Z-ratio, p-value and number of TargetScan 4.2 predicted miR-24
binding sites in the 3'UTR.
[0037] FIG. 29. Presents Supplementary Table 2. Integrated gene
list containing gene annotation, Gene Ontology (G0) processes, and
miR-24 binding sites for all genes analyzed via miR-24 pull-down or
microarray analysis.
[0038] FIG. 30. Presents Supplementary Table 3. Over-represented
subnetworks amongst 100 genes that are miR-24 TargetScan 4.2
predicted targets and are also down-regulated in miR-24
over-expressing cells.
[0039] FIG. 31. Presents Supplementary Table 4. miR-24 pull-down
genes with accession numbers, Z-ratio (enrichment value), p-value
and number of TargetScan 4.2 predicted miR-24 binding sites in the
3'UTR.
[0040] FIG. 32. Presents Supplementary Table 5. Novel potential
miR-24 gene targets based on miR-24 seed detection in coding region
or 5'UTR.
[0041] FIG. 33. Presents Supplementary Table 6. Over-represented
subnetworks within miR-24 pull-down genes.
[0042] FIG. 34. Presents Supplementary Table 7. Distribution of
genes enriched by miR-24 pull-down across cell cycle phases.
[0043] FIG. 35. Presents Supplementary Table 8. Sequence of primers
used for qRT-PCR.
DEFINITIONS
[0044] Combination Therapy: The term "combination therapy", as used
herein, refers to those situations in which two or more different
pharmaceutical agents are administered in overlapping regimens so
that the subject is simultaneously exposed to both agents.
[0045] Expression: As used herein, "expression" of a nucleic acid
sequence refers to one or more of the following events: (1)
production of an RNA template from a DNA sequence (e.g., by
transcription); (2) processing of an RNA transcript (e.g., by
splicing, editing, and/or 3' end formation); (3) translation of an
RNA into a polypeptide or protein; (4) post-translational
modification of a polypeptide or protein.
[0046] Gene: As used herein, the term "gene" has its meaning as
understood in the art. It will be appreciated by those of ordinary
skill in the art that the term "gene" may include gene regulatory
sequences (e.g., promoters, enhancers, etc.) and/or intron
sequences. It will further be appreciated that definitions of gene
include references to nucleic acids that do not encode proteins but
rather encode RNA molecules (e.g., functional RNA molecules, such
as rRNAs and/or tRNAs). For the purpose of clarity we note that, as
used in the present application, the term "gene" generally refers
to a portion of a nucleic acid that encodes an rRNA or a sensitive
fungal gene, as will be clear from context to those of ordinary
skill in the art.
[0047] Gene product or expression product: As used herein, the term
"gene product" or "expression product" generally refers to an RNA
transcribed from the gene (pre- and/or post-processing) or a
polypeptide (pre- and/or post-modification) encoded by an RNA
transcribed from the gene.
[0048] Hybridize: As used herein, the term "hybridize" refers to
the interaction between two complementary nucleic acid sequences.
The phrase "hybridizes under high stringency conditions" describes
an interaction that is sufficiently stable that it is maintained
under art-recognized high stringency conditions. Guidance for
performing hybridization reactions can be found, for example, in
Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y., 6.3.1-6.3.6, 1989 (and in more recent updated editions), and
in Sambrook et al., Molecular Cloning: A Laboratory Manual,
3.sup.rd ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, 2001. Aqueous and nonaqueous methods are described in these
references, and either can be used. Typically, for nucleic acid
sequences over approximately 50-100 nucleotides in length, various
levels of stringency are defined, such as low stringency (e.g.,
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at
50.degree. C. (the temperature of the washes can be increased to
55.degree. C. for medium-low stringency conditions)); 2) medium
stringency hybridization conditions utilize 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 60.degree. C.; 3) high stringency hybridization
conditions utilize 6.times.SSC at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and
4) very high stringency hybridization conditions are 0.5M sodium
phosphate, 0.1% SDS at 65.degree. C., followed by one or more
washes at 0.2.times.SSC, 1% SDS at 65.degree. C.) Hybridization
under high stringency conditions occurs between sequences with a
very high degree of complementarity. One of ordinary skill in the
art will recognize that the parameters for different degrees of
stringency will generally differ based various factors such as the
length of the hybridizing sequences, whether they comprise RNA or
DNA, etc. For example, appropriate temperatures for high, medium,
or low stringency hybridization will generally be lower for shorter
sequences such as oligonucleotides than for longer sequences.
[0049] Identity: As used herein, the term "identity" refers to the
overall relatedness between polymeric molecules, e.g. between
nucleic acid molecules (e.g. DNA molecules and/or RNA molecules)
and/or between polypeptide molecules. Calculation of the percent
identity of two nucleic acid sequences, for example, can be
performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second nucleic acid sequences for optimal alignment and
non-identical sequences can be disregarded for comparison
purposes). In certain embodiments, the length of a sequence aligned
for comparison purposes is at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at
least 95% or 100% of the length of the reference sequence. The
nucleotides at corresponding nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which needs
to be introduced for optimal alignment of the two sequences. The
comparison of sequences and determination of percent identity
between two sequences can be accomplished using a mathematical
algorithm. For example, the percent identity between two nucleotide
sequences can be determined using the algorithm of Meyers and
Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into
the ALIGN program (version 2.0) using a PAM120 weight residue
table, a gap length penalty of 12 and a gap penalty of 4. The
percent identity between two nucleotide sequences can,
alternatively, be determined using the GAP program in the GCG
software package using a NWSgapdna.CMP matrix.
[0050] microRNA (miRNA): As used herein, the term "microRNA" or
"miRNA" refers to an RNAi agent that is approximately 21-23
nucleotides (nt) in length. miRNAs can range between 18-26
nucleotides in length. Typically, miRNAs are single-stranded.
However, in some embodiments, miRNAs may be at least partially
double-stranded. In certain embodiments, miRNAs may comprise an RNA
duplex (referred to herein as a "duplex region") and may optionally
further comprises one or two single-stranded overhangs. In some
embodiments, an RNAi agents comprises a duplex region ranging from
15 to 29 bp in length and optionally further comprising one or two
single-stranded overhangs. An miRNA may be formed from two RNA
molecules that hybridize together, or may alternatively be
generated from a single RNA molecule that includes a
self-hybridizing portion. In general, free 5' ends of miRNA
molecules have phosphate groups, and free 3' ends have hydroxyl
groups. The duplex portion of an miRNA usually, but does not
necessarily, comprise one or more bulges consisting of one or more
unpaired nucleotides. One strand of an miRNA includes a portion
that hybridizes with a target RNA. In certain embodiments of the
invention, one strand of the miRNA is not precisely complementary
with a region of the target RNA, meaning that the miRNA hybridizes
to the target RNA with one or more mismatches. In other embodiments
of the invention, one strand of the miRNA is precisely
complementary with a region of the target RNA, meaning that the
miRNA hybridizes to the target RNA with no mismatches. Typically,
miRNAs are thought to mediate inhibition of gene expression by
inhibiting translation of target transcripts. However, in some
embodiments, miRNAs may mediate inhibition of gene expression by
causing degradation of target transcripts.
[0051] MicroRNA Agent: A "microRNA agent" as that term is used
herein, refers to an entity whose nucleotide sequence is
substantially identical to that of a natural miRNA. As will be
appreciated by those of ordinary skill in the art,
naturally-occurring miRNAs are comprised of RNA. As will be further
appreciated by those of ordinary skill in the art, RNA is a
particularly labile chemical. Furthermore, a variety of strategies
are known for preparing molecules that are structural mimics of RNA
(and therefore have a "sequence" in the same sense as RNA) but that
may, for example, have greater stability and/or somewhat altered
hybridization characteristics. For example, in some embodiments,
such structural mimics include one or more backbone modifications
(e.g., substitution of phosphorothioate backbone structures for
phosphodiester structures found in RNA) and/or one or more base
modifications (e.g., 2'-OMe modifications). In some embodiments,
such structural mimics are encompassed within "microRNA agent" as
that term is used herein.
[0052] miRNA target regulating factor: As used herein, the term
"miRNA target regulating factor" in its broadest sense, refers to
any agent that, when administered to a cell, alters level and/or
activity of an RNA that is also the target of an miRNA. In some
embodiments, miRNA target regulating factors alters the
level/activity to be higher in presence of agent than in absence.
In some embodiments, the level or activity is at least about 1.1,
about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7,
about 1.8, about 1.9, about 2, about 2.5, about 3, about 3.5, about
4, about 4.5, about 5, about 6, about 7, about 8, about 9, about
10, about 11, about 12, about 13, about 14, about 15, about 16,
about 17, about 18, about 19, about 20, to about 200 fold or even
higher in the cell as to regulate the effect of this target as
compared to not administering the siRNA. In some embodiments, miRNA
target regulating factors alters the level/activity to be lower in
presence of agent than in absence. In some embodiments, the level
or activity is at least about 1.1, about 1.2, about 1.3, about 1.4,
about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2,
about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about
6, about 7, about 8, about 9, about 10, about 11, about 12, about
13, about 14, about 15, about 16, about 17, about 18, about 19,
about 20, to about 200 fold or even lower in the cell as to
regulate the effect of this target as compared to not administering
the siRNA. In some embodiments, exemplary miRNA target regulating
factors can include siRNA, shRNA, and/or miRNA. In some embodiments
the siRNA, shRNA and/or miRNA targets RNA. Generally, miRNA target
regulating factors include a portion that is substantially
complementary to a target RNA. In some embodiments, miRNA target
regulating factors are at least partly double-stranded. In some
embodiments, miRNA target regulating factors are single-stranded.
In some embodiments, miRNA target regulating factors may be
composed entirely of natural RNA nucleotides (i.e., adenine,
guanine, cytosine, and uracil). In some embodiments, miRNA target
regulating factors may include one or more non-natural RNA
nucleotides (e.g., nucleotide analogs, DNA nucleotides, etc.).
Inclusion of non-natural RNA nucleic acid residues may be used to
make the miRNA target regulating factors more resistant to cellular
degradation than RNA. In some embodiments, the term "miRNA target
regulating factor" may refer to any RNA, RNA derivative, and/or
nucleic acid encoding an RNA that induces an RNAi effect (e.g.,
degradation of target RNA and/or inhibition of translation). In
some embodiments, the miRNA target regulating factors may comprise
a blunt-ended (i.e., without overhangs) dsRNA that can act as a
Dicer substrate. For example, such an miRNA target regulating
factor may comprise a blunt-ended dsRNA which is >25 base pairs
length, which may optionally be chemically modified to abrogate an
immune response.
[0053] Nucleic acid: As used herein, the term "nucleic acid," in
its broadest sense, refers to any compound and/or substance that
can be incorporated into an oligonucleotide chain. In some
embodiments, "nucleic acid" encompasses RNA as well as single
and/or double-stranded DNA and/or cDNA. Furthermore, the terms
"nucleic acid," "DNA," "RNA," and/or similar terms include nucleic
acid analogs, i.e. analogs having other than a phosphodiester
backbone. For example, the so-called "peptide nucleic acids," which
are known in the art and have peptide bonds instead of
phosphodiester bonds in the backbone, are considered within the
scope of the present invention. The term "nucleotide sequence
encoding an amino acid sequence" includes all nucleotide sequences
that are degenerate versions of each other and/or encode the same
amino acid sequence. Nucleotide sequences that encode proteins
and/or RNA may include introns.
[0054] RNA interference (RNAi): As used herein, the term "RNA
interference" or "RNAi" refers to sequence-specific inhibition of
gene expression and/or reduction in target RNA levels mediated by
an at least partly double-stranded RNA, which RNA comprises a
portion that is substantially complementary to a target RNA.
Typically, at least part of the substantially complementary portion
is within the double stranded region of the RNA. In some
embodiments, RNAi can occur via selective intracellular degradation
of RNA. In some embodiments, RNAi can occur by translational
repression.
[0055] RNAi agent: As used herein, the term "RNAi agent" refers to
an RNA, optionally including one or more nucleotide analogs or
modifications, having a structure characteristic of molecules that
can mediate inhibition of gene expression through an RNAi
mechanism. In some embodiments, RNAi agents mediate inhibition of
gene expression by causing degradation of target transcripts. In
some embodiments, RNAi agents mediate inhibition of gene expression
by inhibiting translation of target transcripts. Generally, an RNAi
agent includes a portion that is substantially complementary to a
target RNA. In some embodiments, RNAi agents are at least partly
double-stranded. In some embodiments, RNAi agents are
single-stranded. In some embodiments, exemplary RNAi agents can
include siRNA, shRNA, and/or miRNA. In some embodiments, RNAi
agents may be composed entirely of natural RNA nucleotides (i.e.,
adenine, guanine, cytosine, and uracil). In some embodiments, RNAi
agents may include one or more non-natural RNA nucleotides (e.g.,
nucleotide analogs, DNA nucleotides, etc.). Inclusion of
non-natural RNA nucleic acid residues may be used to make the RNAi
agent more resistant to cellular degradation than RNA. In some
embodiments, the term "RNAi agent" may refer to any RNA, RNA
derivative, and/or nucleic acid encoding an RNA that induces an
RNAi effect (e.g., degradation of target RNA and/or inhibition of
translation). In some embodiments, an RNAi agent may comprise a
blunt-ended (i.e., without overhangs) dsRNA that can act as a Dicer
substrate. For example, such an RNAi agent may comprise a
blunt-ended dsRNA which is .gtoreq.25 base pairs length, which may
optionally be chemically modified to abrogate an immune
response.
[0056] RNAi-inducing entity: As used herein, the term
"RNAi-inducing entity" encompasses any entity that delivers,
regulates, and/or modifies the activity of an RNAi agent. In some
embodiments, RNAi-inducing entities may include vectors (other than
naturally occurring molecules not modified by the hand of man)
whose presence within a cell results in RNAi and leads to reduced
expression of a transcript to which the RNAi-inducing entity is
targeted. In some embodiments, RNAi-inducing entities are
RNAi-inducing vectors. In some embodiments, RNAi-inducing entities
are compositions comprising RNAi agents and one or more
pharmaceutically acceptable excipients and/or carriers.
[0057] RNAi-inducing vector: As used herein, the term
"RNAi-inducing vector" refers to a vector whose presence within a
cell results in production of one or more RNAs that self-hybridize
or hybridize to each other to form an RNAi agent (e.g. siRNA,
shRNA, and/or miRNA). In various embodiments of the invention this
term encompasses plasmids, e.g., DNA vectors (whose sequence may
comprise sequence elements derived from a virus), or viruses (other
than naturally occurring viruses or plasmids that have not been
modified by the hand of man), whose presence within a cell results
in production of one or more RNAs that self-hybridize or hybridize
to each other to form an RNAi agent. In general, the vector
comprises a nucleic acid operably linked to expression signal(s) so
that one or more RNAs that hybridize or self-hybridize to form an
RNAi agent are transcribed when the vector is present within a
cell. Thus the vector provides a template for intracellular
synthesis of the RNA or RNAs or precursors thereof. For purposes of
inducing RNAi, presence of a viral genome in a cell (e.g.,
following fusion of the viral envelope with the cell membrane) is
considered sufficient to constitute presence of the virus within
the cell. In addition, for purposes of inducing RNAi, a vector is
considered to be present within a cell if it is introduced into the
cell, enters the cell, or is inherited from a parental cell,
regardless of whether it is subsequently modified or processed
within the cell. An RNAi-inducing vector is considered to be
targeted to a transcript if presence of the vector within a cell
results in production of one or more RNAs that hybridize to each
other or self-hybridize to form an RNAi agent that is targeted to
the transcript, i.e., if presence of the vector within a cell
results in production of one or more RNAi agents targeted to the
transcript.
[0058] Short, interfering RNA (siRNA): As used herein, the term
"short, interfering RNA" or "siRNA" refers to an RNAi agent
comprising an RNA duplex (referred to herein as a "duplex region")
that is approximately 19 basepairs (bp) in length and optionally
further comprises one or two single-stranded overhangs. In some
embodiments, an RNAi agents comprises a duplex region ranging from
15 to 29 bp in length and optionally further comprising one or two
single-stranded overhangs. An siRNA may be formed from two RNA
molecules that hybridize together, or may alternatively be
generated from a single RNA molecule that includes a
self-hybridizing portion. In general, free 5' ends of siRNA
molecules have phosphate groups, and free 3' ends have hydroxyl
groups. The duplex portion of an siRNA may, but typically does not,
comprise one or more bulges consisting of one or more unpaired
nucleotides. One strand of an siRNA includes a portion that
hybridizes with a target RNA. In certain embodiments of the
invention, one strand of the siRNA is precisely complementary with
a region of the target RNA, meaning that the siRNA hybridizes to
the target RNA without a single mismatch. In other embodiments of
the invention one or more mismatches between the siRNA and the
targeted portion of the target RNA may exist. In some embodiments
of the invention in which perfect complementarity is not achieved,
any mismatches are generally located at or near the siRNA termini.
In some embodiments, siRNAs mediate inhibition of gene expression
by causing degradation of target transcripts.
[0059] Short hairpin RNA (shRNA): As used herein, the term "short
hairpin RNA" or "shRNA" refers to an RNAi agent comprising an RNA
having at least two complementary portions hybridized or capable of
hybridizing to form a double-stranded (duplex) structure
sufficiently long to mediate RNAi (typically at least approximately
19 bp in length), and at least one single-stranded portion,
typically ranging between approximately 1 and 10 nucleotides (nt)
in length that forms a loop. In some embodiments, an shRNA
comprises a duplex portion ranging from 15 to 29 bp in length and
at least one single-stranded portion, typically ranging between
approximately 1 and 10 nt in length that forms a loop. The duplex
portion may, but typically does not, comprise one or more bulges
consisting of one or more unpaired nucleotides. In some
embodiments, siRNAs mediate inhibition of gene expression by
causing degradation of target transcripts. shRNAs are thought to be
processed into siRNAs by the conserved cellular RNAi machinery.
Thus shRNAs may be precursors of siRNAs. Regardless, siRNAs in
general are capable of inhibiting expression of a target RNA,
similar to siRNAs.
[0060] Small molecule: In general, a "small molecule" is understood
in the art to be an organic molecule that is less than about 5
kilodaltons (Kd) in size. In some embodiments, the small molecule
is less than about 3 Kd, 2 Kd, or 1 Kd. In some embodiments, the
small molecule is less than about 800 daltons (D), 600 D, 500 D,
400 D, 300 D, 200 D, or 100 D. In some embodiments, small molecules
are non-polymeric. In some embodiments, small molecules are not
proteins, peptides, or amino acids. In some embodiments, small
molecules are not nucleic acids or nucleotides. In some
embodiments, small molecules are not saccharides or
polysaccharides.
[0061] Vector: As used herein, "vector" refers to a nucleic acid
molecule capable of mediating entry of (e.g., transferring,
transporting, etc.) a second nucleic acid molecule into a cell. The
transferred nucleic acid is generally linked to (e.g., inserted
into) the vector nucleic acid molecule. A vector may include
sequences that direct autonomous replication, or may include
sequences sufficient to allow integration into cellular DNA. Useful
vectors include, for example
Description of Certain Embodiments
[0062] The present invention provides, among other things, a
discovery that a combination of analyses--biochemical interaction
assays (herein referred to as "pull-down assays") and a second
analysis--together create a powerful system that allows ready
identification of targets of microRNAs. Although some elegant
examples of miRNA gene regulation pathways have emerged by
thoughtful mining of miRNA target prediction algorithms and
differential mRNA expression profiling (2, 48), the unpublished
examples of failures using this approach are probably much more
common. The present invention encompasses the recognition that one
reason for such failures may be that differential expression
profiling often does not reveal miRNA effects (which may well occur
primarily at the level of translation). Furthermore, the present
invention demonstrates unexpectedly that true targets of miRNAs are
often not predicted by available algorithms and other techniques.
The present invention also provides kits for the detection of
miRNAs and identification of drug targets, as well as drug
screening and therapeutic applications.
MicroRNAs
[0063] The present invention provides systems that allow
identification of targets of microRNAs. As will be appreciated by
those of ordinary skill, the inventive methods can be applied to
identify targets of any of a variety of microRNAs. Representative
such miRNAs include, for example, miR-22, miR-125a, miR-24 (e.g.,
miR-24-1; miR-24-2), miR-23 (e.g., miR-23a, miR-23B), miR-27 (e.g.,
miR-27a, miR-27b), miR-17, miR-18, miR-19, miR-20, miR-34a, miR-92,
miR-125, miR-146a, miR-155, miR-181a, 200a, miR-48, miR-84, and
miR-241.
[0064] In some embodiments, the miRNA whose targets are identified
in accordance with the present invention is one whose expression
level increases or decreases during a particular developmental
stage of interest or in response to a particular trigger or event
of interest. For example, in some embodiments, the miRNA is one
whose expression level changes during terminal differentiation. To
give but one specific example, in some embodiments, the miRNA is
up-regulated during terminal differentiation of hematopoeitic
cells.
[0065] In some embodiments, an miRNA whose expression changes
during a particular developmental stage of interest, or in response
to a particular trigger or event of interest, increases or
decreases at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100 fold or more.
[0066] In some embodiments, the miRNA whose targets are identified
in accordance with the present invention is one that regulates cell
cycle progression. In some embodiments, the miRNA suppresses the
expression of cell cycle regulator genes. In some embodiments, the
miRNA is characterized in that its overexpression increases the
number of cells in the G1 phase; in some embodiments, the miRNA is
characterized in that its inhibition causes differentiating cells
to keep proliferating.
[0067] In some embodiments, the miRNA targets genes that initiate
pathways such as synthesis of DNA building blocks; DNA replication;
DNA damage recognition; expression, transcriptional regulation,
and/or post-translational modification of cyclins, cyclin-dependent
kinases, and/or other cell cycle regulators. In some embodiments,
the miRNA targets MYC, E2F, and/or their targets.
[0068] In some embodiments, the miRNA targets genes that are
implicated in progression through the cell cycle, for example,
through G1, the G1/S checkpoint, S, and/or G2/M.
[0069] In some embodiments, the miRNA targets genes that are
involved in DNA repair, including for example, genes (e.g., H2AX)
that sensitize cells to DNA damaging agents.
[0070] In some embodiments, the miRNA is selected from the group
consisting of miR-24 and/or other miRNAs in the same cluster. In
some embodiments, the miRNA is miR-22 or miR-125a. For example, in
some embodiments, the miRNA is selected from the group consisting
of miR-24 (e.g., miR-24-1; miR-24-2), miR-23 (e.g., miR-23a,
miR-23B), and miR-27 (e.g., miR-27a, miR-27b), etc. In some
embodiments, the miRNA is a member of the let-7 family of miRNAs.
In some embodiments, the miRNA is selected from the group
consisting of miR-48, miR-84, and miR-241. In some embodiments, the
miRNA is selected from the group consisting of miR-17, miR-18,
miR-19, miR-20, miR-34a, miR-92, miR-125, miR-146a, miR-155,
miR-181a, 200a. In some embodiments, the miRNA is one that is found
on chromosome 9, or on chromosome 19. In some embodiments, the
miRNA is one that is found in an intergenic region of a chromosome
(e.g., chromosome 19). In some embodiments, the miRNA is a viral
miRNA. In some embodiments, the miRNA is a member of the Herpes
virus family. In some embodiments, the miRNA is miR-K12-11. The
present Examples exemplify the invention with respect to
miR-24.
Pull-Down Technologies
[0071] The present invention combines use of interaction assays, or
pull-down assays, with the analyses (e.g. bioinformatic analyses),
to identify targets of microRNAs.
[0072] According to the present invention, a pull-down assay tests
direct, physical interaction between an miRNA of interest and its
target(s). In some embodiments, pull-down technologies for use in
accordance with the present invention isolate RNAs (e.g. mRNAs)
that are specifically bound to a miRNA of interest.
[0073] For example, in some embodiments, interacting RNAs are
isolated by increasing levels of the miRNA of interest in a cell,
and then identifying RNAs (or other factors) associated with the
overexpressed miRNA. An increase in miRNA level can be achieved by
any of a variety of available means including, for example,
transfection, injection, induction, etc. Those of ordinary skill in
the art will appreciate that pull-down assays may be performed
utilizing a natural miRNA molecule, but will further appreciate
that a variety of strategies are known for preparing molecules that
are structural mimics of RNA (and therefore have a "sequence" in
the same sense as RNA) but that may, for example, have greater
stability and/or somewhat altered hybridization
characteristics.
[0074] For example, in some embodiments, such structural mimics
include one or more backbone modifications (e.g., substitution of
phosphorothioate backbone structures for phosphodiester structures
found in RNA) and/or one or more base modifications (e.g., 2'-OMe
modifications). In some embodiments, such structural mimics are
locked nucleic acids (LNAs; see, for example, U.S. Pat. No.
6,977,295). Use of such miRNA mimics is encompassed by the present
invention; those of ordinary skill will readily appreciate when
discussions of miRNAs herein can relate to such mimics. In some
embodiments, such miRNAs mimics have increased stability as
compared with the natural miRNA. In some embodiments, miRNA mimics
bind with greater affinity and/or specificity to the same target(s)
bound by the natural miRNA. In some embodiments, such greater
affinity and/or specificity is at least about 1.5, 2, 2.5, 3, 3.5,
4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 or more (e.g.
15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 200, 300, 400, 500, 1000
or more) fold higher than that observed with the natural miRNA.
[0075] In some embodiments, an increased level of miRNA is about at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more (e.g. 20, 30, 40, 50,
60, 70, 80, 90, 100 or more) fold compared with levels of
endogenous miRNA. Those of ordinary skill in the art will readily
appreciate that different target RNAs (and/or different amounts of
a given target RNA) may be found in (and therefore identified
and/or isolated from as described herein) different cell types.
[0076] In some embodiments, interacting RNAs are isolated by
increasing levels of the miRNA of interest in a cell that itself
underexpresses the miRNA of interest (e.g. has a low endogenous
expression level as compared with other cells).
[0077] In some embodiments, cells containing overexpressed miRNA
are fixed prior to isolation of miRNA-target RNA complexes. Those
of ordinary skill in the art will be aware of a variety of
techniques for cell fixation. In some embodiments, the cells are
fixed by formaldehyde treatment. In one aspect, the present
invention encompasses the recognition that such fixation can
improve accuracy of miRNA target identification (see FIG. 8).
[0078] In some embodiments, miRNA-target complexes are isolated. In
some embodiments, such isolation includes isolation of a cellular
fraction. In some embodiments, the cellular fraction is or
comprises a polysome fraction. In some embodiments, the present
invention encompasses the recognition that isolation of a cellular
fraction (e.g. a polysome fraction) can improve accuracy of target
identification. In some embodiments, the present invention provides
the recognition that the miRNA profile maybe greater than 80% in
the dense polysome fractions. In some embodiments, the miRNA
profile in polysome fractions may be indistinguishable from the
profile of endogenous miRNA in the same cells (see FIG. 7).
[0079] In some embodiments, the overexpressed miRNA is labeled
(e.g. directly or indirectly). In some embodiments, an miRNA sense
strand is labeled; in some embodiments, and miRNA anti-sense strand
is labeled; in some embodiments, both sense and antisense strands
are labeled. In some embodiments, a label is covalently associated
with the miRNA. In some embodiments, a label is covalently or
non-covalently associated with the 5' end of an miRNA strand. In
some embodiments a label is covalently or non-covalently associated
with the 3' end of an miRNA strand.
[0080] It may be desirable to utilize a label that does not
interfere with activity of the miRNA. For example, in some
embodiments, labeled miRNA is still incorporated into RISC. Ability
to be incorporated into RISC can be assayed by any of a variety of
means including, for example, by (1) microscopy to show
colocalization of the labeled miRNA with processing bodies (P
bodies), and (2) immunoblot analysis of Ago1 and Ago2 enrichment in
pull-down fractions. In some embodiments, labeled miRNA retains
silencing activity, for example when tested on a model silencing
construct. As will be clear to those of ordinary skill in the art,
any of a variety of labels may be utilized in accordance with the
present invention. In some embodiments, the label is one that
facilitates isolation of the miRNA, for example when complexed with
one or more target RNAs. According to the present invention, as
illustrated in the Examples, biotin represents an appropriate and
useful label. In some embodiments of the present invention,
pull-down assays enrich target RNAs by a factor of 2, 3, 4, 5, 6,
7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, or more as compared with their cellular
expression level. Among other things, the present invention
encompasses the recognition that normalization of pulled-down
target RNA levels to cellular levels of the same RNA can materially
facilitate determination of true targets. Those of ordinary skill
in the art will be aware of a variety of strategies for performing
such normalization including, for example, comparison with any of a
variety of controls.
[0081] In some embodiments, specificity of pull-down assays is
assessed. In some embodiments, miRNA-target RNA complex formation
within an intact cell is assessed. In some embodiments, exogenous
miRNAs in excess of that which is overexpressed in the cell are
added to the pull-down assay (e.g., after cell lysis). In some
embodiments, quantitative analysis (e.g. quantitative RT-PCR) is
used to determine the amount of target RNA bound by the miRNA. As
can be evidenced in FIG. 9, the present invention provides the
recognition that a reduction of miRNA-target RNA binding may occur
when exogenous miRNA is added upon lysis.
[0082] The present invention specifically establishes that
pull-down assays without normalization may well be uninformative.
Without wishing to be bound by any particular theory, the present
inventors note that non-normalized pull-down studies often identify
overlapping sets of highly abundant transcripts, such as ribosomal
protein mRNAs, that can be pulled down using any of a variety of
different miRNAs (often including non-specific control miRNAs).
Moreover, the present invention demonstrates that true specific
targets often are not identified in such non-normalized assays.
Specifically, miR-24 validated targets H2AX and CDK6, or let-7
validated targets CDK6, DICER1, MCM4, and CDC20 were not enriched
in non-normalized pull-down experiments. Similarly, a recent study
(40) looking at miR-10a pulled-down-genes, which normalized only to
a control miRNA pull-down and did not take into account cellular
expression, identified 100 putative enriched target genes, most of
which were also highly abundant ribosomal proteins. In fact, 35 of
those 100 genes were pulled down with miR-24 relative to
cel-miR-67, raising questions of specificity.
[0083] In some embodiments of the present invention, target RNAs
are identified in pull-down assays as those that are enriched with
a Z-ratio of at least 1.5. In some embodiments, target RNAs are
identified as those that are enriched with a Z-ratio of at least
2.0. Indeed, in one aspect, the present invention encompasses the
finding that, despite common acceptance of a Z-ratio of 1.5,
increasing the stringency of analysis by requiring a Z-ratio of 2.0
significantly improves accuracy of target identification.
[0084] In some embodiments, pull-down assays utilized in accordance
with the present invention simultaneously or sequentially assess
interaction with at least one factor other than the relevant miRNA.
According to the present invention, such approaches can increase
specificity of assay results as compared with miRNA-only pull-down
assays. For example, in some embodiments, the second factor
comprises a cellular component (other than a target RNA) with which
the miRNA interacts. In some embodiments, the second factor
comprises a cellular component with which all miRNAs interact. For
example, in some embodiments, the second factor comprises one or
more components of RISC. To give but a few specific examples, those
of ordinary skill in the art will appreciate that the RISC complex
can be pulled down using an antibody such as Ago antibody.
[0085] In some embodiments, a second factor is pulled down using a
pull-down reagent that differs from the pull-down reagent used to
pull down the miRNA. For example, in some embodiments, an antibody
is used for one and a different category of binding agent (e.g.,
biotin/streptavidin) is used for the other. In some embodiments,
multiple factors are pulled down.
Analysis of Targets
[0086] Targets (e.g., target RNAs) identified and/or isolated as
described herein may be characterized by any of a variety of means.
In some embodiments, for example, RNAs are subjected to reverse
transcription (RT) and/or polymerase chain reaction (PCR). In some
embodiments, RT and/or PCR are performed under conditions that
permit quantification of the target RNA (quantitative
reverse-transcription-polymerase-chain-reaction, qRT-PCR).
[0087] Target RNAs identified and/or isolated as described herein
may be characterized through sequence analysis (e.g. deep
sequencing). In some embodiments, presence or absence of a
canonical miRNA binding site in the 5'UTR of a putative target RNA
is determined. Among other things, the present invention
demonstrates that not all miRNA targets in fact have canonical
miRNA binding sites in their 5'UTRs. For example, as specifically
exemplified herein, only 39 of the pulled-down genes are predicted
miR-24 target genes by TargetScan 4.2, and most of those do not
have evolutionarily conserved recognition sites. Other similar
algorithms based on evolutionary conservation and seed region
pairing give similar results, although the predicted gene sets are
not identical. This number might be increased by adding 19 genes
that have exact seed matches in their 5'UTR or coding region.
Nonetheless, they still represent a small fraction of the
pulled-down genes. Our high degree of experimental validation of a
subset of pulled-down target genes suggests that most of our set
are true targets. The implication is that a relaxation of the
identical seed pairing requirement (for instance taking into
account G:U wobbles or extensive pairing elsewhere in the sequence)
and/or the requirement of evolutionary conservation for each
particular site built into these algorithms might be desirable.
Analysis of the set of pull-down genes for miR-24 and other miRNAs
might provide a useful data set for training and developing
alternate prediction algorithms
[0088] Target RNAs identified and/or isolated as described herein
may be characterized through analysis of the extent to which their
expression level is affected by expression of the miRNA with which
they interact. The present invention encompasses the finding that
target RNAs often are only modestly affected by levels of their
cognate miRNAs. That is, according to the present invention, the
expression level of many target RNAs is not significantly affected
in response to increases or decreases in expression levels of the
miRNA of interest. For example, it is common for target RNA levels
to remain substantially unchanged, for example within a factor of
two, despite significant changes in miRNA level. Without wishing to
be bound by any particular theory, the present inventors propose
that this common insensitivity to miRNA levels may have contributed
to difficulties encountered in the past by others attempting to
identify and/or validate miRNA targets using gene expression
analysis. The present invention specifically demonstrates that
broad gene expression analysis technologies (e.g., arrays) may be
particularly poorly suited for the identification of miRNA target
RNAs.
[0089] In some embodiments, targets of miRNAs identified and/or
isolated as described herein are cloned (e.g. introduced into a
vector) as is known in the art.
[0090] To give a specific example, in the analysis of miR-24
targets described in the Examples, only 16% of the 269 pull-down
genes were significantly down-regulated by mRNA microarray analysis
after overexpressing miR-24, while 9 of 11 genes in the set had
significantly decreased mRNA by qRT-PCR. This suggests that even
when mRNA levels may be regulated by miRNAs, the degree of mRNA
down-regulation may not be substantial enough and the sensitivity
of mRNA microarrays may not be high enough for this to be an
efficient way of identifying miRNA target genes. Without wishing to
be bound by any particular theory, we note that, since the miR-24
pull-down set includes at least 18 transcription factors or
co-factors (notably E2F2, MYC, MYB, KLF2, VHL), regulated mRNAs may
decline indirectly because of reduced transcription and/or directly
by miRNA-mediated accelerated mRNA decay. It seems likely that
global analysis of differential protein expression, may be
preferable in the analysis of miRNA targets.
[0091] The foregoing notwithstanding, in some embodiments, one or
more target RNAs show expression levels that respond to changes in
level of miRNA of interest. In some embodiments, target RNA levels
are increased or decreased by at least 2 fold or more (e.g., 3, 4,
5, 6, 7, 8, 9, or even 10 fold or more) in response to
corresponding changes in miRNA levels.
[0092] As described below in the Examples, a variety of different
approaches were used to characterize targets of miR-24 as
exemplified herein. For example, potential targets were
characterized by microarray analysis, by qRT-PCR, by responsiveness
to the miRNA in a model gene (luciferase) assay, by protein
expression analysis, etc. Of 269 pull-down genes that were
identified by microarray analysis, 8 of 8 were quantitatively
enriched. The 3'UTRs of 5 of 5 genes (only 2 predicted to be
TargetScan 4.2 targets) were shown by luciferase assay to be
regulated by miR-24; protein expression of 9 of 9 genes (8 in this
manuscript plus H2AX in the accompanying patent application Ser.
No. 61/098,707, entitled "Therapeutic and Diagnostic Strategies",
filed on Sep. 19, 2008) was reduced by at least 2-fold and 9 of 11
genes had significantly reduced mRNA expression in cells
over-expressing miR-24. These confirmatory experiments suggest that
enrichment in the pull-down is very specific and that the
overwhelming majority of identified genes are likely to be true
targets of miR-24.
[0093] In some embodiments of the present invention, target RNAs
identified using pull-down technologies are subjected to network
analysis to identify biological processes that are represented (or
over-represented) among pulled-down RNAs.
[0094] For example, as exemplified herein, genes involved in
various aspects of cell cycle regulation are over-represented among
RNAs enriched in pull-down analyses with miR-24.
Kits and/or Compositions
[0095] The present invention also provides kits or compositions
including components useful in the identification of miRNA target
RNAs and/or drug targets as described herein. Such kits may be of
particular use in both academic and commercial research
applications.
[0096] For example, in some embodiments, inventive such kits
include one or more control miRNAs and/or reagents for labeling
miRNAs and/or for quantification of degree of target RNA enrichment
relative to cellular expression levels. In some embodiments, such
kits include one or more reagents useful in performing reverse
transcription, polymerase chain reaction, nucleic acid sequencing,
analysis of RNA expression levels, etc. In some embodiments, such
kits include one or more antibodies. In some embodiments, such kits
include one or more nucleic acid standards (e.g., size standards,
known miRNAs, etc.). In some embodiments, such kits include
nucleotide analogs useful in preparation of miRNA mimics.
[0097] To give but a few examples, in some embodiments, inventive
kits include, for example, biotin and/or streptavidin reagents
suitable for labeling miRNAs. In some embodiments, such reagents
achieve covalent attachment of the label (e.g., biotin or
streptavidin) to an miRNA. In some embodiments, such reagents
achieve non-covalent attachment of the label to an miRNA. For
example, a labeling reagent may associate a label with an miRNA via
hybridization. To give but one example, in some embodiments,
inventive kits comprise a means for attaching a standard sequence
element to an miRNA (e.g., via expression of the miRNA in a vector
containing the sequence element, direct linkage of a nucleic acid
fragment containing the sequence element, etc.), and further
comprise a label attached to the complement of the sequence
element.
[0098] In some embodiments, inventive kits comprise one or more of
a reverse transcriptase enzyme, deoxyribonucleotides, DNA
polymerase (e.g., thermostable DNA polymerase), chain-terminating
nucleotides, detectable (e.g., fluorescent, radioactive)
nucleotides, one or more buffers, distilled water, etc.
[0099] In some embodiments, the present invention provides kits or
compositions containing one or more agents that regulates mRNA
levels; in some such embodiments, the present invention provides
kits or compositions containing one or more agents that regulate
levels of one or more miRNA targets. In some embodiments, such a
provided kit or compositions will include one or more siRNA, for
example targeting a specific miRNA target. The present invention
therefore provides systems (including methods and compositions) for
regulating an miRNA target RNA through administration of a miRNA
target regulating factor. In some embodiments, miRNA target
regulating factors alters the level/activity to be higher in
presence of agent than in absence. In some embodiments, the level
or activity is at least about 1.1, about 1.2, about 1.3, about 1.4,
about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2,
about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about
6, about 7, about 8, about 9, about 10, about 11, about 12, about
13, about 14, about 15, about 16, about 17, about 18, about 19,
about 20, to about 200 fold or even higher in the cell as to
regulate the effect of this target as compared to not administering
the siRNA. In some embodiments, miRNA target regulating factors
alters the level/activity to be lower in presence of agent than in
absence. In some embodiments, the level or activity is at least
about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6,
about 1.7, about 1.8, about 1.9, about 2, about 2.5, about 3, about
3.5, about 4, about 4.5, about 5, about 6, about 7, about 8, about
9, about 10, about 11, about 12, about 13, about 14, about 15,
about 16, about 17, about 18, about 19, about 20, to about 200 fold
or even lower in the cell as to regulate the effect of this target
as compared to not administering the siRNA. In some embodiments,
exemplary miRNA target regulating factors can include siRNA, shRNA,
and/or miRNA. In some embodiments the siRNA, shRNA and/or miRNA
targets RNA. In some embodiments, the mixture comprises a RNA
mimic, which is a sequence that is analogous to another RNA
sequence. In some embodiments the mixture comprises a small
molecule agent or moiety that regulates the levels of the target
miRNA. In some embodiments, these components will be administered
together. In some embodiments, these components will be
administered separately.
[0100] In certain embodiments, inventive such kits contain all of
the components necessary to perform a relevant assay (e.g.,
detection assay, regulation assay, . . . ), including all controls,
directions for performing assays, and any necessary software for
analysis and presentation of results.
[0101] In some embodiments, components of inventive kits are
provided in individual containers and multiple such containers are
provided together in a common housing.
Exemplification
Materials and Methods
Cell Culture and Differentiation
[0102] HepG2 cells were grown in DMEM supplemented with 10% FCS.
HL60 and K562 cells were grown in RPMI-1640 supplemented with 10%
FCS. K562 cells (0.5.times.10.sup.6 cells/ml) were treated with TPA
(16 nM, 2 or 4 d) or hemin (100 .mu.M, 5 d) for differentiation
into megakaryocytes or erythrocytes, respectively. To induce
macrophage or monocyte differentiation, HL60 cells
(0.5.times.10.sup.6 cells/ml) were treated with TPA (16 nM, 3 d) or
vitamin D3 (25 nM, 5 d), respectively. Differentiation was verified
by flow cytometry staining for CD41a, CD61, and CD14 (antibodies
from BD Biosciences), by benzidine staining for hemoglobin and by
microscopic analysis of morphology.
RNA Isolation and Quantitative RT-PCR
[0103] Total RNA was isolated using Trizol reagent (Invitrogen) and
reverse transcribed using random hexamers and superscript II
reverse transcriptase (Invitrogen). qRT-PCR was performed in
triplicate samples using the SYBR Green master mix (Applied
Biosystems) and the BioRad iCycler. Results were normalized to
GAPDH. miRNA quantitative PCR was done in triplicate using the
TaqMan MicroRNA Assay from Applied Biosystems as per the
manufacturer's instructions and normalized to U6. Sequences of
primers are listed in Supplementary Table 8. HepG2 cells were
reverse transfected with miRNA mimics using Neofx (Ambion, Inc) as
per manufacturer's instructions. K562 cells were transfected with
miRNA mimics or antisense oligonucleotides using Amaxa
nucleofection following the manufacturer's protocol.
Transfection of miRNA Mimics, Antisense Oligonucleotides, siRNAs,
and Expression Plasmids
[0104] HepG2, WI-38 and IMR-90 cells were reverse transfected using
Neofx (Ambion, Inc) as per the manufacturer's instructions. K562
cells were transfected using Amaxa nucleofection following the
manufacturer's protocol. siRNAs targeting GFP (D-001940-01-05),
E2F2 (On-targetplus SMARTpool L-003260-00-005) or MYC
(On-targetplus SMARTpool L-003282-00-0005) were purchased from
Dharmacon and transfected into K562 cells (1.times.10.sup.6 cells)
for 48 hr using Amaxa. In some experiments K562 cells
(1.times.10.sup.6 cells) were transfected with miR-24 or cel-miR-67
miRNA mimics (100 nM or indicated concentrations, Dharmacon), with
or without a plasmid expressing HA-tagged E2F2 or eGFP (5 .mu.g)
for 48 hr using Amaxa. To determine the effect of miR-24 knockdown
on E2F2 and MYC expression, K562 cells (1.times.10.sup.6
cells/well) were transfected in triplicate wells with miR-24 or
control ASO (100 nM, Ambion) using Amaxa nucleofection following
the manufacturer's protocol and 72 hr later treated with TPA (16
nM) for 6 hr. The cells were then harvested followed by qRT-PCR and
Western blot analysis for MYC and E2F2.
Biotin Pull-Down Experiments
[0105] HepG2 cells (2.5.times.10.sup.5/well) were reverse
transfected with 3'-biotinylated miR-24 (Dharmacon) or
3'-biotinylated control miRNA (cel-miR-67) at a final concentration
of 30 nM using Neofx (Ambion, Inc) in six-well plates in
sextuplicate wells following the manufacturer's protocol.
Twenty-four hours later, the cells were trypinized and pelleted at
500.times.g. After washing twice with PBS and resuspension in 0.5
ml lysis buffer (20 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl.sub.2,
0.3% NP-40, 50 U of RNase OUT (Invitrogen), complete mini-protease
inhibitor cocktail (Roche Applied Science)), and incubation on ice
for 5 min, the cytoplasmic extract was isolated by centrifugation
at 10,000.times.g for 10 min. Streptavidin-coated magnetic beads
(50 .mu.A, Invitrogen) were blocked for 2 hr at 4.degree. C. in
lysis buffer containing 1 mg/ml yeast tRNA and 1 mg/ml BSA (Ambion)
and washed twice with 1 ml lysis buffer. Cytoplasmic extract was
then added to the beads and incubated for 4 h at 4.degree. C.,
following which the beads were washed five times with 1 ml lysis
buffer. RNA bound to the beads (pull-down RNA) or from 10% of the
extract (input RNA), was isolated using Trizol LS reagent
(Invitrogen). The level of mRNA in the miR-24 or control pull-down
was quantified by qRT-PCR and normalized to its abundance in the
input RNA.
miRNA Microrray
[0106] miRNA microarrays were performed as described in (S1).
Microarray Analysis
[0107] HepG2 cells (2.5.times.10.sup.5/well) were reverse
transfected in triplicate in six-well plates with either miR-24
mimics or control miRNA mimics (cel-miR-67) at a final
concentration of 30 nM using NeoFx (Ambion). Total RNA isolated 48
hr post-transfection (independently for two experiments) was
amplified, labeled and hybridized to Illumina arrays (Refseq-8).
Raw hybridization intensity data were log-transformed and
normalized to yield Z-ratios, which in turn were used to calculate
a Z-ratio value for each gene. The Z-ratio was calculated as the
difference between the observed gene Z-ratios for the experimental
and the control comparisons, divided by the standard deviation
associated with the distribution of these differences (S2). Z-ratio
values of >1.5 or 5-1.5 were chosen as cut-off values, defining
increased and decreased expression, respectively. To identify the
mRNAs directly bound to miR-24, biotin pull-down assays were
performed from two independent experiments (as described above) and
the isolated RNA was subjected to microarray analysis as above. For
each pull-down, Z-ratios>2.0 were chosen as cut-off. The
complete microarray data set is available at:
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17828.
TargetScan Analysis of miR-24 Target Genes
[0108] To determine whether a gene was also a predicted target of
miR-24, the presence of miR-24 binding sites was analyzed using
TargetScan 4.2 (http://www.targetscan.org). To determine whether a
gene is a predicted target of miR-24, the presence of miR-24
binding sites was analyzed using TargetScan 4.2
(http://www.targetscan.org) (Lewis et al., 2003) or rna22
(http://cbcsrv.watson.ibm.com/rna22_targets.html (Miranda et al.,
2006). miR-24 binding sites in the miR-24 down-regulated mRNAs
(Z-ratio>1.5) that had a sequence complementary to the miR-24
seed were identified by using PITA
(http://132.77.150.113/pubs/mir07/mir07_prediction.html). The
miR-24 mature miRNA sequence was obtained from miRBase
(http://microrna.sanger.ac.uk/sequences/). The 3' UTR sequences in
FASTA format were obtained from the UCSC Genome Browser [1] using
RefSeq version (Release 34, [2]). UTR coordinate intervals were
filtered through a Perl script to remove redundant UTRs from
transcript variants and non-reference genomic sequences yielding a
final set of 22,231 sequences (background set) after filtering.
Occurrence and frequencies of the target nucleotide sequences
(UGAGCC, CUGAGCC, and ACUGAGCC) were established for both the
background set as well as the subset of 3'UTR sequences present in
the miR-24-target gene set. For each target sequence we compared
the number of matches in the UTR sequences of both the target and
background set to the number of all possible N-mer matches of the
same size as the target sequence. The number of matches in the
target UTR sequences was contrasted to their background
distribution using a chi-square test in the R environment. Of the
249 target genes, 219 genes had an annotated, non-redundant UTR
sequence.
Gene Ontology (GO) Analysis
[0109] The Gene Ontology (GO) project provides structured
controlled vocabularies, or ontologies, to describe genes relative
to their biological processes. Each biological process consists of
a series of events achieved by one or more molecular functions. The
ontologies are stored in directed acyclic graphs where each node
represents a biological process and each subsequent node
corresponds to a more specialized term. Over-represented Gene
Ontology (GO) biological processes were determined using a MetaCore
tool which utilizes the hypergeometric distribution to calculate
the statistical significance (p-value) for a subset of genes
showing enrichment in a biological process. The value is equivalent
to the probability of a subset of genes from a specific experiment
(ie, miR-24 pulldown) to arise by chance given the number of total
genes associated with the biological process.
Network Visualization and Analysis
[0110] We developed a graphical representation of the molecular
relationships between proteins from miR-24 pulldown and miR-24
TargetScan 4.2 targets down-regulated in miR-24 over-expressing
cells. Network analysis of miR-24 pulldown genes and miR-24
TargetScan 4.2 targets down-regulated in miR-24 over-expressing
cells was performed using the MetaCore Analytical Suite (GeneCo
Inc., St Joseph, Mich., http://www.genego.com) and Ingenuity
Pathways Analysis (Ingenuity Inc. www.ingenuity.com). Proteins are
represented as nodes, and the biological relationship between two
nodes is represented as an edge (line). Nodes are displayed using
shapes that represent the functional class of the gene product. The
tools were used for functional and pathway analysis utilizing
support for edges in the networks from at least 1 reference from
the literature, from a textbook, or from canonical information
stored in the network generation software database--manually
curated for human protein-protein interactions, protein-DNA, and
protein-compound interactions.
[0111] To identify unconventional miR-24 binding sites, the coding
and 5'-UTR regions were downloaded from UCSC
(http://genome.ucsc.edu), for genes satisfying the following
criteria: (1) down-regulation after miR-24 over-expression
(Z-ratio>1.5) or enrichment by miR-24 pulldown (Z-ratio>2.0);
and (2) not identified as a miR-24 target by TargetScan 4.2. The
miR-24 mature miRNA sequence was obtained from miRBase
www.mirbase.com). The seed region was defined as any 7-mer
contained within positions 2-9 of the sequence (5'-TACGATCGA-3').
Using Perl scripts, we enumerated instances of this seed region in
any of the 5'UTR and/or coding regions. We analyzed all alternative
splice variants and enumerated potential miR-24 recognition sites
using the isoform that yielded maximum seed regions.
Cell Cycle Analysis
[0112] HepG2 cells were reverse transfected with miR-24 mimics or
control miRNA mimics as described above and 2 days later, treated
with nocodazole (100 ng/ml) to synchronize cells in G2/M phase of
the cell cycle. After 16 hr, cells were stained with propidium
iodide and analyzed by flow cytometry using a FACScaliber
instrument (Becton Dickinson) and Cellquest Pro software following
the manufacturer's protocol. To analyze changes in miR-24
expression with cell cycle progression, K562 cells were arrested in
G2/M phase by treatment with nocodazole (100 ng/ml) for 16 hr, and
then washed to remove nocodazole and grown in complete medium in
the absence of nocodazole. Cells were collected at indicated times
and analyzed for cell cycle distribution by propidium iodide
staining and flow cytometry using FlowJo software and by qRT-PCR
for miR-24 expression.
Luciferase Assay
[0113] HepG2 cells were reverse transfected (as above) in
triplicate with 30 nM miR-24 mimic or control miRNA mimic. Two days
later, cells were transfected using Lipofectamine 2000 (Invitrogen)
with psiCHECK2 (Promega) vector (0.5 .mu.g/well) containing a
single copy of the predicted MREs or the full-length 3'UTR of
indicated genes cloned into the multiple cloning site (NotI and
XhoI) of Renilla luciferase or control. After 24 hr luciferase
activities were measured using the Dual Luciferase Assay System
(Promega) and Top count NXT microplate reader (Perkin Elmer) per
manufacturer's instructions. Data were normalized to Firefly
luciferase. The 3'UTR of miR-24 target genes was PCR amplified
using human genomic DNA as template and primers containing the NotI
and XhoI restriction enzyme sites at the 5' end. The PCR products
were digested with NotI and XhoI and cloned into the 3'UTR of
Renilla luciferase of pSICHECK2. Individual wild-type and mutant
MREs were cloned into pSICHECK2 by annealing the forward and
reverse oligonucleotides containing NotI and XhoI sticky ends,
followed by phosphorylation (using T4 polynucleotide kinase (New
England Biolabs)) and ligation (quick DNA ligase (New England
Biolabs)). The wild-type short fragment in CCNA2 3'UTR (containing
WT CCNA2 MRE1) was cloned by PCR and the mutant short CCNA2 3'UTR
fragment (containing MT CCNA2 MRE1) was cloned by oligonucleotide
annealing as mentioned above.
Thymidine Incorporation Assay
[0114] To measure the effects of miR-24 on cell proliferation, K562
cells (1.times.10.sup.6 cells/well) were transfected with miR-24
ASO (100 nM) or control ASO using Amaxa nucleofection following the
manufacturer's protocol and 36 hr later treated with TPA (16 nM)
for 2 hr. The cells in duplicate wells were then incubated with
.sup.3H-Thymidine (2 pCi/well) for 2 hr and [.sup.3H]-incorporation
measured using a liquid scintillation counter (Beckman). The ratio
of [.sup.3H]-incorporation in miR-24 ASO-transfected cells relative
to that in cells treated with control ASO from 3 independent
experiments was compared.
Immunoblot
[0115] K562 cells (1.times.10) were transfected with miR-24 mimics
or control miRNA mimics (cel-miR-67) as above and 48 h later whole
cell lysates were prepared using RIPA buffer (150 mM NaCl, 1%
NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0).
Protein samples were quantified using Bradford reagent (BioRad) and
resolved on 10% SDS-PAGE gels and analyzed by immunoblot probed
with antibodies to MYC (Santa Cruz Biotechnology), E2F2 (Sigma),
Cyclin A (Santa Cruz Biotechnology), Chek1, PCNA, BRCA1, AURKB and
CDK4 (Cell Signaling Technology). a-Tubulin and HuR (Santa Cruz
Biotechnology) served as internal controls. All antibodies were
used at a dilution of 1:500. Western blots were quantified by
densitometry relative to a-tubulin.
Results
[0116] As described herein, the role of miRNAs during terminal
hematopoietic cell differentiation, was analyzed by microarray in 2
human leukemia cell lines--K562 cells differentiated to
megakaryocytes using 12-O-tetradecanoylphorbol-13-acetate (TPA) or
to erythrocytes with hemin, and HL60 cells differentiated to
macrophages using TPA or to monocytes using vitamin D3. Only a few
miRNAs were consistently up-regulated (by at least 40%) in all 4
systems of terminal differentiation--miR-22, miR-125a, and members
of the two miR-24 clusters--miR-24, miR-23a, miR-23b and miR-27a.
Consistent with other information (including, for example, as
described herein), miR-24 stood out as the most up-regulated miRNA.
The only member of the 2 miR-24 clusters that was not consistently
up-regulated was miR-27b, whose hybridization signal was
substantially lower for all conditions than the other cluster
members, suggesting that hybridization to that probe was
inefficient.
[0117] qRT-PCR analysis established that miR-24 transcript was in
fact upregulated, and further showed a 2- to 8-fold increase during
differentiation into megakaryocytes, erythrocytes, macrophages and
monocytes. miR-24 also increased 3-fold in HL60 cells
differentiated to granulocytes using DMSO. Expression of the
chromosome 19 miR-24 cluster primary transcript, encoding miR-23a,
miR-27a and miR-24, increased in both cell lines within 6 h of TPA
treatment, peaked at -12 hr and remained elevated for at least 2 d,
suggesting that the observed increase in mature miR-24 was due to
increased transcription. The Dicer-cleaved miRNA showed a slightly
delayed increase following TPA induction of K562 or HL60 cells,
becoming significant at 12-16 hr. Mature miR-24 levels remained
elevated for as long as was measured (4 d).
[0118] Because cessation of cell proliferation is a hallmark of
terminal differentiation, we first examined whether proliferation
is altered by either inhibiting or enhancing miR-24 function by
transfecting cells with miR-24 2'-OMe antisense oligonucleotide
(ASO) or miRNA mimics, respectively. When K562 cells were
transfected with miR-24 ASO, miR-24 was dramatically and
specifically reduced by qRT-PCR 36 hr later (FIG. 10B). DNA
replication, measured by thymidine incorporation, doubled in cells
transfected with miR-24 ASO compared to cells transfected with
control ASO (FIG. 10C). When K562 cells were differentiated with
TPA for 4 hr, thymidine incorporation declined by 60%. However, in
cells transfected with miR-24 ASO and treated with TPA, thymidine
uptake was indistinguishable from that of the control
ASO-transfected, but TPA-untreated, cells (FIG. 10C). Therefore,
miR-24 ASO fully restored proliferation to differentiating K562
cells. To examine whether miR-24 also inhibits cell proliferation
in nontransformed cells, we next antagonized miR-24 in early
passage WI-38 and IMR-90 normal diploid fibroblasts. Antagonizing
miR-24 in WI-38 and IMR-90 cells dramatically reduced miR-24 (FIG.
10D) and increased thymidine uptake>2-fold 48 hr after
transfection (FIG. 10E). Conversely, overexpressing miR-24 in HepG2
cells synchronized with nocodazole, which typically leads to
mitotic arrest and only 8% of cells in G1, increased G1 cells
3-fold (22%; miR-24 versus cel-miR-67, p<0.001) (FIG. 10F).
[0119] We next analyzed how miR-24 expression changes during normal
cell-cycle progression using K562 cells released at various times
from nocodazole treatment, which synchronized them in G2/M
(Bar-Joseph et al., 2008; O'Donnell et al., 2005) (FIGS. 10G and
10H). Before release, 90% of cells were in G2/M; 8 hr later, 65%
were in G1; and 12 hr after removing nocodazole, 45% were in S
phase. miR-24 was low in G2/M, increased >3-fold by 8 hr when
most cells were in G1, and then declined by 12 hr as cells
progressed into S phase. These results suggest that miR-24 is most
highly expressed in G1. Taken together with our finding that cells
transduced with miR-24 mimics accumulate in G1, these results
suggest that miR-24 regulates cell-cycle progression mostly by
blocking or delaying the G1/S transition.
[0120] The conventional approach to identify potential miRNA
targets is to analyze gene expression following miRNA
over-expression or knockdown (14, 15, 21). We therefore transfected
HepG2 cells, which have low endogenous miR-24 expression (FIG. 2A),
with a dsRNA that mimics Dicer-cleaved miR-24 increasing miR-24
expression-80-fold compared with cells transfected with control
miRNA (FIG. 2B). Total RNA, isolated 48 hr later from cells
transfected with miR-24 or control miRNA (cel-miR-67), mimic, was
amplified, labeled and hybridized to Illumina mRNA microarrays. 248
genes were down-regulated at least 2-fold by miR-24 over-expression
(Z-ratio>1.5), of which 100 were also predicted TargetScan 4.2
targets (22) (FIG. 2C, Suppl. Table 1,2). Amongst these 100
targets, only 20 genes have predicted miR-24 3'UTR binding sites
that are conserved in human, mouse, rat and dog, while 80 genes
have poorly conserved sites. The microarray data was validated by
performing qRT-PCR for 9 randomly chosen down-regulated genes
(TOP1, MBD6, H2AFX UBD, CNDP2, PER2, BCL2L12, STX16, ZNF317) that
spanned the range of significantly down-regulated genes (Z-ratios,
1.6-6.1). Six genes (CNDP2, PER2, STX16, UBD, BCL2L12 and ZNF317)
were predicted miR-24 targets by TargetScan 4.2 and 3 were not. All
9 genes were significantly down-regulated and the extent of
down-regulation correlated well with the degree of reduced
expression in the microarray data (FIG. 2D).
[0121] To determine what fraction of the downregulated genes are
likely direct targets of miR-24, we used two approaches. First, we
compared our experimental list of downregulated transcripts with
TargetScan predictions. One hundred downregulated genes were also
predicted by TargetScan 4.2 (Lewis et al., 2003) (FIG. 2C). Among
these 100 targets, however, only 20 have predicted miR-24 3'UTR
miRNA recognition sites (MRE) conserved in human, mouse, rat, and
dog. Second, we examined the frequency of a 3'UTR sequence
perfectly complementary to the miR-24 seed (hexamer [positions
2-7], heptamer [positions 2-8], and octamer [positions 2-9]). The
down regulated genes were highly enriched for miR-24 seed matches
(FIG. 2G). Just more than half of the 219 miR-24-down-regulated
transcripts that have an annotated 3'UTR contain a 3'UTR
complementary hexamer sequence (53%, p=2.times.10.sup.-16 relative
to the background frequency of the seed in the known
transcriptome); 32% have a heptamer match (p=7.times.10.sup.-15);
and 8% have an octamer seed match (p=0.0002). This significant
enrichment of predicted miR-24 target genes and the high frequency
of genes containing perfect seed matches suggest that a substantial
proportion of the downregulated genes may be direct miR-24
targets.
[0122] We next looked at whether the set of 100 down-regulated
genes, which were also predicted miR-24 targets, are enriched for
specific biological processes. A functional enrichment and network
analysis (GeneGo Inc) revealed statistically significant enrichment
for 49 processes, many of which are overlapping (FIG. 2E,F; Suppl.
Table 3). The top 3 most enriched GO processes involve DNA repair
(DNA damage checkpoint, double strand break repair by homologous
recombination, and recombinational repair; each enriched with
significance of p=0.0001). Also amongst the most significantly
enriched processes were categories involved in cell cycle
regulation (regulation of cell cycle, p=0.0002; cell cycle arrest,
p=0.0007; cell cycle, p=0.001; DNA recombination, p=0.001).
[0123] We previously found that miR-24 interferes with the DNA
damage response in terminally differentiated hematopoietic cells,
predominantly by reducing expression of the histone variant H2AFX,
which recruits and retains DNA repair factors at double-strand
breaks (Lal et al., 2009). In addition, multiple GO processes
involved in cell-cycle regulation were also highly enriched
(regulation of cell cycle, p=0.0002; DNA integrity checkpoint,
p=0.0003; cell-cycle arrest, p=0.0007; cell cycle, p=0.001; DNA
recombination, p=0.001). This was not surprising based on the
effect of miR-24 on cell-cycle progression. When networks were
developed to identify known directly interacting proteins from
these overrepresented biological processes, there was one cluster
of six genes centered around MYC (c-myc) and three other small
clusters involving two or three genes (FIG. 15A); the other 87
genes in the data set lack any previously annotated direct
interactions. Because the TargetScan algorithm might miss some
important miRNA-regulated genes, we also constructed a direct
interaction network from the 248 downregulated mRNAs. The direct
interaction network constructed from all significantly
downregulated mRNAs was a highly interactive set of 68 interacting
genes, many of which are important in cell-cycle regulation. The
major connected network of miR-24-downregulated genes is shown in
FIG. 3B; there were also some smaller networks (Figure S2B). Key
nodes of the major network are MYC (22 interactions), E2F2 (six
interactions), VHL (six interactions), CDC2 (six interactions),
CCNB1 (five interactions), and CDKN1B (five interactions). The MYC
and E2F2 transcription factors play a central role in regulating
G1/S transition and progression through S. They inhibit cell
differentiation and apoptosis and promote cellular transformation
(Bracken et al., 2004; Lebofsky and Walter, 2007). MYC regulates
the transcription of other genes in the network, including E2F2,
CDKN1B, CCNB1, CDC2, CDCA7, and RRM2. E2F2 also regulates the
transcription of other genes in the network, including CDC2, MYC,
RRM2, and the mini-chromosome maintenance proteins MCM4 and MCM1 0
that are essential for initiating DNA replication. These analyses
support our experimental findings that miR-24 regulates cell-cycle
progression and DNA repair.
[0124] When network diagrams were developed to identify directly
interacting genes from these over-represented biological processes,
one cluster of genes centered around MYC (c-myc) involving 14 genes
was identified (FIG. 2F); the other 86 genes in the data set lack
any previously published direct interactions. MYC is a
transcription factor that plays a central role in cell cycle
progression, apoptosis and cellular transformation (23, 24). In
particular MYC regulates the transcription of other genes in the
network, including CDKNIB (p27KIP1), a cyclin dependent kinase
(CDK) inhibitor that inhibits progression through G1 (25).
Therefore this analysis suggests that miR-24 may regulate cell
cycle progression and DNA repair during terminal differentiation
and its effect on MYC may play an important role in these
processes.
[0125] To validate the role of miR-24 in regulating the mRNAs that
were enriched in the pull-down, we began with MYC, since it was a
key node of both networks (FIG. 2F, 4C). To validate the
association of MYC mRNA and miR-24, streptavidin pull-downs were
performed from HepG2 cells transfected for 24 hr with Bi-miR-24 or
control Bi-miRNA (cel-miR-67) (FIG. 5A). MYC mRNA, but not mRNA
encoding the housekeeping gene UBC, was enriched -2.5 fold by
qRTPCR in the Bi-miR-24 streptavidin pull-down. The MYC 3'UTR is
488 nucleotides long and contains a poorly conserved 7-mer exact
match to the miR-24 seed, at positions 462-468 (FIG. 5B). To
determine the effect of miR-24 on MYC expression, we transfected
HepG2 and K562 cells with miR-24 mimics and 48 hr later, measured
MYC mRNA by qRT-PCR normalized to GAPDH. Over-expression of miR-24
decreased MYC mRNA by -2-4 fold in HepG2 and K562 cells (FIG.
5C,D). UBC mRNA, a negative control gene, did not change
significantly. The decrease in MYC mRNA was associated with a
similar decrease (85%) in MYC protein when miR-24 was
over-expressed for 72 hr in K562 cells (FIG. 5E). Further evidence
that the decrease in MYC expression by over-expressing miR-24 was
direct was provided by measuring changes in luciferase activity
upon miR-24 co-transfection from a reporter containing the MYC
3'UTR. Luciferase activity was unchanged from control reporters,
but was reduced 2.2-fold by miR-24 expression (FIG. 5F).
Collectively, these findings suggest that miR-24 binds to the MYC
3'UTR and down-regulates its expression.
[0126] We next sought to identify miR-24 MREs in the MYC 3'UTR.
TargetScan 4.2 predicts a single MRE containing a poorly conserved
7-mer exact seed match at positions 462-468 (although the recent
TargetScan 5.0 algorithm does not list MYC as a miR-24 target),
whereas rna22, an algorithm that does not require a seed match
(Miranda et al., 2006), identifies six potential miR-24 MREs in the
488 nt MYC 3'UTR, including the TargetScan 4.2-predicted MRE (MRE6)
(FIG. 5B). miR-24 overexpression specifically and significantly
reduced luciferase activity by 1.9- and 3.9-fold for MRE3 and MRE6,
respectively, but the other MREs had no significant effect on
luciferase activity (FIG. 5H). MRE3 has no seed but has extensive
complementarity with the 3' end of miR-24. Point mutations of MRE3
and MRE6 that disrupted miR-24 binding restored luciferase activity
(FIGS. 5G and 5I). These findings suggest that miR-24 binds to two
partially complementary sites (MRE3 and MRE6) in the MYC 3'UTR.
[0127] To gain a better understanding of miR-24 regulated genes, we
developed a direct approach to isolate miRNA-bound mRNAs. We
initially tried to optimize the biochemical method developed by S.
Cohen and colleagues (18) to pull-down mRNAs bound to HA-tagged
Ago1 in RISC in HepG2 and HeLa cells. However, we were unable to
obtain more than 2-fold enrichment for miR-24 in cells
over-expressing miR-24, compared to cells transfected with
cel-miR-67 (data not shown).
[0128] We next modified a method (26) of capturing miRNA-mRNA
complexes using streptavidin-coated beads from cells transfected
with miR-24 biotinylated at the 3'-end of the antisense strand or
control biotinylated miRNA (FIG. 3A). Biotinylated (Bi-)miR-24 had
unimpaired gene silencing activity in a luciferase assay in which a
fully complementary miR-24 sequence was engineered into the
luciferase 3'UTR (FIG. 3B), suggesting that the biotinylated active
strand was incorporated into RISC and bound to target genes as well
as the unmodified miRNA mimic. Moreover, in miR-24-transfected
HepG2 cells harvested 24 hr after transfection, miR-24 was
enriched-500-fold in the miR-24 pull-down compared to pull-down
from control biotinylated miRNA-transfected cells, as assessed by
qRT-PCR (FIG. 3C), suggesting that the pull-down conditions were
specific.
[0129] To improve the assay conditions, qRT-PCR was used to
quantify pulled-down mRNAs in K562 cells transfected with miR-24 or
cel-miR-67 harvested at different times after transfection. Two
target genes, CDK6, a previously validated miR-24 target with 3
predicted miR-24 binding sites (27), and H2AX, which has 2
predicted evolutionarily conserved miR-24 binding sites and is
down-regulated 2.3-fold in miR-24 over-expressing cells (Suppl.
Table 1), were chosen as likely positive controls, while the
housekeeping gene UBC was used as a negative control. Both H2AX and
CDK6 mRNAs, but not UBC mRNA, were enriched in miR-24 vs cel-miR-67
transfected cells at all times tested (6, 12 and 24 hr) (FIG. 3D).
The enrichment was greatest (2-3 fold) at 24 hr, which was chosen
for subsequent experiments. The specificity of the pull-down was
verified in HepG2 cells where H2AX and CDK6 mRNAs were reproducibly
enriched 4- and 2.5-fold, respectively, in cells transfected with
biotinylated miR-24 compared to control cells, and 2 housekeeping
genes (SDHA and UBC) were unchanged (FIG. 3E). The general
applicability of the pull-down to enrich for miRNA target genes was
verified for another miRNA, let-7. Streptavidin pull-down also
enriched for 2 well validated let-7 targets, HRAS and CDK6 (15, 28,
29), in let-7a-transfected HepG2 cells (FIG. 3F).
[0130] With the pull-down method validated, we next analyzed by
mRNA microarray the enrichment for mRNAs pulled down from duplicate
samples of biotinylated miR-24-transfected HepG2 cells, normalized
to total cellular RNA. 269 mRNAs were enriched >2-fold
(Z-ratio>2) in the miR-24 pull-downs (FIG. 4A, Suppl. Table
2,4). Although a Z-ratio of 1.5 is generally considered
significant, we excluded genes with Z-ratios of 1.5-2 from further
analysis because we were unable to validate by qRT-PCR analysis 3
of 3 randomly chosen genes with this level of enrichment. All 7 of
the genes we tested with a Z-ratio>2 were validated (FIG. 6A).
Of note, only a minority of pulled down mRNAs were predicted
TargetScan 4.2 targets (39; 14%) and only 8 genes had conserved
binding sites. An additional 19 genes had a perfect seed match
potential binding site in the coding region and 4 of these also had
a miR-24 binding site in their 5'UTR (Suppl. Table 5). Also only a
small minority of the pulled down mRNAs decreased by >2-fold
(42; 16%) when miR-24 was over-expressed, suggesting that many of
the mRNAs bound by miR-24 may be regulated to a greater extent by
inhibiting translation rather than by substantially altering mRNA
stability. 22 genes satisfied all 3 criteria.
[0131] An enrichment and network analysis of the 269 genes enriched
by at least 2-fold in the miR-24 pull-down experiment found a
highly significant over-representation of genes annotated as
involved in various aspects of cell cycle regulation (FIG. 4B,C;
Suppl. Table 6). Roles in cell cycle regulation could be attributed
to the 9 most over-represented GO processes, which were each
significantly over-represented with p-values ranging from 8E-18 to
6E-13; the 10.sup.th most significantly overrepresented GO process
was DNA damage response (p=6E-13). Therefore both approaches
strongly suggested a role for miR-24 in regulating both cell cycle
progression and DNA repair.
[0132] Two trends emerged when comparing the over-represented
processes identified by miR-24 target prediction and mRNA
down-regulation (`set 1`), with the processes prominent in the
miR-24 pull-down genes (`set 2`). First, the miR-24 pulldown genes
are highly enriched for a coherent set of processes that involve
interrelated pathways associated with cellular proliferation.
Second, for each cell cycle or DNA repair process identified, the
significance was overwhelmingly greater in set 2 of pull-down genes
than in set 1. A high score in the analysis (defined as -log
[p-value]) suggests that the network is highly saturated with
identified genes and that there are few nodes in the network not
identified in the experiment. For example for DNA replication, 12%
of all genes annotated to this GO process (31 of 267 annotated
genes) were in the pull-down data set 2 (P=8E-18, score 17). The
most significant GO process in set 1, DNA damage checkpoint,
included 4 of 44 genes (9%) and had a score of 4. Interestingly
this GO process was the 44.sup.th most significant process
over-represented in set 1, but the pulldown captured more of the
checkpoint genes (7 of 44, 16%) and hence had higher significance
(p=7E-06, score 5).
[0133] This analysis, particularly of the genes that were pulled
down with miR-24, suggests that miR-24 might act as a master
regulator of cell proliferation, suppressing many key genes that
control various aspects of cell cycle progression. Regulation
likely occurs at multiple levels. For example, not only is the
transcription factor E2F2 a miR-24 pulled-down gene target, but so
are many of the genes whose transcription it regulates (CCNA2,
PCNA, MCM2, MCM3, MYC, MYB, AURKB, RRM2, BRCA1, CHEK1). In fact, 28
of the miR-24 pull-down genes (-10%) are genes known to be
regulated by E2F transcription factors, representing 22% of the 130
known E2F-dependent genes compiled in a recent review (30).
Similarly, MYC is in the pull-down set of genes and so are many
genes known to be transcriptionally regulated by MYC (including
AURKB, TYMS, CDCA7, CEBPB, CDK4).
[0134] A recent genome-wide survey used microarray analysis of gene
expression in synchronized cells to identify 480 of 18,400
transcripts (2.6%), whose expression varies with the cell cycle in
primary fibroblasts (31). In line with the postulated role of
miR-24 in regulating the cell cycle, an analysis of the pull-down
genes shows that a large fraction (62/269, 23%) are periodic--8 are
preferentially expressed in M/G1, 25 in G1/S and 29 in G2/M (Suppl.
Table 7). This highly significant selective enrichment for cycling
genes, many of which control cell cycle transitions, supports the
idea that miR-24 regulates cell proliferation.
[0135] A direct interaction network, constructed from miR-24
pull-down genes, revealed a highly interactive network of genes
whose products regulate cell cycle checkpoints, transition through
G1, S and G2/M, and DNA repair (FIG. 4C). The interaction network
of the pull-down genes contains 67 genes and is much more connected
than the similar analysis of `set l` genes in which only 14 genes
demonstrate previously published direct interactions and the
network only has one node centered around MYC (FIG. 2F). The genes
in the pull-down network that have the most annotated interactions
with other pull-down genes are a "who's who" of genes involved in
cell cycle regulation: MYC (29 interactions), PCNA (12), BRCA1
(12), CDKN1B (p27KIP1, 8), CCNB 1 (cyclin B1, 7), E2F2 (6), CDK4
(6) and CCNA2 (cyclin A2, 6). Key nodes of the miR-24 pull-down
network include important genes that regulate multiple stage of
cell cycle progression. These include a CDK active in regulating G1
to S progression (CDK4) and cyclin A2 and cyclin B1, which regulate
progression through S and G2/M transitions, and the CDK2
phosphatase and activator CDC25A (32). It should be noted that
although the mRNA for CDK6, the other CDK that can substitute for
CDK4 and regulate G1 to S transition, was not significantly
enriched in the pull-down microarray analysis, CDK6 mRNA was
enriched in the pull-down by qRT-PCR (FIG. 3E) and is regulated by
miR-24 (27). Other genes that regulate progression through G1 and S
include the transcription factors MYC and E2F2 that participate in
regulating G1/S transition and progression through S (33).
[0136] Important genes required for DNA replication were also
pulled-down with miR-24, including ORC1L, which binds to origins of
replication to recruit the pre-replication complex. Of note 5 of
the 6 MCM genes in the pre-replication complex were also pulled
down, as was the licensing factor CDT1, required to initiate
replication (34), and PCNA, which forms a moving platform to
recruit replication enzymes to the replication fork, and 2 genes
that make up its RFC chaperone complex (RFC4 and RFC5) (35). In
addition the pulldown captured genes encoding a key enzyme required
for nucleotide synthesis (thymidylate synthetase, TYMS) and many
DNA replication enzymes, the primase PRIM 1, the DNA polymerases
POLA2 and POLE, the topoisomerase TOP2A required to relieve
torsional stress generated during DNA replication, and the repair
genes needed to remove Okazaki fragments, FEN 1 and EXO 1.
[0137] In addition to inhibiting transition to mitosis by targeting
the A and B cyclins, genes associated with key steps in mitosis
were also pulled-down. These included mRNAs for genes involved in
chromosomal attachment to the mitotic spindle (CENPA), mitotic
spindle formation, stability and regulation (CDCA8, aurora B kinase
that regulates the kinetochore and chromosomal segregation (36)),
microtubule dynamics (STMN1), and the anaphase promoting complex
(APC) adaptor CDC20.
[0138] Silencing the genes mentioned above would all be expected to
inhibit cell division. In addition to these genes, miR-24 also
interacted with mRNAs for genes that encode for cell cycle
progression inhibitors. Prominent in the network is the cyclin D
inhibitor CDKN 1 B (p27KIP 1), an inhibitor of the CDK required for
progression through G1 (25). Previous work also showed that
p161NK4A, an inhibitor of the cyclin D CDKs, is regulated by
miR-24, although it was not enriched in the pull-down microarray
analysis.
[0139] Prominent in the network are other genes involved in
arresting the cell cycle, especially in response to DNA damage,
notably CHEK1, which participates in the G2/M checkpoint and is
activated by ATR in response to unresolved DNA damage (37); BRCA1,
which participates in a surveillance complex that activates
double-strand break repair (38); PCNA involved in repairing
replication-mediated DNA damage; FEN1, a flap endonuclease (39)
involved in base excision repair (BER). In addition to these
checkpoint proteins, many of the miR-24-bound genes are key players
in multiple DNA repair pathways, including BER (UNG, FEN1, EXO1),
H2AFX, a histone variant phosphorylated at sites of double-stranded
DNA damage (40); and XBP1, a transcription factor that up-regulates
DNA repair genes (41). There are other examples of regulating both
a gene required for cell cycle progression and its inhibitor--CDT1
and GMNN; CDC20 and both MAD2L1 and the F box gene FBXO5, and
another F box gene SKP1 and its target p27KIP1 (42). This suggests
that the role of miR-24 in regulating the cell cycle may be
complex. However if cells are unable to exit G1 and replicate their
DNA, it may be economical to suppress the expression of the
inhibitory genes that guard the genome from propagating damaged
DNA.
[0140] To validate the miR-24 pull-down genes further, we analyzed
the streptavidin pull-down samples from HepG2 cells transfected
with either biotinylated miR-24 or cel-miR-67 by qRT-PCR for 6
genes enriched in the pull-down microarrays (FIG. 4C) that are
important in cell cycle progression and/or DNA repair (E2F2, H2AX,
PCNA, AURKB, CCNA2, BRCA1 and CHEK1) and 3 genes not enriched (SDHA
and the E2F2 homologs E2F1 and E2F3). The qRT-PCR analysis
replicated the microarray results (FIG. 6A). Of note, of the 6
miR-24-associated genes studied, only E2F2 and H2AX mRNAs were
significantly reduced (e.g., at least about 2-fold) by microarray
analysis after miR-24 over-expression.
[0141] To validate that these genes are direct targets of miR-24,
the 3'UTR of E2F1, E2F2, E2F3, CCNA2, CDK4 and BRCA1 were cloned
downstream of luciferase, and luciferase activity was measured
following co-transfection with miR-24 or the control miRNA mimic.
The 3'UTR of E2F2, CCNA2, CDK4 and BRCA1, but not of the control
genes (E2F1 and E2F3), significantly repressed luciferase activity
in a miR-24-dependent manner (FIG. 6B). mRNA expression of 10
pull-down genes and 3 control genes was also compared by qRT-PCR in
miR-24 or cel-miR-67 over-expressing cells. Eight of the 10
pull-down genes had significantly reduced mRNA coincident with
miR-24 over-expression, but the difference was >2-fold only for
E2F2, CDK4, AURKB and TOP1 (FIG. 6C). A different, but overlapping,
set of 4 genes in this group (E2F2, CDC2, FEN1, TOP1) were
identified as significantly down-regulated by miR-24 by microarray
(Suppl. Tables 1, 2). If these results are typical of the extent of
mRNA down-regulation by miRNA over-expression, they suggest that
mRNA microarray is not sensitive enough to identify most miRNA
target genes, consistent with the low overlap between pull-down and
down-regulated genes (FIG. 4A). Of note, qRT-PCR analysis
identified all 3 E2F paralogs as significantly down-regulated by
over-expressing miR-24, although the two E2F genes that are not
miR-24 target genes were not identified by the less sensitive
microarray analysis. The down-regulation of E2F1 and E2F3 may be
secondary to E2F2 down-regulation since the E2F-family of
transcription factors are known to regulate each other (30, 43, 44)
or may be mediated by MYC, since MYC and E2F1 have been shown to
activate each other's transcription (45).
[0142] Although target gene mRNA levels might not be altered by
miRNA over-expression, reduced protein levels are expected. Protein
expression of all seven pull-down genes examined (E2F2, CCNA2,
PCNA, CHEK1, BRCA1, AURKB, CDK4), quantified by densitometry of
immunoblots, decreased by 53-98% in cells transfected with miR-24
mimics, compared to cells transfected with the control mimics (FIG.
6D). H2AX protein was also reduced by 88% (accompanying
manuscript). These analyses demonstrate that the miR-pulldown
specifically identifies miR-24 target genes. Moreover, most target
genes show some reduction in mRNA levels. However, these modest
changes in mRNA may be difficult to capture by current microarray
technology.
[0143] If miR-24 inhibits cell cycle progression and DNA
replication as postulated, then antagonizing miR-24 should enhance
cell proliferation, while overexpressing miR-24 should cause cell
cycle arrest. To test this hypothesis, K562 cells, which express
higher levels of endogenous miR-24 than HepG2 cells (FIG. 2A), were
transfected with miR-24 2'-OMe antisense oligonucleotide (ASO) to
knock down miR-24. miR-24, but not control miRNAs, was dramatically
reduced 36 hr later (FIG. 6E). DNA replication, measured by
thymidine incorporation, doubled in cells transfected with miR-24
ASO compared to cells transfected with a control ASO (FIG. 6F).
When K562 cells were treated with terminal differentiation-inducing
TPA for 4 hr, thymidine incorporation declined by 60%. However, in
cells transfected with miR-24 ASO and treated with TPA, thymidine
uptake was indistinguishable from that of the control
ASO-transfected but TPA untreated cells. Therefore miR-24 ASO fully
restored replicative capacity to differentiating K562 cells.
Conversely, over-expression of miR-24 in synchronized HepG2 cells
led to a substantial increase in G1 cells (22.4% in miR-24
transfected cells vs 8.2% in cel-miR-67-controls, p<0.001) (FIG.
6G). These results suggest that miR-24 is a major regulator of cell
cycle progression, operating mostly to block or delay the G1/S
transition. Although miR-24 regulates genes involved in progression
through G1, S and G2/M, its effect on progression through G1 and S
may be more important.
[0144] We next examined the effect of miR-24 on E2F2 because E2F2
is downregulated by miR-24 overexpression by microarray, is a key
node in the gene interaction network (FIG. 11), and plays a crucial
role in regulating progression through G1, where
miR24-overexpressing cells pile up (Polager and Ginsberg, 2008).
qRT-PCR analysis confirmed that E2F2 mRNA was significantly
downregulated by overexpressing miR-24 (FIG. 6C). In addition, the
related E2F family members, E2F1 and E2F3, were also significantly
decreased, although these two genes were not identified by the less
sensitive microarray analysis. E2F1 and E2F3 downregulation may be
secondary to E2F2 downregulation because the E2F family of
transcription factors regulates each other (Bracken et al., 2004;
Vernell et al., 2003) or may be mediated by MYC, given that MYC and
E2F1 have been shown to transactivate each other (Fernandez et al.,
2003). As expected, E2F2 protein (FIG. 6D) was also substantially
reduced (9-fold).
[0145] The E2F transcription factors activate the transcription of
many genes essential for DNA replication, cell-cycle progression,
and DNA repair. If miR-24 overexpression downregulates E2F2, E2F2
target gene mRNAs would also be expected to decline after ectopic
miR-24 expression. The effect of ectopic miR-24 expression in HepG2
cells on transcripts of 10 E2F targets that are important for
cell-cycle progression and DNA repair (AURKB, BRCA1, CCNA2, CDC2,
CHEK1, FEN1, PCNA, RRM2, MCM4, and MCM10) was analyzed 48 hr later.
Eight of the ten transcripts, with the exception of BRCA1 and PCNA,
were significantly reduced (>40%) (FIG. 6C). A subset of these
genes (CDC2, MCM4, MCM10, RRM2, and FEN1) was also significantly
downregulated by miR-24 overexpression by microarray (Table S1).
mRNA microarray may not be sensitive enough to identify some genes
whose expression is suppressed, either directly or indirectly, by a
miRNA. Protein levels of E2F2 and all seven miR-24 target genes
examined (AURKB, BRCA1, CCNA2, CDC2, CHEK1, FEN1, and PCNA),
quantified by densitometry of immunoblots, decreased by at least
2-fold (FIG. 6D). A possible explanation for the lack of
correlation between the mRNA and protein levels of BRCA1 and PCNA
is that mRNA levels were measured 48 hr after transfection of one
cell type (HepG2), whereas protein levels were assayed 72 hr
posttransfection in K562 cells. In fact, BRCA1, but not PCNA, mRNA
was reduced by 2-fold when K562 cells were transfected with miR-24
for 3 days (FIG. 18).
[0146] Because miR-24 overexpression reduced MYC protein by 85%
(FIG. 5E), MYC target genes should also be downregulated.
Consistent with this hypothesis, 11 known MYC-regulated genes (A
TAD3A, ACTL6A , ARHGEF7, CCNB1, CDCA7, EXOSC8, E2F2, METAP2, N-PAC,
RRM2, and UBE2C) were significantly downregulated in
miR-24-overexpressing HepG2 cells by mRNA microarray (Table S1).
Among MYC-regulated genes, CDK4 is an important mediator of MYC's
effects on cellular proliferation (Hermeking et al., 2000).
Although CDK4 mRNA was not significantly altered by microarray,
CDK4 mRNA declined 4-fold after ectopic expression of miR-24 in
HepG2 cells by more sensitive qRT-PCR assay (FIG. 6C), and CDK4
protein became undetectable (FIG. 6C). Therefore, miR-24
overexpression decreases the levels of many genes that are
important in cell-cycle progression.
[0147] In these experiments, we overexpressed miR-24 80-fold above
the level in undifferentiated K562 cells, whereas the physiological
increase after TPA treatment of K562 cells is only 8-fold. To
determine whether these genes are regulated by a physiological
increase in miR-24, these experiments were repeated by transfecting
K562 cells with varying miR-24 mimic concentrations (2-50 nM).
Transfection of 2 nM miR-24 did not significantly alter miR-24,
whereas 10 and 50 nM miR-24 increased miR-24 levels by 4- and
28-fold, respectively (FIG. 12A). E2F2, MYC, and three of four
other E2F2-regulated mRNAs (AURKB, CCNA2, and H2AX, but not PCNA)
(FIG. 18) and protein levels of all seven genes tested (E2F2,
AURKB, CHEK1, CCNA2, CDK4, MYC, and PCNA) were all significantly
reduced by a 4-fold increase in miR-24 (FIG. 12B). Therefore, the
genes that were identified by mRNA microarray as down regulated
after ectopic miR-24 expression are likely physiologically relevant
direct and/or indirect miR-24 targets.
[0148] None of the three E2F paralogs (E2F1, E2F2, and E2F3) are a
predicted target of miR24, and their 3'UTRs do not contain a miR-24
seed match sequence. We nonetheless tested whether the E2F 3'UTRs
might be directly regulated by miR-24 by luciferase assay. The
E2F2, but not the E2F1 or E2F3, 3'UTR significantly repressed
luciferase activity in a miR-24-dependent manner (FIG. 6B),
suggesting that E2F2 is a direct miR-24 target. rna22 identified
five candidate E2F2 3'UTR miR-24 MREs (FIGS. 13A and 16). miR-24
significantly suppressed luciferase activity of a reporter gene
containing the E2F2 MRE1. E2F2 MRE1 does not have a seed match in
the 3'UTR, even if G:U wobbles are allowed, but has extensive
complementarity to miR-24 elsewhere. Point mutations that disrupt
base pairing between miR-24 and E2F2 MRE1 rescued luciferase
expression, verifying that miR24 specifically recognizes the E2F2
MRE1.
[0149] To verify further that MYC and E2F2 are direct targets of
miR-24, we also looked at changes in expression of luciferase
reporter genes 24 hr after transduction of HepG2 cells with miR-24
mimic. At this early time, thymidine uptake of HepG2 cells does not
significantly change (FIG. 17A), but ectopic miR-24 still
suppresses luciferase reporters encoding MYC MRE3 or MRE6 or E2F2
MRE1 (FIG. 17B). The identification of seedless E2F2 and MYC MREs
confirms previous studies showing that MREs lacking a seed with
good downstream complementarity can contribute to miRNA gene
regulation (Didiano and Hobert, 2006, 2008; Vella et al.,
2004).
[0150] Because seedless MREs contributed to the regulation of E2F2
and MYC by miR-24, we next investigated whether some of the E2F2-
and MYC-regulated genes, whose transcripts declined in response to
miR-24, might also be direct miR-24 targets even though they might
lack a predicted MRE. We selected eight genes (AURKB, BRCA1, CCNA2,
CHEK1, CDC2, CDK4, FEN1, and PCNA) that play important roles in
cell-cycle progression and cloned their entire 3'UTRs into the
luciferase reporter. The 3'UTR of six out of eight genes (AURKB,
BRCA1, CCNA2, CDC2, CDK4, and FEN1, but not CHEK1 or PCNA) was
significantly repressed by miR-24, suggesting that these genes may
be direct targets (FIG. 6B). To confirm that these genes are direct
miR-24 targets, we next sought to identify the miR-24 MREs that
regulate their expression. Among the six genes whose 3'UTR was
specifically repressed by miR-24, BRCA1 is the only gene that is
predicted by TargetScan. The BRCA1 3'UTR contains a nonconserved
perfect 7-mer seed match sequence that functions as a miR-24 MRE by
luciferase assay (FIGS. 13B and 13C). To identify potential miR-24
MREs in these E2F2- and MYC-regulated genes, we used the rna22 or
PITA algorithms, which allow G:U wobbles or seed mismatches. These
algorithms identified one candidate MRE for AURKB; five for BRCA1
(which included the TargetScan BRCA1 site); and three sites each
for CCNA2, CDC2, CDK4, and FEN1 (FIG. 19). miR-24 significantly
repressed luciferase activity of one MRE for five out of six of
these reporter genes (AURKB MRE1, BRCA1 MRE5, CDC2 MRE1, CDK4 MRE1,
and FEN1 MRE1) (FIGS. 13B, 13D, and 20). A1 though CCNA2 MRE1
appeared to be inactive, a longer fragment (181 nucleotides) from
the CCNA2 3'UTR (that included only CCNA2 MRE1) significantly
repressed luciferase expression in a miR-24-dependent manner when
cloned into the luciferase vector 3'UTR (FIG. 13D). Point mutations
that disrupt base pairing between miR-24 and the five minimal MREs
and the CCNA2 MRE within the extended sequence rescued luciferase
expression, verifying that these MREs are regulated by miR-24
(FIGS. 13B-13D). Therefore, we have identified and verified by
mutation seven seedless miR-24 MREs in genes important in
cell-cycle progression.
[0151] Because both MYC and E2F2 are important cell-cycle
progression regulators, we next examined their contributions to the
increased cellular proliferation from antagonizing miR-24 by
knocking down MYC and/or E2F2 in K562 cells cotransfected with
miR-24 ASO (FIGS. 14A and 21). Introducing miR-24 ASO into K562
cells doubled thymidine incorporation (as in FIG. 1C). E2F2
knockdown completely abrogated the proliferative effect of miR-24
ASO, but MYC knockdown had no significant effect. Moreover, E2F2
downregulation by miR-24 is physiologically relevant. When K562
cells were terminally differentiated to megakaryocytes with TPA,
the decrease in E2F2 mRNA and protein was completely blocked by
inhibiting miR-24 (FIGS. 14B and 14C). Conversely, ectopic
expression of miR-24-insensitive E2F2 lacking the 3'UTR restored
proliferation to miR-24-treated K562 cells (FIGS. 14D and 14E).
Therefore, the miR-24 antiproliferative effect is largely mediated
by its downregulation of E2F2.
[0152] Antagonizing miR-24 elevated MYC protein levels in untreated
K562 cells, and the downregulation of MYC mRNA in TPA-treated K562
cells could be partially rescued by antagonizing miR-24 (FIGS. 22A
and 22B). However, antagonizing miR-24 did not restore MYC protein
to differentiating cells, suggesting that, although miR-24
suppresses MYC expression, downregulation of MYC protein during
postmitotic differentiation is also controlled by
miR-24-independent changes in protein stability. This may help to
explain why MYC siRNAs had no significant effect on proliferation
of cells transduced with miR-24 ASO (FIG. 14A).
[0153] miR-24 and its clustered miRNAs are among only a handful of
miRNAs consistently up-regulated during hematopoietic terminal
differentiation. Here, we show that miR-24 suppresses expression of
several key genes that regulate cell-cycle progression.
Overexpressing miR-24 increases the percentage of cells in the G1
phase, whereas antagonizing it causes differentiating cells to keep
proliferating. The antiproliferative effect of miR-24 is not
restricted to tumor cells (HepG2 and K562 cells) but also occurs in
human diploid fibroblasts.
[0154] miRNAs can regulate expression of hundreds of genes.
Genomewide analysis of miRNA target genes has been assessed
following miRNA overexpression or knockdown for only a handful of
miRNAs (Chang et al., 2007; Johnson et al., 2007; Lim et al.,
2005). Using this approach for miR-24 enabled us to identify 248
candidate genes that might be either directly or indirectly
regulated by miR-24. Of these downregulated genes, 40% are
predicted miR-24-regulated genes by TargetScan, and 53% have a
3'UTR hexamer sequence complementary to the miR-24 seed, suggesting
that a large proportion of miR-24-downregulated genes may be direct
targets.
[0155] To make sense of the set of 248 genes downregulated by
miR-24 overexpression, we used bioinformatics to identify
overrepresented processes and direct interacting protein networks
within this gene set. This type of analysis, which surprisingly
does not seem to have been applied to understanding miRNA
regulation, led to the hypothesis that miR-24 might regulate
cell-cycle progression during postmitotic differentiation by
targeting MYC and/or E2F2, given that they constituted nodes of the
major interaction network of the downregulated gene set. Both MYC
and E2F2 are directly regulated by miR-24, but neither of these
genes is a predicted miR-24 target. MYC, which has a 3'UTR hexamer
seed sequence, is regulated both by a seed-containing MRE and a
noncanonical seedless MRE. E2F2 lacks any miR-24 seed match.
However, E2F2 turned out to be the key gene for miR-24 inhibition
of the cell cycle because overexpressing miR-24-insensitive E2F2
completely restored proliferation.
[0156] The GO analysis of miR-24-downregulated genes also suggested
that miR-24 might regulate DNA repair. We recently verified this
prediction by showing that overexpression of miR-24 enhances
sensitivity to DNA damage (Lal et al., 2009). The key miR-24 target
for this biological effect is H2AFX, which has two seed-bearing
predicted MREs.
[0157] An unbiased analysis, which did not filter out genes whose
3'UTR lack seed binding sites, was critical for enabling us to
identify E2F2 as the key miR-24 target gene for cell-cycle
regulation. In addition to MYC and E2F2, we found five other
miR-24-downregulated genes whose 3'UTR was inhibited by miR-24
through seedless MREs. These genes (AURKB, CCNA2, CDK4, CDC2, and
FEN1) are also transcriptionally regulated by E2Fs or MYC and play
crucial roles in cell-cycle progression. Our results suggest that,
in addition to genes containing miR-24 perfect seed matches,
seedless MREs are also important. Indeed, seedless MREs are
critical for miR-24 function because the antiproliferative effect
of miR-24 can be recapitulated by silencing or obliterated by
overexpressing the seedless E2F2 gene. However, the importance of
recognition of seedless versus seed-bearing MREs could vary between
miRNAs. An assessment of this question could be determined by
experimental testing of a large set of randomly chosen genes, whose
protein or mRNA is downregulated by miRNA overexpression or
increased by miRNA inhibition. In addition to seedless 3'UTR MREs,
we previously identified a coding region miR-24 MRE in p161NK4A
(Lal et al., 2008). Other recent studies also identified coding
region MREs (Duursma et al., 2008; Tay et al., 2008). Taken
together, these results suggest that target gene identification
might be improved by not disregarding noncanonical MREs.
[0158] miR-24 directly regulates not only critical nodes of the
interactome of cell-cycle regulatory genes, but also genes
downstream of these nodes. This multitiered gene regulation may
guarantee that cell-cycle arrest is not easily evaded. In fact, we
have preliminary data suggesting that miR-24 may directly regulate
many additional periodic genes, including others that lack a
canonical seed-bearing MRE. Because expression of many of these
genes is suppressed in nondividing cells, we were careful to show
that miR-24-mediated gene suppression occurs before
miR-24-transduced cells have stopped dividing (FIG. 17), so their
downregulation is a cause, not consequence, of cell-cycle
arrest.
[0159] MYC and E2F regulate progression through G1. Regulating the
transition to S phase may be the major site of miR-24 action
because miR-24-treated cells accumulate in G1. Because MYC and E2F2
promote each other's transcription, miR-24 may prevent the
reciprocal activation of these genes by regulating both of them.
The dramatic downregulation of both proteins in
miR-24-overexpressing cells could, therefore, be a combined effect
of posttranscriptional and transcriptional regulation. Other miR-24
targets that are also transcriptionally regulated by MYC or E2F2
are implicated in controlling progression through G1, the G1/S
checkpoint, S, and G2/M. For example, overexpressing miR-24
downregulated the mRNA and protein levels of the E2F regulated
genes, CCNA2 and CDC2, which act together to promote G1/S and G2/M
transitions. CCNA2 binds to and activates CDC2, thereby promoting
G1/S and G2/M transition. Although the mRNA for CDK6, which
regulates G1-to-S transition, was not significantly changed in our
microarrays, we previously showed that CDK6 is directly regulated
by miR-24 (Lal et al., 2008). CDK6 may be an example of a target
gene regulated primarily by translational inhibition. p161NK4A is
another direct target of miR-24 that is translationally regulated
by miR-24 and, therefore, not downregulated in the microarrays (Lal
et al., 2008). In addition to genes, which act at the G1/S
transition, cells transfected with miR-24 mimics also have
decreased expression of genes that principally act at other phases
of the cell cycle. Important genes required for DNA replication in
S phase were also downregulated by miR-24, including MCM4 and MCM
10 in the prereplication complex; RRM2, a ribonucleotide reductase
that catalyzes deoxyribonucleotide synthesis from ribonucleotides;
PCNA, which forms a moving platform to recruit replication enzymes
to the replication fork; and FEN1, a flap endonuclease involved in
rejoining Okasaki fragments. Other downregulated genes act
principally to facilitate mitosis, including AURKB and CCNB 1.
Therefore, miR-24 may put the breaks on cell division at multiple
steps in cell-cycle progression.
[0160] Silencing the genes mentioned above would be expected to
inhibit cell division. However, suppressing other
miR-24-downregulated genes would promote cell-cycle progression,
especially in the context of DNA damage. These genes include CHEK1,
which participates in the G2/M checkpoint and is activated by ATR
in response to unresolved DNA damage, and BRCA1, which is in a
surveillance complex that activates double-strand break repair.
Prominent in the downregulated gene interaction network are the
cyclin D inhibitor CDKN1B (p27KIP1) and VHL, a tumor suppressor
protein. In addition, p161NK4A, a CDK inhibitor, is a validated
direct miR-24 target (Lal et al., 2008). Thus, the role of miR-24
in regulating the cell cycle may be complex. If cells are unable to
exit G1 and replicate their DNA, it may be economical to suppress
the inhibitory genes that guard the genome from propagating damaged
DNA. However, in some contexts, depending on the transcripts
expressed in a particular cell, miR-24 might actually promote cell
proliferation by suppressing these cell-cycle inhibitory genes. In
fact, inhibiting miR-24 decreases proliferation of A549 lung cancer
cells but has the opposite effect on HeLa cells (Cheng et al.,
2005).
[0161] miR-24 is most highly expressed in G1. This is consistent
with our finding that miR-24 regulates the G1/S transition. The E2F
family of transcription factors regulates progression through this
checkpoint. It therefore makes sense that miR-24 acts, in large
part, by directly targeting E2F2 (and thereby indirectly
suppressing E2F1 and E2F3 expression). The pattern of miR-24
expression is consistent with the known cell-cycle variation of E2F
family members (Sears et al., 1997). When miR-24 is high in G1,
E2F1 and E2F2 are low; the E2F family begins to be expressed in
late G1 and peaks in S phase when miR-24 is turned down. The E2Fs
continue to be expressed in G2 and M (where they also have
important functions) when miR-24 levels remain low.
[0162] miR-24 is not the only miRNA that regulates the cell cycle
and targets the E2F family (FIG. 14F). For instance, the miR-1792
cluster directly downregulates the E2F family (O'Donnell et al.,
2005; Petrocca et al., 2008). However, unlike miR-24, whose
expression varies inversely with E2F expression, miR-1792 appears
to be expressed uniformly except in quiescent cells. Moreover, E2F2
downregulation should antagonize the dominant effect of these
miRNAs to promote cell proliferation. Thus, E2F downregulation is
likely not a defining effect of miR-1 792 but, rather, a secondary
effect that fine-tunes its major proliferative effect. Because
miR-24 suppresses MYC and E2F expression and both MYC and the E2F
family activate miR-1 792 and miR-1 06b25 transcription (O'Donnell
et al., 2005; Petrocca et al., 2008), miR-24 also likely inhibits
proliferation by indirectly suppressing transcription of these
cell-cycle-promoting miRNAs. A recent paper also suggests another
layer of complexity to the miR-24, miR-1792, MYC, and E2F network
(Gao et al., 2009). MYC suppresses the transcription of miR-23b,
which is encoded with miR-24. Although one recent study suggests
that miR-24 and miR-23b are independently transcribed (Sun et al.,
2009), MYC might also regulate miR-24 transcription. It is worth
noting that E2F1 mRNA and protein do not correlate during the cell
cycle, consistent with posttranscriptional regulation by miRNAs
(O'Donnell et al., 2005). Because E2F2 mRNA has kinetics similar to
E2F1 (Sears et al., 1997), miRNA-dependent regulation of
translation may be operating, possibly for all E2Fs that promote
G1/S transition.
[0163] The integrated effect of miR-24 on a highly interacting set
of key genes acts as a switch to stop cell division, rather than as
a fine-tuning rheostat. It will be interesting to understand how
expression of these two miR-24 gene clusters is regulated and to
understand the function of the clustered miRNAs (miR-23 and
miR-27). The only other miRNAs consistently up-regulated during
terminal differentiation are miR-22 and miR-125a (a mammalian
ortholog of lin-4) (Lal et al., 2009). There are suggestions in the
literature that these genes might also regulate important pathways
of cell differentiation (Choong et al., 2007; Wu and Belasco,
2005).
[0164] miR-24 directly regulates both cell proliferation and DNA
repair Enhancing miR-24 function in cancer cells by introducing
miR-24 mimics might be an attractive therapeutic, given that it
could potentially block dysregulated cell proliferation and also
sensitize cancer cells to DNA damage from chemo- and
radiotherapy.
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EQUIVALENTS
[0258] Having now described some illustrative embodiments of the
invention, it will be apparent to those skilled in the art that the
foregoing is merely illustrative and not limiting, having been
presented by way of example only. Numerous modifications and other
illustrative embodiments are within the scope of one of ordinary
skill in the art and are contemplated as falling within the scope
of the invention. In particular, although many of the examples
presented herein involve specific combinations of method acts or
system elements, it should be understood that those acts and those
elements may be combined in other ways to accomplish the same
objectives. Acts, elements and features discussed only in
connection with one embodiment are not intended to be excluded from
a similar role in other embodiments.
Sequence CWU 1
1
100122RNAArtificial SequenceSynthetic oligonucleotide miR-24
1uggcucaguu cagcaggaac ag 22222RNAHomo
sapiensmisc_feature(1)..(22)MRE1 (58-79) 2acuuguuuca aaugcaugau ca
22322RNAHomo sapiensmisc_feature(1)..(22)MRE2 (46-67) 3aaucaccuau
gaacuuguuu ca 22422RNAHomo sapiensmisc_feature(1)..(22)MRE3
(255-276) 4uguuucucug uaaauauugc ca 22522RNAHomo
sapiensmisc_feature(1)..(22)MRE4 (95-116) 5ccuuggcuga gucuugagac ug
22622RNAHomo sapiensmisc_feature(1)..(22)MRE5 (141-162) 6gccucaaauu
ggacuuuggg ca 22723RNAHomo sapiensmisc_feature(1)..(23)MRE6
(447-468) 7cuggcaaaua uaucauugag cca 23822RNAHomo
sapiensmisc_feature(1)..(22)MRE3 (255-276) 8uguuucucug uaaauauugc
ca 22922RNAHomo sapiensmisc_feature(1)..(22)MRE3 (255-276)
9ugaaugucug uacacauuug ga 221023RNAHomo
sapiensmisc_feature(1)..(23)MRE6 (447-468) 10cuggcaaaua uaucauugag
cca 231122RNAHomo sapiensmisc_feature(1)..(22)MRE6 (447-468)
11cuggcaaaua uauccgaaca ag 221222RNAHomo
sapiensmisc_feature(1)..(22)E2F2 MRE1 12gugggugcuc ugggcugaac ca
221322RNAHomo sapiensmisc_feature(1)..(22)E2F2 MRE1 13gacggaggag
acuugacuag gu 221422RNAHomo sapiensmisc_feature(1)..(22)AURKB MRE1
14cguguguuug uaugucugug ua 221522RNAHomo
sapiensmisc_feature(1)..(22)AURKB MRE1 15ggacagggaa gaacugacuc gu
221622RNAHomo sapiensmisc_feature(1)..(22)BRCA1 MRE5 16agguggaggu
ugcagugagc ca 221723RNAHomo sapiensmisc_feature(1)..(23)BRCA1 MRE5
17agguggaggu ugcagccuua gca 231822RNAHomo
sapiensmisc_feature(1)..(22)CCNA2 MRE1 18aucaauuugc ugacuugggc au
221922RNAHomo sapiensmisc_feature(1)..(22)CCNA2 MRE1 19aacaaggacg
acucgacucg au 222022RNAHomo sapiensmisc_feature(1)..(22)CDC2 MRE1
20cuuggcuuuc gagucugagu uu 222122RNAHomo
sapiensmisc_feature(1)..(22)CDC2 MRE1 21gauggggaug gauugacucg gu
222222RNAHomo sapiensmisc_feature(1)..(22)CDK4 MRE1 22cuuugccuuu
aucucugagg cu 222322RNAHomo sapiensmisc_feature(1)..(22)CDK4 MRE1
23gauagggaug aucugacucg gu 222422RNAHomo
sapiensmisc_feature(1)..(22)FEN1 MRE1 24caccuggcaa ucagcugagu ug
222522RNAHomo sapiensmisc_feature(1)..(22)FEN1 MRE1 25gaccaggcaa
acuugacucg gu 222622RNAHomo sapiensmisc_feature(1)..(22)E2F2 MRE1
26gugggugcuc ugggcugaac ca 222722RNAHomo
sapiensmisc_feature(1)..(22)E2F2 MRE2 27gcuccugugg aaacaggagc ca
222822RNAHomo sapiensmisc_feature(1)..(22)E2F2 MRE3 28uggcuccuga
gcugacugac ug 222922RNAHomo sapiensmisc_feature(1)..(22)E2F2 MRE4
29acuccugacc ucaagugauc ca 223022RNAHomo
sapiensmisc_feature(1)..(22)E2F2 MRE5 30cacaucucca gcugagcugc cg
223122RNAHomo sapiensmisc_feature(1)..(22)AURKB MRE1 31cguguguuug
uaugucugug ua 223222RNAHomo sapiensmisc_feature(1)..(22)BRCA1 MRE1
32uguucacaaa ggcagagagu ca 223322RNAHomo
sapiensmisc_feature(1)..(22)BRCA1 MRE2 33ucucaaaugu uggaguggaa ca
223422RNAHomo sapiensmisc_feature(1)..(22)BRCA1 MRE3 34gugacaguga
gacuguggcu ca 223522RNAHomo sapiensmisc_feature(1)..(22)BRCA1 MRE4
35gccugaaaag gacuucuggc ua 223623RNAHomo
sapiensmisc_feature(1)..(23)BRCA1 MRE5 36agguggaggu ugcagugagc caa
233722RNAHomo sapiensmisc_feature(1)..(22)CCNA2 MREI 37aucaauuugc
ugacuugggc au 223822RNAHomo sapiensmisc_feature(1)..(22)CCNA2 MRE2
38auuuuccuaa gcaacuggau ca 223922RNAHomo
sapiensmisc_feature(1)..(22)CCNA2 MRE3 39aaaauguguc agcuaugagu aa
224022RNAHomo sapiensmisc_feature(1)..(22)CDK4 MRE1 40cuuugccuuu
aucucugagg cu 224122RNAHomo sapiensmisc_feature(1)..(22)CDK4 MRE2
41uucccuucug gacacugaga gg 224222RNAHomo
sapiensmisc_feature(1)..(22)CDK4 MRE3 42cauuucucua cacuaagggg ua
224322RNAHomo sapiensmisc_feature(1)..(22)CDC2 MREI 43cuuggcuuuc
gagucugagu uu 224422RNAHomo sapiensmisc_feature(1)..(22)CDC2 MRE2
44gcuuaucuug gcuuucgagu cu 224522RNAHomo
sapiensmisc_feature(1)..(22)CDC2 MRE3 45caugccaaaa uuugcuaagu cu
224622RNAHomo sapiensmisc_feature(1)..(22)FEN1 MRE1 46caccuggcaa
ucagcugagu ug 224722RNAHomo sapiensmisc_feature(1)..(22)FEN1 MRE2
47ugacugauua cuggcugugu cu 224822RNAHomo
sapiensmisc_feature(1)..(22)FEN1 MRE3 48gacccaccuu gagagagagc ca
224920DNAArtificial SequenceSynthetic GAPDH forward primer
49tgcaccacca actgcttagc 205021DNAArtificial SequenceSynthetic GAPDH
reverse primer 50ggcatggact gtggtcatga g 215120DNAArtificial
SequenceSynthetic SDHA forward primer 51tgggaacaag agggcatctg
205222DNAArtificial SequenceSynthetic SDHA reverse primer
52ccaccactgc atcaaattca tg 225319DNAArtificial SequenceSynthetic
UBC forward primer 53atttgggtcg cggttcttg 195421DNAArtificial
SequenceSynthetic UBC reverse primer 54tgccttgaca ttctcgatgg t
215522DNAArtificial SequenceSynthetic c-myc forward primer
55tcttccccta ccctctcaac ga 225625DNAArtificial SequenceSynthetic
c-myc reverse primer 56ttcctcatct tcttgttcct cctca
255720DNAArtificial SequenceSynthetic CDK4 forward primer
57cccgaagttc ttctgcagtc 205820DNAArtificial SequenceSynthetic CDK4
reverse primer 58ctggtcggct tcagagtttc 205920DNAArtificial
SequenceSynthetic CDK6 forward primer 59gcccgcatct atagtttcca
206020DNAArtificial SequenceSynthetic CDK6 reverse primer
60tatgcagcca acactccaga 206120DNAArtificial SequenceSynthetic H2AFX
forward primer 61agcaaactca actcggcaat 206220DNAArtificial
SequenceSynthetic H2AFX reverse primer 62actccccaat gcctaaggtt
206320DNAArtificial SequenceSynthetic UBD forward primer
63gctcagtggc acaagtgaaa 206420DNAArtificial SequenceSynthetic UBD
reverse primer 64ctgccatcat cttcccatct 206520DNAArtificial
SequenceSynthetic CNDP2 forward primer 65gggcttatga gtgacctgga
206620DNAArtificial SequenceSynthetic CNDP2 reverse primer
66gtgcttttgg accttgggta 206720DNAArtificial SequenceSynthetic PER2
forward primer 67aaatggatcc cccttgaatc 206820DNAArtificial
SequenceSynthetic PER2 reverse primer 68agcaccacct ggtgtacctc
206920DNAArtificial SequenceSynthetic BCL2L12 forward primer
69cgcctcctct tttcctcttt 207020DNAArtificial SequenceSynthetic
BCL2L12 reverse primer 70ggctgtgtac tctggggaaa 207120DNAArtificial
SequenceSynthetic STX16 forward primer 71ccagactttg agaggccaag
207220DNAArtificial SequenceSynthetic STX16 reverse primer
72ccacccggct aatttttgta 207320DNAArtificial SequenceSynthetic
ZNF317 forward primer 73gactggacct cccgtatgaa 207420DNAArtificial
SequenceSynthetic ZNF317 reverse primer 74tcacgtgact ctccaagctg
207520DNAArtificial SequenceSynthetic HRAS forward primer
75ggaagcaggt ggtcattgat 207620DNAArtificial SequenceSynthetic HRAS
reverse primer 76atggcaaaca cacacaggaa 207720DNAArtificial
SequenceSynthetic E2F1 forward primer 77taccccaact ccctctaccc
207820DNAArtificial SequenceSynthetic E2F1 reverse primer
78gtctccctcc ctcactttcc 207920DNAArtificial SequenceSynthetic E2F2
forward primer 79gagctcactc agaccccaag 208020DNAArtificial
SequenceSynthetic E2F2 reverse primer 80aacaggctga agccaaaaga
208120DNAArtificial SequenceSynthetic E2F3 forward primer
81gggtatgcgt gggtgtatgt 208220DNAArtificial SequenceSynthetic E2F3
reverse primer 82agtgtgtgtg aggggaggag 208320DNAArtificial
SequenceSynthetic PCNA forward primer 83cggatacctt ggcgctagta
208422DNAArtificial SequenceSynthetic PCNA reverse primer
84cacagctgta ctcctgttct gg 228520DNAArtificial SequenceSynthetic
AURKB forward primer 85tctgctctta gggctcaagg 208620DNAArtificial
SequenceSynthetic AURKB reverse primer 86tgccacacat tgtcttcctc
208720DNAArtificial SequenceSynthetic CCNA2 forward primer
87cacagcatgc acaacagtca 208822DNAArtificial SequenceSynthetic CCNA2
reverse primer 88agaaaacaaa ggcagtcttt ca 228920DNAArtificial
SequenceSynthetic BRCA1 forward primer 89tcatgccagc tcattacagc
209020DNAArtificial SequenceSynthetic BRCA1 reverse primer
90taagccaggc tgtttgcttt 209121DNAArtificial SequenceSynthetic CHEK1
forward primer 91tgcagaacca gttgatgttt g 219220DNAArtificial
SequenceSynthetic CHEK1 reverse primer 92acagctgtca ctgggttggt
209322DNAArtificial SequenceSynthetic CDC2 forward primer
93tgagtttctt tccatggatc tg 229420DNAArtificial SequenceSynthetic
CDC2 reverse primer 94caatcccctg taggatttgg 209520DNAArtificial
SequenceSynthetic FEN1 forward primer 95aaccccgaac caagctttag
209620DNAArtificial SequenceSynthetic FEN1 reverse primer
96gggccacatc agcaattagt 209720DNAArtificial SequenceSynthetic TOP1
forward primer 97gagatgaaag tccggcagag 209820DNAArtificial
SequenceSynthetic TOP1 reverse primer 98gtgtccgctg tttctccttc
209920DNAArtificial SequenceSynthetic pri-miR-24 forward primer
99agggcttagc tgcttgtgag 2010019DNAArtificial SequenceSynthetic
pri-miR-24 reverse primer 100caaggccaga ggaggtgag 19
* * * * *
References