U.S. patent application number 16/687501 was filed with the patent office on 2020-03-05 for composition and method of using mir-302 precursors as drugs for treating alzheimer's diseases.
The applicant listed for this patent is Te-Jen LAI, Hsin-Hua LI, Chih-Li LIN, Shi-Lung LIN. Invention is credited to Te-Jen LAI, Hsin-Hua LI, Chih-Li LIN, Shi-Lung LIN.
Application Number | 20200071700 16/687501 |
Document ID | / |
Family ID | 59387170 |
Filed Date | 2020-03-05 |
View All Diagrams
United States Patent
Application |
20200071700 |
Kind Code |
A1 |
LIN; Chih-Li ; et
al. |
March 5, 2020 |
COMPOSITION AND METHOD OF USING MIR-302 PRECURSORS AS DRUGS FOR
TREATING ALZHEIMER'S DISEASES
Abstract
This invention generally relates to a composition and method of
using recombinant microRNAs (miRNA) and their hairpin-like
precursors (pre-miRNA) as therapeutic drugs for treating
Alzheimer's diseases (AD). More specifically, the present invention
relates to the use of man-made miRNA miR-302 precursors
(pre-miR-302) for AD therapy in humans. These pre-miR-302 molecules
can be mass produced in prokaryotes as a form of DNA
expression-competent DNA vectors and/or hairpin-like RNAs. As
prokaryotic cells do not transcribe or process hairpin-like RNAs,
the present invention also teaches a method for expressing
pre-miRNAs in prokaryotes, i.e. pro-miRNA, using a novel
hairpin-like RNA transcription mechanism newly found in
prokaryotes. Additionally, since miR-302 is a well-known embryonic
stem cell (ESC)-specific factor in humans, our novel findings of
this invention can be further used to advance the designs and
development of novel regenerative medicine for treating many other
ageing-related degenerative diseases, such as Parkinson's diseases,
osteoporosis, diabetes, and cancers.
Inventors: |
LIN; Chih-Li; (Taichung
City, TW) ; LI; Hsin-Hua; (Taichung City, TW)
; LIN; Shi-Lung; (Arcadia, CA) ; LAI; Te-Jen;
(Taichung City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIN; Chih-Li
LI; Hsin-Hua
LIN; Shi-Lung
LAI; Te-Jen |
Taichung City
Taichung City
Arcadia
Taichung City |
CA |
TW
TW
US
TW |
|
|
Family ID: |
59387170 |
Appl. No.: |
16/687501 |
Filed: |
November 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15048964 |
Feb 19, 2016 |
10519440 |
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16687501 |
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14527439 |
Oct 29, 2014 |
9637747 |
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15048964 |
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14502608 |
Sep 30, 2014 |
9783811 |
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14527439 |
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62262280 |
Dec 2, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/141 20130101;
C12N 5/10 20130101; C12N 15/635 20130101; C12N 15/113 20130101;
C12N 15/63 20130101; C12N 2310/531 20130101; C12N 1/38 20130101;
C12N 5/0619 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 5/0793 20060101 C12N005/0793; C12N 5/10 20060101
C12N005/10; C12N 15/63 20060101 C12N015/63 |
Claims
1. A method of protecting human brain neurons from A.beta.-induced
neurotoxicity in Alzheimer's diseases with at least one
hairpin-like RNA mimics of microRNA precursor (hairpin-like
pre-miRNA mimics), comprising: (a) formulating at least one
hairpin-like pre-miRNA mimic with glycylglycerin or its chemical
derivatives, wherein the at least one hairpin-like pre-miRNA mimic
contains SEQ. ID. NO.2; and (b) delivering at least one of the
formulated hairpin-like pre-miRNA mimic into at least one
neuron.
2. The method as defined in claim 1, wherein said hairpin-like
pre-miRNA mimics are miR-302 precursors (pre-miR-302) in a
structural conformation selected from the group consisting of
microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA
(siRNA), their precursors and homologs, and a combination
thereof.
3. The method as defined in claim 1, wherein said hairpin-like
pre-miRNA mimics consist of SEQ. ID. NO3, SEQ. ID. NO. 4, SEQ. ID.
NO. 5, and SEQ. ID. NO.6.
4. The method as defined in claim 1, wherein the expression of said
hairpin-like pre-miRNA mimics produces miR-302a, miR-302b, miR-302c
and miR-302d in the treated neurons.
5. The method as defined in claim 1, further comprising a step of
inducing Akt signaling activation.
6. The method as defined in claim 5, wherein said Akt signaling
activation improves insulin resistance in the treated neurons.
7. The method as defined in claim 6, further comprising a step of
suppressing p-307 IRS-1 serine phosphorylation, increasing IRS-1
tyrosine phosphorylation, or both.
8. The method as defined in claim 5, wherein said Akt signaling
activation further stimulates Nanog expression to increase
sensitivity of insulin signaling.
9. The method as defined in claim 1, further comprising a step of
inducing Nrf2/HO-1 expression to reduce A.beta.-induced
intracellular ROS accumulation and apoptosis.
10. The method as defined in claim 1, further comprising a step of
inducing CD34-positive adult stem cell expansion.
Description
PRIORITY
[0001] The present invention is a continuation application of U.S.
application Ser. No. 15/048,964, filed on Feb. 19, 2016, which
claims priority to the U.S. Provisional Application Ser. No.
62/262,280 filed on Dec. 2, 2015. U.S. application Ser. No.
15/048,964 is a continuation-in-part of U.S. patent application
Ser. No. 14/502,608 filed on Sep. 30, 2014, which is a divisional
of U.S. patent application Ser. No. 13/572,263, filed on Aug. 10,
2010. U.S. application Ser. No. 15/048,964 is also a
continuation-in-part of U.S. patent application Ser. No. 14/527,439
filed on Oct. 29, 2014, which is a divisional of U.S. patent
application Ser. No. 13/572,263, filed on Aug. 10, 2010. Each of
the above applications is hereby incorporated by reference in its
entirety as if fully set forth herein.
FIELD OF INVENTION
[0002] This invention generally relates to a composition and method
of using recombinant microRNAs (miRNA) and their hairpin-like
precursors (pre-miRNA) as therapeutic drugs for treating
Alzheimer's diseases (AD). More specifically, the present invention
relates to the use of man-made miRNA miR-302 precursors
(pre-miR-302) for AD therapy in humans. These pre-miR-302 drugs can
be produced in prokaryotes as a form of expression-competent DNA
vectors and/or hairpin-like structured RNAs. As prokaryotic cells
do not transcribe or process hairpin-like RNAs, which resemble a
transcriptional termination code in prokaryotes, and further in
view of the lack of several essential eukaryotic enzymes such as
type-II RNA polymerases (Pol-2) and RNaseIII Dicers in prokaryotes,
the present invention herein further teaches a method for
expressing pre-miRNAs in prokaryotes, or called pro-miRNA, using a
novel hairpin-like RNA transcription mechanism newly found in
prokaryotes (as disclosed in our priority U.S. patent application
Ser. No. 13/572,263). In addition, since miR-302 is a well-known
embryonic stem cell (ESC)-specific factor in humans, our findings
of this invention can be further used to advance the designs and
development of novel regenerative medicine for treating many other
ageing-related degenerative diseases, such as Parkinson's diseases,
diabetes, osteoporosis, and cancers.
BACKGROUND
[0003] Insulin resistance represents a loss or reduction in its
normal functionality on target tissues and hence affects our
cognitive and memory functions, ultimately leading to the onset of
Alzheimer disease (AD) (Cholerton et al, 2011). Insulin resistance
has been linked to several previously identified risk factors that
accelerate the cognitive dysfunction and ageing process, including
diabetes, obesity, hypertension, hyperlipidemia, and metabolic
syndrome (Spielman et al, 2014). Particularly, brains exhibit
defective insulin receptor (IR) and insulin receptor substrate-1
(IRS-1) show alteration or aberrant activation of insulin signaling
in progression of AD, the most common cause of dementia (Williamson
et al, 2012). These findings suggest that neuronal insulin
signaling becomes dysfunction in the AD brains similar to the
dementia symptoms of Type 2 diabetes. The pathogenesis of AD is
initially triggered by the presence of extracellular amyloid-.beta.
(A.beta.) peptides, which impair mitochondrial membrane potential
(MMP) and contribute to an increase in the accumulation of
intracellular reactive oxygen species (ROS), ultimately leading to
neuronal cell death (Butterfield DA, 2002; Li et al, 2015). It has
been well established that A.beta. deposition may play a pathogenic
role in age-associated AD pathogenesis (Lesne et al, 2013). In
addition, our previous studies have indicated that A.beta. induces
p-Ser307 IRS-1 expression and inhibits IRS-1 tyrosine
phosphorylation and its downstream target protein kinase B (PKB,
also called Akt) (Kornelius et al, 2015). Subsequently, A.beta.
further suppresses Ser9 phosphorylation of glycogen synthase kinase
3.beta. (GSK3.beta.), which is one of the enzymes responsible for
causing tau hyperphosphorylation and neurotoxicity (Hernandez et
al, 2013). These findings all indicate that insulin signaling plays
a key regulatory role in A.beta.-induced neurotoxicity and neuronal
cell death in AD patients.
[0004] Cell survival is maintained by external factors such as
growth factors, the lack of which often causes apoptosis. The Akt
signaling pathway has been reported as a major downstream effector
of growth factor-mediated cell survival mechanisms that inhibit
apoptosis (Bhat and Thirumangalakudi, 2013). To this, Akt functions
to promote cell survival by inactivating certain pro-apoptotic
mediators such as Bid, a pro-apoptotic member of the Bcl-2 family
involved in the induction of death receptor-mediated apoptosis
(Majewski et al, 2004). Also, Akt signaling can reduce oxidative
stress via activating the nuclear factor erythroid 2-related factor
2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway (Surh et al,
2008), subsequently leading to prevention of A.beta.-induced
neurotoxicity (Kwon et al, 2015). As a result, both of these
reported Akt-mediated protective mechanisms against cell apoptosis
and oxidative stress may be useful for preventing neurodegeneration
and mitochondrial dysfunction in human brains. Interestingly, in
human embryonic stem cells (hESC), microRNA miR-302 has been found
to mediate Akt activation through downregulating phosphatase and
tensin homolog (PTEN) in order to maintain the pluripotency of
hESCs (Alva et al, 2011). Moreover, Akt signaling also regulates
the pluripotency-associated gene Nanog to maintain stem cell
self-renewal and anti-ageing (Kuijk, 2010; Han et al, 2012). Taken
together, based on all the above findings, we have proposed that
miR-302 may be able to stimulate the activation of the Akt
signaling pathway in neurons, so as to prevent A.beta.-induced
neurotoxicity in AD patients. Yet, neurons as one type of somatic
cells normally do not express miR-302.
[0005] MicroRNA (miRNA) miR-302 is the most abundant non-coding RNA
species specifically found in human embryonic stem cells (hESCs)
and induced pluripotent stem cells (iPSCs). Our previous studies
have shown that ectopic expression of miR-302 in mammalian somatic
cells is able to reprogram the somatic cells to hESC-like iPSCs (as
demonstrated in Lin et al., 2008, 2010 and 2011; EP 2198025; U.S.
Ser. Nos. 12/149,725; 12/318,806; 12/792,413). Moreover, we have
also observed that introduction of miR-302 into mammalian cells can
further stimulate the expression of many other miRNA species, such
as miR-92, miR-93, miR-367, miR-369, miR-371.about.373, miR-374,
miR-517, and the whole miR-520 familial members (Lin et al., 2008,
2010 and 2011; EP 2198025; U.S. Ser. Nos. 12/149,725; 12/318,806;
12/792,413). Further analyses using the online "TARGETSCAN" and
"PICTAR-VERT" programs, published in the Sanger Institute miRBase
website (http://www.mirbase.org/), revealed that miR-302 shares
over 400 target genes with these stimulated miRNAs, suggesting that
they may play a similar or partially functional role like miR-302.
Based on ours and many other previous reports, these shared target
genes include, but not limited, members of RAB/RAS-related
oncogenes, ECT-related oncogenes, pleiomorphic adenoma genes, E2F
transcription factors, cyclin D binding Myb-like transcription
factors, HMG-box transcription factors, Sp3 transcription factors,
transcription factor CP2-like proteins, NFkB activating protein
genes, cyclin-dependent kinases (CDKs), MAPK/JNK-related kinases,
SNF-related kinases, myosin light chain kinases, TNF-alpha-induce
protein genes, DAZ-associated protein genes, LIM-associated
homeobox genes, DEAD/H box protein genes, forkhead box protein
genes, BMP regulators, Rho/Rac guanine nucleotide exchange factors,
IGF receptors (IGFR), endothelin receptors, left-right
determination factors (Lefty), cyclins, p53 inducible nuclear
protein genes, RB-like 1, RB binding protein genes, Max-binding
protein genes, c-MIR cellular modulator of immune recognition,
Bc12-like apoptosis facilitator, protocadherins, TGF.beta.
receptors, integrin .beta.4/.beta.8, inhibin, ankyrins, SENP1,
NUFIP2, FGF9/19, SMAD2, CXCR4, EIF2C, PCAF, MECP2, histone
acetyltransferase MYST3, nuclear RNP H3, and many nuclear receptors
and factors. Notably, the majority of these target genes are highly
involved in embryonic development and cancer tumorigenecity. Hence,
it is conceivable that miR-302 can stimulate these downstream
homologous miRNAs, such as miR-92, miR-93, miR-367,
miR-371.about.373, miR-374, and miR-520s, to enhance and/or
maintain its functionality.
[0006] Particularly, we noted that miR-302, miR-92.about.93,
miR-367, miR-371.about.374, and miR-520s are all hESC-specific
miRNAs that are abundantly expressed in hESCs and iPSCs (Lin et al,
2008; EP 2198025; U.S. Ser. No. 12/149,725), all of which are also
useful for designing and developing novel regenerative medicine. To
achieve this goal, stem cells such as hESCs and iPSCs can be used
as a treasure box as well as a tool for us to screen, search,
extract, and produce novel effective drug-like ingredients that are
useful for designing and developing many pharmaceutical and
therapeutic applications, including but not limited, for
stimulating tissue/organ regeneration, for repairing and/or
rejuvenating damaged/aged cells/tissues, for treating
ageing-associated degenerative diseases (i.e. Alzheimer's diseases,
Parkinson's diseases, osteoporosis, diabetes and cancers), and for
preventing tumor and/or cancer formation/progression/metastasis. As
a result, it is conceivable that we can use these hESC-specific
miRNAs as candidate drugs for developing novel therapies and
treating human diseases in vivo. To fulfill this goal, we need a
method for producing a significantly large amount of hairpin-like
miRNAs and their precursors (pre-miRNAs) using modern DNA
recombination and amplification technologies with bacterial cells;
yet, it has been widely known that hairpin-like DNA/RNA structures
resemble signals of intrinsic transcription termination mechanisms
in prokaryotes (McDowell et al., Science 1994) and hence make it
impossible for prokaryotic cells to transcribe hairpin-like RNAs,
such as small hairpin RNAs (shRNA), microRNAs (miRNA) and the
related precursors (i.e. pre-miRNA). To this problem, neither the
first finder of miR-302-Houbaviy et al. (Developmental Cell (2003)
5, 351-358) nor the next follower Kim et al. (WO 2005/056797) could
provide any solution for it.
[0007] Furthermore, as learning from current textbooks, a person of
ordinary skill in the art must know that prokaryotic and eukaryotic
transcription machineries are different and thus are not compatible
to each other in many aspects. For example, based on most current
understandings, eukaryotic RNA polymerases do not directly bind to
gene promoter sequences and hence require additional accessory
proteins to help it to initiate RNA transcription, whereas
prokaryotic RNA polymerases can form a holoenzyme that binds
directly to gene promoters, so as to initiate RNA transcription.
However, because the holoenzyme can not process through a DNA
sequence with a high degree of secondary structures, such as a
hairpin DNA, the prokaryotic promoters naturally do not contain any
hairpin-like structure, which otherwise resembles a transcription
termination code in prokaryotes (McDowell et al, 1994). In
addition, it is a common sense for a person of ordinary skill in
the art to understand that eukaryotic messenger RNA (mRNA) is
transcribed in the nucleus by type II RNA polymerases (pol-2) and
then processed and exported to the cytoplasm for protein synthesis,
whereas prokaryotic RNA transcription and protein translation take
place simultaneously off the same piece of DNA in the same place
(cytoplasm) because prokaryotic cells such as bacteria and archaea
do not possess any nucleus-like structure. Due to these
differences, it makes prokaryotes difficult or even impossible to
produce eukaryotic RNAs and the related peptides/proteins using
eukaryotic RNA promoters, which tend to contain DNA motifs with
specific secondary structures in the 5'-untranslational regions
(5'-UTR).
[0008] Prior arts attempt at producing mammalian gene products in
bacterial cells, such as U.S. Pat. No. 7,959,926 to Buechler and
U.S. Pate. No. 7,968,311 to Mehta, used bacterial or bacteriophage
promoters. Since prokaryotes do not contain any splicing machinery
such as spliceosome to process introns, the intron-less
complementary DNA (cDNA) of a desired gene was made and cloned into
a plasmid vector driven by a bacterial or bacteriophage promoter.
Then, the vector so obtained was introduced into a competent strain
of bacterial cells, such as Escherichia coli (E. coli), for
expressing the gene transcripts (i.e. mRNAs) and subsequently
translating the mRNAs into proteins. Nevertheless, the bacterial
and bacteriophage promoters, such as Tac, Lac, Tc, T1, T3, T7, and
SP6 RNA promoters, are not pol-2 promoters and their transcription
activities tend to be an error-prone process, which causes
mutations and can not express hairpin-like miRNAs or shRNAs as
reported by McDowell et al (Science 1994). In addition, Mehta
further taught that glycerol/glycerin might be used to increase the
efficiency of bacterial transformation; yet, no teaching was
related to enhancement of RNA transcription, in particular pol-2
promoter-driven hairpin-like RNA transcription. Due to lack of
compatibility between eukaryotic and prokaryotic transcription
systems, these prior arts were still limited by the use of
prokaryotic RNA promoters for expressing gene cDNA in prokaryotes
and none of them were useful for expressing hairpin-like RNAs, such
as miRNAs and shRNAs.
[0009] Using a novel hairpin-RNA transcription mechanism newly
found in prokaryotes (Lin et al, U.S. patent application Ser. Nos.
13/572,263, 14/502,608, and 14/527,439), we now can overcome the
prokaryotic transcription termination mechanisms and thus induce
over-expression of hairpin-like microRNA precursors (pre-miRNA) and
shRNAs in prokaryotic cells, particularly useful for expressing
human miR-302 familial microRNAs (miR-302a, b, c, d, e, and f) and
their precursors (pre-miR-302). By adding certain transcriptional
inducer chemicals into bacterial culture medium, we are able to
transform prokaryotes to adopt eukaryotic pol-2 and/or viral
pol-2-like promoters for transcribing our desired hairpin RNAs and
the related miRNAs/shRNAs thereof. The advantages of this
production method are: first, cost-effective production due to the
fast and cheap growth of single-cell prokaryotes such as bacterial
cells; second, easy handling because of no need for culturing
dedicate hybridomas or mammalian cells; third, high product quality
in view of the improved reading fidelity of pol-2 promoter-driven
transcription; fourth, industrial level bulk production for desired
hairpin RNAs and their related miRNAs/shRNAs as well as the
introduced vectors all at once in prokaryotes; and last, multiple
task capacity in that the desired RNAs and other desired
peptides/proteins can be produced together but separately isolated
and purified from the resulting bacterial extracts and/or lysates
for further applications. Therefore, taken together, a composition
and method for producing hairpin RNAs and/or their related
miRNAs/shRNAs using eukaryotic RNA promoter-driven transcription in
prokaryotes is highly desirable for the need of mass production of
hairpin-like RNA drugs.
SUMMARY OF THE INVENTION:
[0010] Our previous invention European patent No 2198025 has
demonstrated the use of miR-302-like small hairpin RNAs (shRNAs)
and/or short interfering RNAs (siRNAs) to reprogram mammalian
somatic cells to hESC-like induced pluripotent stem cells (iPSCs).
These miR-302-like shRNAs/siRNAs possess the same functional
structures as native miR-302 molecules and are all share the same
17-nucleotide seed sequence of 5'-UAAGUGCUUC CAUGUUU-3' (SEQ. ID.
NO.1) in order to specifically and concurrently target over 400
genes in humans. In our special designs and methods, these
miR-302-like shRNAs/siRNAs are transcribed from a recombinant
miR-302 familial gene (SEQ. ID. NO.2, as shown in FIG. 13A), of
which the transcripts can be further processed into precursors
(i.e. pre-miRNAs) of miR-302a (pro-miR-302a, SEQ. ID. NO.3),
miR-302b (pro-miR-302b, SEQ. ID. NO.4), miR-302c (pro-miR-302c,
SEQ. ID. NO.5), and (pro-miR-302d, SEQ. ID. NO.6), as shown in FIG.
13B. As a result, the present invention further discloses a novel
composition and method of using these miR-302-like shRNAs/siRNAs
for treating diabetes-associated Alzheimer's diseases (AD) in
humans.
[0011] In one preferred embodiment, the desired miR-302-like
shRNA/siRNA molecules of the present invention are derived from a
vector-based expression composition, such as plasmid and/or viral
vector, which can be delivered into at least a targeted cell type,
tissue and/or organ, in particular brain and/or pancreas, for
releasing the desired miR-302-like shRNAs/siRNAs for AD therapy. In
another preferred embodiment, the desired miR-302-like
shRNAs/siRNAs can be produced in a mass amount in vitro and then
further purified and used for in vivo delivery into at least a
targeted cell type, tissue and/or organ, in particular brain and/or
pancreas, for AD therapy. For in vivo treatments, the
delivery/transfection method includes, but not limited, all kinds
of injection, lipid-/glycerin-/chemical-mediated
infusion/perfusion, peptide-/sugar-/liposome-/chemical-mediated
transfection, antigen-/antibody-/receptor-mediated endocytosis,
transposon-/retrotransposon-mediated cell penetration,
adenoviral/retroviral/lentiviral infection, and a combination
thereof. For facilitating in-vivo delivery efficiency, the desired
miR-302-like shRNAs/siRNAs can be further formulated with at least
a kind of lipid-/liposome-, peptide-/protein-, sugar-, and/or
glycylglycerin-based molecules, and/or the combination thereof,
which are able to stabilize the structural integrity of
shRNAs/siRNAs as well as to enhance drug penetration rates in
vivo.
[0012] In our experimental design, which is provided here as an
example of practical evidence, FIGS. 1A and 1B show the basic
construct of a miR-302-expressing lentiviral plasmid vector (called
pLenti-EF1alpha-RGFP-miR302 or pLVX-GFP-miR302) that has been
tested for treating ageing-related diseases (i.e. cancers, diabetes
and AD) in animal models in vivo as well as been used for
performing mass production of miR-302-like microRNAs/shRNAs/siRNAs
in prokaryotes (such as E. coli and Lactobacillus spp bacterial
cells). As demonstrated in FIG. 1A, components of the
miR-302-expressing plasmid vector can be re-arranged to be located
in different regions of the vector or even deleted for providing
more compact and effective delivery into targeted cells. In view of
this example, a person of ordinary skill in the art would
understand that any vector with similar structural features can be
used for achieving the same functional purpose as the present
invention. Additionally, after vector delivery into the targeted
cells, the natural processes of miR-302 generation from a
miR-302-expressing plasmid/vector are further demonstrated in FIG.
1B.
[0013] For using prokaryotes to produce hairpin-like
microRNAs/shRNAs, the recombinant miR-302 familial gene (SEQ. ID.
NO.2; FIG. 13A) must be placed in the 5'-UTR of its encoding gene
(i.e. RGFP), as shown in FIGS. 1A and 1B. Because prokaryotic cells
do not contain any splicing machinery such as spliceosomes to
process in-frame introns, the original vectors used in our prior
inventions, such as EP 2198025; U.S. Ser. Nos. 12/149,725;
12/318,806; 12/792,413 to Lin, can not be used for produce
hairpin-like microRNAs/shRNAs in prokaryotes. Also, since a
hairpin-like DNA/RNA structure resembles the stop signal of
intrinsic transcription termination mechanisms in prokaryotes
(McDowell et al., 1994), our design of the recombinant miR-302
familial gene located in the 5'-UTR of the RGFP gene can not be
transcribed in prokaryotes without adding any chemical inducer like
MOPS, glycerin, and/or ethanol (FIGS. 3, 4 and 5). To overcome this
problem, our claimed priority invention, U.S. patent application
Ser. Nos. 13/572,263, 14/502,608, and 14/527,439 to Lin, had found
a novel hairpin-RNA transcription mechanism existing in prokaryotic
cells. As shown in FIGS. 2, 3, 4, 5 and 6, by adding certain
transcription inducers such as 3-morpholinopropane-l-sulfonic acid
(MOPS), ethanol, and/or glycerin (or called glycerol) in bacterial
culture, we can further transform the prokaryotes to adopt
eukaryotic pol-2 and viral pol-2-like promoters for transcribing
hairpin-like RNAs (i.e. the recombinant miR-302 familial gene, SEQ.
ID. NO.2; FIG. 13A), so as to achieving mass production of the
miR-302-like shRNA/siRNA molecules. These eukaryotic pol-2 and
viral pol-2-like promoters include, but not limited, mammalian
EF1alpha and/or cytomegalovirus (CMV) promoters, as shown in FIG.
1A.
[0014] In experiments, competent cells (i.e. E. coli) are
transformed or transfected by a vector with a structural design
similar to pLenti-EF1alpha-RGFP-miR302 and then cultivated in
Luria-Bertani (LB)-based culture broth at about 37.degree. C. with
frequent agitation at about 150.about.300 rpm. After >8-hour
incubation, the transformed cells grown in LB broth supplemented
with about 0.05%.about.8% (v/v) MOPS and/or about 0.05%.about.4%
(v/v) glycerin show abundant expression of red RGFP proteins that
can stain the LB broth into red, whereas other blank controls
without any inducer addition fail to produce any RGFP, as shown in
FIG. 2 and Example 1. The presence of red fluorescent RGFP
indicates that both its RNAs and proteins are successfully
produced. To further confirm the specificity of RNA transcription
induced by the chemical inducers, such as MOPS and/or glycerin, two
strains of transformed competent cells are prepared as follows: one
carries a pLVX-GFP-miR302+367 plasmid vector that has a modified
CMV promoter-driven green fluorescent protein (AcGFP) gene encoding
the whole miR-302.about.miR-367 cluster in its 5'-UTR and the other
carries the aforementioned pLenti-EF1alpha-RGFP-miR302 vector (FIG.
1A). After .gtoreq.8-hr incubation in culture medium/broth
supplemented with .gtoreq.0.1% (v/v) MOPS, the cells transformed
with pLVX-GFP-miR302+367 produce green AcGFP only, while the other
cells transformed with pLenti-EF1alpha-RGFP-miR302 show red RGFP
(FIG. 3). This result clearly indicates that the chemicals like
MOPS and glycerin can induce specific hairpin-like RNA expression
through both eukaryotic pol-2 promoter-driven and pol-2-like viral
promoter-driven transcription mechanisms. Based on our practical
evidence shown in FIGS. 2, 3 and 4, these "transcription inducer"
chemicals include, but not limited, ethanol, glycerin (glycerol),
MOPS and their chemical isoforms as well as derivatives, such as
2-(N-morpholino)ethanesulfonic acid (MES), and
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
mannitol, and/or a mixture thereof. The quantitative levels of the
induced RGFP protein production can be measured by Western blot
analysis, as shown in FIG. 5, in which Bacterial RuvB protein is
served as a house-keeping standard to normalize the RGFP
expression. In addition, the quantitative levels of the induced
hairpin-like miR-302/pre-miR-302 expression can also be measured by
Northern blot analysis, as shown in FIG. 6. Due to the structural
similarity of all microRNAs (miRNAs) and shRNAs, it is obvious for
a person of ordinary skill in the art to use the vector design of
the present invention for producing other kinds of miRNAs, shRNAs
and/or their precursors/homologs in prokaryotes.
[0015] Due to our discovery of the hairpin-RNA transcription
mechanism in prokaryotic cells, the present invention is able to
use either miRNA/shRNA-expressing viral vectors (i.e.
pLenti-EF1alpha-RGFP-miR302; FIG. 1A) or hairpin-like shRNA/siRNA
molecules (i.e. pro-miR-302a, b, c, and d; FIG. 13B) so obtained
for disease therapy, in particular AD therapy as demonstrated in
the present invention. In eukaryotic cells, these hairpin-like
miRNAs/shRNAs can be further processed into mature microRNA
molecules (i.e. miR-302a, b, c and d) for eliciting their
functions. Alternatively, some non-coding RNAs (ncRNAs), such as
short interfering RNAs (siRNAs) and small hairpin RNAs (shRNAs),
can be designed to mimic native microRNAs. These ncRNAs preferably
contain at least a sequence sharing 30% to 100% homology to a
microRNA or a part of its precursor (pre-miRNA). Also, these
shRNAs/siRNAs can be manually designed to contain perfectly matched
hairpin-stem regions, while native microRNA precursors (pre-miRNAs
or pri-miRNAs) often contain mismatched base pairs. Given that most
of microRNAs function as specific gene silencers and may play a
variety of distinctive roles in many physiological and pathological
mechanisms, including but not limited to biological development,
stem cell generation, nuclear reprogramming, cell differentiation,
cell cycle regulation, tumor suppression, immunological defense,
apoptosis, rejuvenation, wound healing, and many other more, the
potential applications in theses pharmaceutical and therapeutical
fields are therefore highly expected.
Induction of Eukaryotic Promoter-Driven miRNA/shRNA Expression in
Prokaryotes.
[0016] As aforementioned, the lentiviral
pLenti-EF1alpha-RGFP-miR302 plasmid vector contains a recombinant
miR-302 familial cluster gene (SEQ. ID. NO.2; FIG. 13A) located in
the 5'-UTR of its encoding RGFP gene (FIGS. 1A and 1B); as a
result, the induced expression of the RGFP gene will also generate
miR-302 molecules (miR-302a, b, c and d; SEQ. ID. NOs.3-6; FIG.
13B), as shown in the schematic mechanism of FIG. 1B. Due to lack
of RNA splicing machinery (e.g. spliceosomes) in prokaryotic cells,
the miR-302 molecules so obtained will remain as hairpin-like
microRNA precursors (pre-miR-302s and pri-miR-302s), as shown in
FIG. 6, which are useful for being isolated and delivered into
eukaryotic cells for eliciting the desired function of miR-302.
Using this production method, both of the vector and miR-302
molecules can be simultaneously amplified in the transformed
prokaryotic cells, such as E. coli. The method for isolating the
amplified pLenti-EF1alpha-RGFP-miR302 vector DNA and the
transcribed miR-302 molecules are disclosed in Examples 5 and 6,
respectively. The methods for delivering/transfecting miR-302,
pre-miR-302 and/or its encoding expression vector (i.e. pLenti-EF
lalpha/CMV-RGFP/GFP-miR302; FIG. 1A) into prokaryotic and/or
eukaryotic cells can be selected from the group consisting of
injection, microinjection, lipid-/glycerin-/chemical-mediated
infusion/perfusion, peptide-/sugar-/liposome-/chemical-mediated
transfection, antigen-/antibody-/receptor-mediated endocytosis,
transposon-/retrotransposon-mediated cell penetration, viral
infection, gene gun penetration, electroporation, and a combination
thereof.
[0017] After vector delivery into cells (Example 1), we observed
the induced expression of red RGFP and green GFP, respectively, in
the transformed cells cultivated in LB broth supplemented with
either MOPS or glycerin, or ethanol, or in combination, but not in
LB broth without any inducer added (FIGS. 3, 4 and 5), indicating
that this transcriptional induction effect is highly dependent on
these chemical inducers and hence no transcriptional leakage can be
found in blank negative controls. The expression of RGFP protein
was confirmed by Western blot analysis, as shown in FIG. 5. After
confirming the induced RGFP expression, we further measured the
induced miR-302 expression levels in the pLenti-EF
lalpha-RGFP-miR302-transformed cells with or without inducer
addition. The result of induced hairpin-like miR-302/pre-miR-302
expression was confirmed by Northern blot analysis, as shown in
FIG. 6. In agreement with the result of induced RGFP expression,
the miR-302 expression was detected only in transformed cells
treated with MOPS, glycerin and/or ethanol, but not in blank
negative controls, indicating that in the absence of any chemical
inducer no transcription activity can function through a gene
promoter containing a hairpin-like structure in prokaryotic cells,
as reported by McDowell et al (Science 1994). Hence, the
hairpin-like miR-302 expression shown in FIG. 6 is a specific
result induced by the added transcription inducers of the present
invention, not a random transcription leakage event.
Functional Applications of the Present Invention in Neural Stem
Cell Generation.
[0018] MicroRNA miR-302 has been found to reprogram mammalian
somatic cells to embryonic stem cell (ESC)-like induced pluripotent
stem cells (iPSC) (Lin, 2008, 2010, 2011; U.S. patent application
Ser. Nos. 12/149,725 and 12/318,806 to Lin). Using these iPSCs,
many stem cell-associated applications and therapies have been
developed for advancing modern regenerative medicine. Yet, miR-302
is only abundantly found in human ESCs rather than differentiated
tissue cells. Also, isolation of miR-302 from human ESCs is highly
debatable, costly and tedious. To solve these problems, the present
invention provides a simple, cheap, fast and inducible composition
and method for mass production of hairpin-like miR-302 molecules
and/or their precursors/homologs in prokaryotes. Moreover, the
isolation of miR-302 and/or its precursors from prokaryotic cells
is relatively easy and cost-effective, as shown in FIG. 6 and
Example 6 of the present invention.
[0019] We have used the pLenti-EF1alpha-RGFP-miR302-transformed E.
coli cells to produce and isolate high quantity and quality of the
pLenti-EF1alpha-RGFP-miR302 vector and pre-miR-302s, as shown in
Examples 5 and 6. The use of pLenti-EF lalpha-RGFP-miR302 had been
shown to produce human ESC-like iPSCs in view of our previous U.S.
patent application Ser. Nos. 12/149,725 and 12/318,806. Also, the
iPSCs so obtained can be further differentiated into neuron cells
as demonstrated in our previous studies (Lin et al, 2008, 2010 and
2011). In FIG. 7 and Example 2 of the present invention, we further
used a high concentration (.gtoreq.600 .mu.g/mL) of pre-miR-302s
obtained by the method of the present invention to reprogram human
keratinocytes to iPSCs, which then expressed strong ESC marker
Oct4. Further analysis using bisulfate DNA sequencing assays showed
that global DNA demethylation did occur in the nuclei of these
iPSCs, particularly in the promoter regions of Oct4 and Sox2 genes,
two of the most important reprogramming factors and markers in
human ESCs and iPSCs (FIG. 8 and Example 8). As global DNA
demethylation and Oct4 expression are widely known to be the first
and most important sign of stem cell pluripotency (Simonsson and
Gurdon, (2004) Nat Cell Biol. 6:984-990), the present invention may
also provide a composition and method for inducing iPSC derivation
using isolated miR-302 and/or pre-miR-302 molecules. To this
application, the methods for delivering miR-302 and/or pre-miR-302
molecules into mammalian cells can be selected from the group of
microinjection, lipid-/glycerin-/chemical-mediated
infusion/perfusion, peptide-/sugar-/liposome-/chemical-mediated
transfection, antigen-/antibody-/receptor-mediated endocytosis,
transposon-/retrotransposon/caspase-mediated cell penetration,
viral infection, gene gun penetration, electroporation, and a
combination thereof.
[0020] The applications of isolated miR-302 and/or pre-miR-302
molecules may further include the induction and expansion of
CD34-positive adult stem cells. As shown in FIGS. 9A and 9B, our
recent studies in wound healing therapy using a novel
miR-302-formulated drug revealed that treatments of relatively low
concentrations (50.about.500 m/mL) of the isolated
miR-302/pre-miR-302 molecules not only greatly enhance scar-less
wound healing but also induce CD34-positive adult stem cell
expansion around the wounded area in pig skins in vivo. Based on
the miR-302-treated (glycylglycerin-formulated
miR-302s/pre-miR-302s+antibiotic ointment) result of FIG. 9B in
comparison with that of control (only antibiotic ointment) result
of FIG. 9A, it clearly showed a .gtoreq.40-fold increase of
CD34-positive adult stem cell populations (labeled by green
fluorescent antibodies) in vivo after miR-302 treatments. The
currently known CD34-positive adult stem cell types include, but
not limited, skin, hair, muscle, blood (hematopoietic),
mesenchymal, and neural stem cells. As a result, since miR-302 can
be used to induce CD34-positive adult stem cell expansion in vivo,
this therapeutic effect may also help to re-grow and/or revive
functional neurons for treating AD in patients.
MiR-302 Protects SK-N-MC Cells against A.beta.-induced
Apoptosis.
[0021] Recent studies have demonstrated the crucial functions of
miR-302 in regulating oxidative stress-induced apoptosis. To
address whether miR-302 exerts any protective effect on neuronal
cells against A.beta.-induced apoptosis, we transfected human
neuronal SK-N-MC cells with a cytomegalovirus (CMV)-promoter-driven
miR-302 expression lentivector as previously reported (Lin et al,
2008, 2010 and 2011), and then exposed to A.beta. (2.5 .mu.M) for
24 hours. After that, the miR-302-transfected cells were identified
by the presence of a co-expressed AcGFP green fluorescent protein
under an inverted fluorescent microscope (FIG. 14A) and the
expression of miR-302 was further confirmed by RT-qPCR (n=3,
p<0.01, FIG. 14B) and miRNA microarray analysis (FIG. 12),
showing successful transcription of the whole miR-302 familial
cluster (i.e. miR-302a, b, c, and d). Notably, FIG. 14C further
demonstrated that A.beta. treatment triggered massive cell death in
control cell groups, whereas miR-302-transfected cells showed
marked attenuation of such A.beta.-induced cell death (n=3,
p<0.01). To determine which kind of cell death induced by
A.beta., we further examined the nuclei fragmentation by DAPI
staining. As shown in FIGS. 14D and 14E, A.beta. treatment
disrupted nucleus margin and significantly increased the apoptotic
cell population in the control groups compared to those of
miR-302-transfected cells (n=3, p<0.01). In addition, FIG. 14F
revealed that A.beta. treatment markedly increased the cleavage
formation of both caspase 3 and PARP in control cells but not in
miR-302-transfected cells (n=3, p<0.01), further confirming this
point. Taken together, our data strongly suggest that miR-302 plays
a protective role in preventing A.beta.-induced cell apoptosis.
[0022] Activation of Akt Signaling Is Involved in miR-302-mediated
Neuroprotection.
[0023] We have previously reported that restoration of insulin
sensitivity in neurons leads to Akt activation and so as to inhibit
A.beta.-induced apoptosis (Kornelius et al, 2015). To determine
whether miR-302 expression can restore neuronal insulin sensitivity
and prevent A.beta.-induced neurotoxicity, we used western blot
analyses to measure the expression levels of major insulin
signaling-related proteins, such as pSer307-IRS-1, tyrosine
phosphorylation of IRS-1, and their downstream target pSer473-Akt.
As shown in FIG. 15A, A.beta. treatment in control cells
significantly increased p-307 IRS-1 serine phosphorylation (n=3,
p<0.05) while decreasing IRS-1 tyrosine phosphorylation (n=3,
p<0.01), both of which are considered as hallmarks of insulin
resistance; yet, in miR-302-transfected cells this A.beta.-induced
insulin resistance was markedly attenuated (n=3, p<0.05).
Moreover, A.beta. treatment also led to a significant decrease of
p-Ser 473-Akt in control groups but not in miR-302-transfected
cells (n=3, p<0.01) (FIG. 15A). To further elucidate the
protective role of PI3K/Akt signaling in miR-302-transfected cells,
we applied a PI3K inhibitor LY294002. As a result, FIG. 15B
revealed that co-treatment of A.beta. (2.5 .mu.M) and LY294002 (20
.mu.M) could disrupt miR-302-mediated Akt signaling (n=3,
p<0.01) and thus resulted in a marked reduction of the viable
cell population, as determined by MTT assay (n=3, p<0.01, FIG.
15C). All these findings suggest that miR-302 prevents
A.beta.-induced neurotoxicity and neuronal death via activating
PI3K/Akt signaling. Alternatively, A.beta.-impaired insulin
signaling may also lead to an increase of GSK3.beta. activity as
well as tau hyperphosphorylation, a relevant step in AD
pathogenesis. To this, we found that miR-302 expression could
stimulate Akt signaling to slightly increase p-Ser 9-GSK30 levels
and hence may provide a mild inhibitory effect on tau
hyperphosphorylation (n=3, p<0.05) (FIG. 15D). As a result, FIG.
15D also showed that co-treatment of A.beta. and LY294002 totally
abolished the inhibitory effect of miR-302 on p-Ser 9-GSK30
expression and tau phosphorylation in control cells compared to
those of miR-302-transfected cells (n=3, p<0.05). Taken
together, our data demonstrate that miR-302 may exert its
protective effects mainly through activating and/or restoring the
Akt/GSK3.beta. signaling pathway.
MiR-302 Attenuates A.beta.-induced Oxidative Stress through
Akt-upregulated Nrf2/HO-1
[0024] To determine whether miR-302-mediated Akt activation can
prevent A.beta.-induced intracellular ROS accumulation, we
performed a fluorometric assay to measure the concentration of
hydrogen peroxide accumulated in the cells. As shown in FIG. 16A,
A.beta. treatment stimulated a significant elevation of
intracellular superoxide radical anions in control groups but not
in miR-302-transfected cells (n=3, p<0.01). Co-treatment of
A.beta. (2.5 .mu.M) and insulin (1 .mu.M) could restore the normal
levels of intracellular superoxide radical anions in control groups
(p<0.05), indicating that miR-302-mediated Akt activation did
inhibit A.beta.-induced ROS. Furthermore, Nrf2, a redox-sensitive
transcription factor, may also confer protection against ROS damage
by upregulating antioxidant-response elements, such as HO-1. Since
PI3K/Akt signaling has been reported to elevate HO-1 expression and
Nrf2-dependent transcription (Kwon et al, 2015), we further
elucidate this possible anti-oxidant effect of miR-302 by Western
blot assays. As a result, FIG. 16B revealed that A.beta. treatment
reduced both Nrf2 and HO-1 expressions in control groups but not in
miR-302-transfected cells (n=3, p<0.05). To further confirm the
source of this effect, further treatment of LY294002 (20 .mu.M)
with A.beta. (2.5 .mu.M) also decreased Nrf2 expression in
miR-302-transfected cells (n=3, p<0.05) (FIG. 16C), indicating
that miR-302 regulates Nrf2 expression via the PI3K/Akt signaling
pathway. Moreover, activation of Akt signaling significantly
restored the Nrf2 expression after co-treatment of A.beta. (2.5
.mu.M) and insulin (1 .mu.M) in control groups (n=3, p<0.05)
(FIG. 16C), further suggesting that miR-302-mediated Akt activation
can prevent A.beta.-induced ROS accumulation through the
upregulation of Nrf2 and HO-1.
[0025] To investigate the miR-302 effect on A.beta.-mediated
mitochondria dysfunction and apoptosis, we examined MMP with JC-1
staining assays and the expression of apoptotic-associated marker
truncated Bid (tBid) and anti-apoptotic-associated marker Bcl-2
with western blotting assays. As shown in FIG. 16D, control cells
displayed a significant deficiency of mitochondrial membrane
depolarization in response to A.beta. treatment (n=3, p<0.05),
which was however not found in miR-302-transfected cells, as
indicated by the concurrent loss of cytoplasmic red J-aggregate
fluorescence and elevation of diffused green fluorescence. Yet,
this miR-302-mediated protective effect on MMP integrity could be
totally abolished by co-treatment of A.beta. (2.5 .mu.M) and
LY294002 (20 .mu.M) for 24 hours (n=3, p<0.05), indicating the
involvement of Akt/PI3K signaling. In addition, A.beta. treatment
resulted in a marked increase of tBid expression (p<0.01) and
decrease of Bcl-2 (p<0.05) in control groups, but not in
miR-302-transfected cells (FIG. 16E). All these findings clearly
suggest that miR-302-mediated Akt activation can inhibit
A.beta.-induced oxidative stress, mitochondria dysfunction and
apoptosis via upregulating Nrf2 activities.
MiR-302 Regulates Akt Signaling by Targeting PTEN and Inducing
Nanog Expression
[0026] After having determined the important role of miR-302 in
activating Akt signaling to prevent A.beta.-induced neurotoxicity,
we further investigate the molecular mechanism underlying such
miR-302-mediated Akt activation. Recent studies have indicated that
miR-302 promotes pluripotency through Akt signaling by targeting
PTEN (Alva et al, 2011). To search the miR-302 target site in PTEN,
we performed screening analyses using a prediction program,
TargetScan (http://www.targetscan.org/), and identified a specific
miR-302 binding site located in the 3'UTR of human PTEN gene (FIG.
17A). As our western blotting data have shown a significantly
decrease of PTEN expression in miR-302-transfected cells (n=3,
p<0.05) (FIG. 17B), it suggests that miR-302 may target this
3'UTR binding site to suppress PTEN expression. Also, since
knockdown of PTEN can increase the pluripotency-associated gene
Nanog expression (Kuijk et al, 2010), which is further mediated by
PI3K/Akt signaling in ESCs (Alva et al, 2011), we herein examined
the miR-302 effects on PTEN, pSer473 Akt, and Nanog expressions
with western blot assays. As a result, FIG. 17C showed a marked
elevation of Nanog expression only detected in miR-302-transfected
cells (n=3, p<0.05), while A.beta. treatment (2.5 .mu.M for 24
hours) stimulated a significant increase of PTEN as well as
decreases of pSer473 Akt and Nanog expressions in control groups
but not in miR-302-transfected cells (n=3, p<0.05) (FIG. 17D).
Interestingly, further studies revealed that blocking Akt signaling
with LY294002 (20 .mu.M for 24 hours) could restore
A.beta.-mediated inhibitory effects on pSer473 Akt and Nanog
expressions in miR-302-transfected cells (n=3, p<0.05) (FIG.
17E), demonstrating that miR-302 activates Akt signaling to induce
Nanog expression.
[0027] To determine whether Nanog plays a protective role in
A.beta. treatment, we further performed shRNA-mediated knockdown of
Nanog in miR-302-transfected cells. As shown in FIG. 17F,
downregulation of Nanog resulted in an increase of p-Ser307 IRS-1
expression as well as a decrease of both tyrosine phosphorylation
and p-Ser 473-Akt/p-Ser 9-GSK3.beta. levels in miR-302-transfected
cells after A.beta. treatment. Taken together, our results strongly
suggest that miR-302 may confer protection against A.beta.-induced
neurotoxicity by downregulating PTEN to activate Akt and the
downstream Nanog signaling.
In Vitro and in Vivo Expression Patterns of Naong and miR-302 (from
LARP7 Gene).
[0028] We observed that impaired Nanog expression is associated
with A.beta.-disrupted insulin sensitivity. To investigate this
point, we first performed RT-qPCR to show that A.beta. treatment
significantly decreased Nanog mRNA expression in control neurons in
vitro (n=3, p<0.05, FIG. 18A). Then, we further addressed the
relevance of this finding to human AD patients in vivo by measuring
the mRNA expression levels of Nanog in AD patients' PBMCs. A
detailed overview of the testing subjects' characteristics is
summarized in Table 1. A number of AD patients (n=7) had moderated
dementia by MMSE and CASI measurement scales, which can
differentiate between AD patients and age-matched healthy controls
(n=6). As a result, both scales of MMSE and CASI were decreased in
these AD patients (Table. 1). To this, FIG. 18B further showed that
the level of Nanog mRNA was significantly decreased in AD patients
compared to normal age-match controls (p<0.05). This observation
confirmed our therapy goal of miR-302 treatments in that AD
patients exhibit reduced Nanog expression, which contributes to the
pathogenesis of AD-associated neurodegeneration and therefore can
be a valid therapy target for the miR-302 treatments of the present
invention in AD patients.
[0029] In addition, the miR-302 familial gene is known to be
encoded in the human LARP7 gene on the chromosome 4 of human
genome. To determine whether the endogenous level of miR-302 was
affected by A.beta.-induced neurotoxicity during the progression of
AD, we examined the expression of miR-302-encoding LARP7 gene by
RT-qPCR with a special primer directed against the joining region
of exons 8 and 9. As a result, FIG. 18C showed that A.beta.
treatment markedly decreased LARP7 mRNA expression in control
neurons in vitro (n=3, p<0.05). Further detection of LARP
expression in AD patients' PBMCs also revealed that the expression
of LARP7 mRNA was significantly reduced in AD patients compared to
normal age-match controls (FIG. 18D, p<0.05). These results
proved that endogenous LARP7/miR-302 expression likely plays an
important role in preventing the progression of AD.
[0030] In conclusion, as summarized in FIG. 19, impairment of
insulin signaling not only presents a serious threat to neuron
survival but also plays a critical role in ageing-related diseases
such as AD. Our present invention, for the first time, demonstrated
that miR-302 can regulate cell survival and anti-ageing processes
via activating the Akt signaling pathway, which confers protection
against A.beta.-induced neurotoxicity in human neuronal cells. We
herein concluded that: (i) miR-302 silences PTEN to activate Akt
signaling, which the stimulates Nrf2/HO-1 elevation and hence
attenuates A.beta.-induced apoptosis, and (ii) miR-302-mediated Akt
activation also stimulates Nanog expression to suppress p-Ser307
IRS-1 expression and thus enhance IRS-1 tyrosine phosphorylation
and p-Ser 473-Akt/p-Ser 9-GSK3.beta. formation. Conceivably, both
of these newly identified miR-302 effects are useful for developing
AD-related therapies.
[0031] Although the invention has been described with references to
all of the above examples, it will be understood that modifications
and variations are encompassed within the spirit and scope of the
invention.
A. Definitions
[0032] To facilitate understanding of the invention, a number of
terms are defined below: Nucleic Acid: a polymer of
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), either
single or double stranded.
[0033] Nucleotide: a monomeric unit of DNA or RNA consisting of a
sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic
base. The base is linked to the sugar moiety via the glycosidic
carbon (1' carbon of the pentose) and that combination of base and
sugar is a nucleoside. A nucleoside containing at least one
phosphate group bonded to the 3' or 5' position of the pentose is a
nucleotide. DNA and RNA are consisted of different types of
nucleotide units called deoxyribonucleotide and ribonucleotide,
respectively.
[0034] Oligonucleotide: a molecule comprised of two or more
monomeric units of DNA and/or RNA, preferably more than three, and
usually more than ten. An oligonucleotide longer than 13 nucleotide
monomers is also called polynucleotiude. The exact size will depend
on many factors, which in turn depends on the ultimate function or
use of the oligonucleotide. The oligonucleotide may be generated in
any manner, including chemical synthesis, DNA replication, RNA
transcription, reverse transcription, or a combination thereof.
[0035] Nucleotide Analog: a purine or pyrimidine nucleotide that
differs structurally from adenine (A), thymine (T), guanine (G),
cytosine (C), or uracil (U), but is sufficiently similar to
substitute for the normal nucleotide in a nucleic acid
molecule.
[0036] Nucleic Acid Composition: a nucleic acid composition refers
to an oligonucleotide or polynucleotide such as a DNA or RNA
sequence, or a mixed DNA/RNA sequence, in either a single-stranded
or a double-stranded molecular structure.
[0037] Gene: a nucleic acid composition whose oligonucleotide or
polynucleotide sequence codes for an RNA and/or a polypeptide
(protein). A gene can be either RNA or DNA. A gene may encode a
non-coding RNA, such as small hairpin RNA (shRNA), microRNA
(miRNA), rRNA, tRNA, snoRNA, snRNA, and their RNA precursors as
well as derivatives. Alternatively, a gene may encode a
protein-coding RNA essential for protein/peptide synthesis, such as
messenger RNA (mRNA) and its RNA precursors as well as derivatives.
In some cases, a gene may encode a protein-coding RNA that also
contains at least a microRNA or shRNA sequence.
[0038] Primary RNA Transcript: an RNA sequence that is directly
transcribed from a gene without any RNA processing or
modification.
[0039] Precursor messenger RNA (pre-mRNA): primary RNA transcripts
of a protein-coding gene, which are produced by eukaryotic type-II
RNA polymerase (Pol-II) machineries in eukaryotes through an
intracellular mechanism termed transcription. A pre-mRNA sequence
contains a 5'-untranslated region (UTR), a 3'-UTR, exons and
introns.
[0040] Intron: a part or parts of a gene transcript sequence
encoding non-protein-reading frames, such as in-frame intron,
5'-UTR and 3'-UTR.
[0041] Exon: a part or parts of a gene transcript sequence encoding
protein-reading frames (cDNA), such as cDNA for cellular genes,
growth factors, insulin, antibodies and their analogs/homologs as
well as derivatives.
[0042] Messenger RNA (mRNA): assembly of pre-mRNA exons, which is
formed after intron removal by intracellular RNA splicing
machineries (e.g. spliceosomes) and served as a protein-coding RNA
for peptide/protein synthesis. The peptides/proteins encoded by
mRNAs include, but not limited, enzymes, growth factors, insulin,
antibodies and their analogs/homologs as well as derivatives.
[0043] Complementary DNA (cDNA): a single-stranded or
double-stranded DNA that contains a sequence complementary to an
mRNA sequence and does not contain any intronic sequence.
[0044] Sense: a nucleic acid molecule in the same sequence order
and composition as the homologous mRNA. The sense conformation is
indicated with a "+", "s" or "sense" symbol.
[0045] Antisense: a nucleic acid molecule complementary to the
respective mRNA molecule. The antisense conformation is indicated
as a "-" symbol or with an "a" or "antisense" in front of the DNA
or RNA, e.g., "aDNA" or "aRNA".
[0046] Base Pair (bp): a partnership of Watson-Crick base pairing
between adenine (A) and thymine (T) or between cytosine (C) and
guanine (G) in a double-stranded DNA molecule. In RNA, uracil (U)
is substituted for thymine (T) and another partnership of
non-Watson-Crick base pairing between guanine (G) and uracil (U)
also occurs. Generally the partnership is achieved through hydrogen
bonding. For example, a sense nucleotide sequence "5'-A-T-C-G-U-3"
can form complete base pairing with its antisense sequence
"5'-A-C-G-A-T-3" or "5'-A-U-G-A-T".
[0047] 5'-end: a terminus lacking a nucleotide at the 5' position
of successive nucleotides in which the 5'-hydroxyl group of one
nucleotide is joined to the 3'-hydroyl group of the next nucleotide
by a phosphodiester linkage. Other groups, such as one or more
phosphates, may be present on the terminus.
[0048] 3'-end: a terminus lacking a nucleotide at the 3' position
of successive nucleotides in which the 5'-hydroxyl group of one
nucleotide is joined to the 3'-hydroyl group of the next nucleotide
by a phosphodiester linkage. Other groups, most often a hydroxyl
group, may be present on the terminus.
[0049] Template: a nucleic acid molecule being copied by a nucleic
acid polymerase. A template can be single-stranded, double-stranded
or partially double-stranded, depending on the polymerase. The
synthesized copy is complementary to the template, or to at least
one strand of a double-stranded or partially double-stranded
template. Both RNA and DNA are synthesized in the 5' to 3'
direction. The two strands of a nucleic acid duplex are always
aligned so that the 5' ends of the two strands are at opposite ends
of the duplex (and, by necessity, so then are the 3' ends).
[0050] Nucleic Acid Template: a double-stranded DNA molecule,
double stranded RNA molecule, hybrid molecules such as DNA-RNA or
RNA-DNA hybrid, or single-stranded DNA or RNA molecule.
[0051] Conserved: a nucleotide sequence is conserved with respect
to a pre-selected (referenced) sequence if it non-randomly
hybridizes to an exact complement of the pre-selected sequence.
[0052] Homologous or Homology: a term indicating the similarity
between a polynucleotide and a gene or mRNA sequence. A nucleic
acid sequence may be partially or completely homologous to a
particular gene or mRNA sequence, for example. Homology may be
expressed as a percentage determined by the number of similar
nucleotides over the total number of nucleotides.
[0053] Complementary or Complementarity or Complementation: a term
used in reference to matched base pairing between two
polynucleotides (i.e. sequences of an mRNA and a cDNA) related by
the aforementioned "base pair (bp)" rules. For example, the
sequence "5'-A-G-T-3" is complementary to the sequence
"5'-A-C-T-3", and also to "5'-A-C-U-3". Complementation can be
between two DNA strands, a DNA and an RNA strand, or between two
RNA strands. Complementarity may be "partial" or "complete" or
"total". Partial complementarity or complementation occurs when
only some of the nucleic acid bases are matched according to the
base pairing rules. Complete or total complementarity or
complementation occurs when the bases are completely or perfectly
matched between the nucleic acid strands. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, as well as in detection methods that
depend on binding between nucleic acids. Percent complementarity or
complementation refers to the number of mismatch bases over the
total bases in one strand of the nucleic acid. Thus, a 50%
complementation means that half of the bases were mismatched and
half were matched. Two strands of nucleic acid can be complementary
even though the two strands differ in the number of bases. In this
situation, the complementation occurs between the portion of the
longer strand corresponding to the bases on that strand that pair
with the bases on the shorter strand.
[0054] Complementary Bases: nucleotides that normally pair up when
DNA or RNA adopts a double stranded configuration.
[0055] Complementary Nucleotide Sequence: a sequence of nucleotides
in a single-stranded molecule of DNA or RNA that is sufficiently
complementary to that on another single strand to specifically
hybridize between the two strands with consequent hydrogen
bonding.
[0056] Hybridize and Hybridization: the formation of duplexes
between nucleotide sequences which are sufficiently complementary
to form complexes via base pairing. Where a primer (or splice
template) "hybridizes" with target (template), such complexes (or
hybrids) are sufficiently stable to serve the priming function
required by a DNA polymerase to initiate DNA synthesis. There is a
specific, i.e. non-random, interaction between two complementary
polynucleotides that can be competitively inhibited.
[0057] Posttranscriptional Gene Silencing: a targeted gene knockout
or knockdown effect at the level of mRNA degradation or
translational suppression, which is usually triggered by either
foreign/viral DNA or RNA transgenes or small inhibitory RNAs.
[0058] RNA Interference (RNAi): a posttranscriptional gene
silencing mechanism in eukaryotes, which can be triggered by small
inhibitory RNA molecules such as microRNA (miRNA), small hairpin
RNA (shRNA) and small interfering RNA (siRNA). These small RNA
molecules usually function as gene silencers, interfering with
expression of intracellular genes containing either completely or
partially complementarity to the small RNAs.
[0059] Gene Silencing Effect: a cell response after a gene function
is suppressed, consisting but not limited of cell cycle
attenuation, GO/G1-checkpoint arrest, tumor suppression,
anti-tumorigenecity, cancer cell apoptosis, and a combination
thereof.
[0060] Non-coding RNA: an RNA transcript that cannot be used to
synthesize peptides or proteins through intracellular translation
machineries. Non-coding RNA includes long and short regulatory RNA
molecules such as microRNA (miRNA), small hairpin RNA (shRNA),
small interfering RNA (siRNA) and double strand RNA (dsRNA). These
regulatory RNA molecules usually function as gene silencers,
interfering with expression of intracellular genes containing
either completely or partially complementarity to the non-coding
RNAs.
[0061] MicroRNA (miRNA): single-stranded RNAs capable of binding to
targeted gene transcripts that have partial complementarity to the
miRNA. MiRNA is usually about 17-27 oligonucleotides in length and
is able to either directly degrade its intracellular mRNA target(s)
or suppress the protein translation of its targeted mRNA, depending
on the complementarity between the miRNA and its target mRNA.
Natural miRNAs are found in almost all eukaryotes, functioning as a
defense against viral infections and allowing regulation of gene
expression during development of plants and animals.
[0062] Precursor MicroRNA (Pre-miRNA): hairpin-like single-stranded
RNAs containing stem-arm and stem-loop regions for interacting with
intracellular RNaseIII endoribonucleases to produce one or multiple
microRNAs (miRNAs) capable of silencing a targeted gene or genes
complementary to the microRNA sequence(s). The stem-arm of a
pre-miRNA can form either a perfectly (100%) or a partially
(mis-matched) hybrid duplexes, while the stem-loop connects one end
of the stem-arm duplex to form a circle or hairpin-loop
conformation. In the present invention, however, precursor of
microRNA may also includes pri-miRNA.
[0063] Small interfering RNA (siRNA): short double-stranded RNAs
sized about 18-27 perfectly base-paired ribonucleotide duplexes and
capable of degrading target gene transcripts with almost perfect
complementarity.
[0064] Small or short hairpin RNA (shRNA): single-stranded RNAs
that contain a pair of partially or completely matched stem-arm
nucleotide sequences divided by an unmatched loop oligonucleotide
to form a hairpin-like structure. Many natural miRNAs are derived
from hairpin-like RNA precursors, namely precursor microRNA
(pre-miRNA).
[0065] Vector: a recombinant nucleic acid composition such as
recombinant DNA (rDNA) capable of movement and residence in
different genetic environments. Generally, another nucleic acid is
operatively linked therein. The vector can be capable of autonomous
replication in a cell in which case the vector and the attached
segment is replicated. One type of preferred vector is an episome,
i.e., a nucleic acid molecule capable of extrachromosomal
replication. Preferred vectors are those capable of autonomous
replication and expression of nucleic acids. Vectors capable of
directing the expression of genes encoding for one or more
polypeptides and/or non-coding RNAs are referred to herein as
"expression vectors" or "expression-competent vectors".
Particularly important vectors allow cloning of cDNA from mRNAs
produced using a reverse transcriptase. A vector may contain
components consisting of a viral or a type-II RNA polymerase
(Pol-II or pol-2) promoter, or both, a Kozak consensus translation
initiation site, polyadenylation signals, a plurality of
restriction/cloning sites, a pUC origin of replication, a SV40
early promoter for expressing at least an antibiotic resistance
gene in replication-competent prokaryotic cells, an optional SV40
origin for replication in mammalian cells, and/or a tetracycline
responsive element. The structure of a vector can be a linear or
circular form of single- or double-stranded DNA selected form the
group consisting of plasmid, viral vector, transposon,
retrotransposon, DNA transgene, jumping gene, and a combination
thereof.
[0066] Promoter: a nucleic acid to which a polymerase molecule
recognizes, perhaps binds to, and initiates RNA transcription. For
the purposes of the instant invention, a promoter can be a known
polymerase binding site, an enhancer and the like, any sequence
that can initiate synthesis of RNA transcripts by a desired
polymerase.
[0067] Eukaryotic Promoter: a sequence of nucleic acid motifs which
are required for RNA transcription and can be recognized by
eukaryotic type II RNA polymerases (pol-2), pol-2 equivalent,
and/or pol-2-like viral polymerases.
[0068] Type-II RNA Polymerase (Pol-II or pol-2) Equivalent: a
eukaryotic transcription machinery selected from the group
consisting of mammalian type-II RNA polymerases (Pol-II or pol-2)
and Pol-II-like viral RNA polymerases.
[0069] Type-II RNA Polymerase (Pol-II or pol-2) Promoter: a RNA
promoter that is recognized and used by eukaryotic type-II RNA
polymerases (Pol-II or pol-2) which transcribe eukaryotic messenger
RNAs (mRNAs) and/or microRNAs (miRNAs). For example, but not
limited, mammalian EF1alpha promoter is a pol-2 promoter.
[0070] Pol-II-like Viral Promoter: a viral RNA promoter capable of
using the eukaryotic pol-2 or equivalent transcription machinery
for its gene expression. For example, but not limited,
cytomegaloviral (CMV) promoter and retroviral long terminal repeat
(LTR) promoter are pol-2-like viral promoters.
[0071] Cistron: a sequence of nucleotides in a DNA molecule coding
for an amino acid residue sequence and including upstream and
downstream DNA expression control elements.
[0072] RNA Processing: a cellular mechanism responsible for RNA
maturation, modification and degradation, including RNA splicing,
intron excision, exosome digestion, nonsense-mediated decay (NMD),
RNA editing, RNA processing, and a combination thereof.
[0073] Antibiotic Resistance Gene: a gene capable of degrading
antibiotics selected from the group consisted of penicillin G,
streptomycin, ampicillin (Amp), neomycin, G418, kanamycin,
erythromycin, paromycin, phophomycin, spectromycin, tetracycline
(Tet), doxycycline (Dox), rifapicin, amphotericin B, gentamycin,
chloramphenicol, cephalothin, tylosin, and a combination
thereof.
[0074] Restriction/Cloning Site: a DNA motif for restriction enzyme
cleavage including but not limited to AatII, AccI, AflIII, AgeI,
ApaI/LI, AseI, Asp718I, BamHI, BbeI, BcII/II, BglII, BsmI, Bsp1201,
BspHI/LU111/1201, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, Clal, Csp61,
Dpnl, DraI/II, EagI, Ecl136II, EcoRI/RII/47111/RV, EheI, FspI,
HaeIII, HhaI, HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI,
MaeII/III, MfeI, MluI, MscI, MseI, NaeI, NarI, NcoI, NdeI, NgoMI,
NotI, NruI, NsiI, PmlI, Ppu10I, PstI, PvuI/II, RsaI, SacI/II, SalI,
Sau3AI, SmaI, SnaBI, SphI, SspI, StuI, TaiI, TaqI, XbaI, XhoI, XmaI
cleavage site.
[0075] Gene Delivery: a genetic engineering method selected from
the group consisting of polysomal transfection, liposomal
transfection, chemical transfection, electroporation, viral
infection, DNA recombination, transposon insertion, jumping gene
insertion, microinjection, gene-gun penetration, and a combination
thereof.
[0076] Genetic Engineering: a DNA recombination method selected
from the group consisting of DNA restriction and ligation,
homologous recombination, transgene incorporation, transposon
insertion, jumping gene integration, retroviral infection, and a
combination thereof.
[0077] Cell Cycle Regulator: a cellular gene involved in
controlling cell division and proliferation rates, consisting but
not limited of CDK2, CDK4, CDK6, cyclins, BMI-1, p14/p19Arf,
p15Ink4b, p16Ink4a, p18Ink4c, p21Cip1/Waf1, and p27Kip1, and a
combination thereof.
[0078] Tumor Suppression: a cellular anti-tumor and anti-cancer
mechanism consisting but not limited of cell cycle attenuation,
G0/G1-checkpoint arrest, tumor suppression, anti-tumorigenecity,
cancer cell apoptosis, and a combination thereof.
[0079] Targeted Cell: a single or a plurality of human cells
selected from the group consisting of a somatic cell, a tissue, a
stem cell, a germ-line cell, a teratoma cell, a tumor cell, a
cancer cell, and a combination thereof.
[0080] Cancerous Tissue: a neoplastic tissue derived from the group
consisting of skin cancer, prostate cancer, breast cancer, liver
cancer, lung cancer, brain tumor/cancer, lymphoma, leukemia and a
combination thereof.
[0081] Antibody: a peptide or protein molecule having a
pre-selected conserved domain structure coding for a receptor
capable of binding a pre-selected ligand.
[0082] Pharmaceutical or therapeutic Application: a biomedical
utilization and/or apparatus useful for stem cell generation, stem
cell research and/or therapy development, cancer therapy, disease
treatment, wound healing and tissue regeneration treatment,
high-yield production of drug and/or food supplies, and a
combination thereof.
[0083] Prokaryote or Prokaryotic Cell: an one-cell organism that
lacks a distinct membrane-bound nucleus and has its genetic
materials in the form of a continuous strand of DNA, such as
bacteria.
[0084] Eukaryote or Eukaryotic Cell: an one-cell or multiple-cell
organism whose cells contain a nucleus and other structures
(organelles) enclosed within membranes, such as yeast, plant and
animal cells.
[0085] Transcription Inducer: a chemical agent that can induce
and/or enhance hairpin-like RNA transcription from a eukaryotic
pol-2 or pol-2-like viral promoter in prokaryotic cells. For
example, a transcription inducer contains, but not limited, a
chemical structure similar to 3-morpholinopropane-1-sulfonic acid
(MOPS), ethanol and/or glycerin, as well as their functional
analogs, such as mannitol, 2-(N-morpholino)ethanesulfonic acid
(MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES), or a mixture thereof.
B. Compositions and Applications
[0086] A composition for using hairpin-like RNA mimics of microRNA
precursors (pre-miRNA) capable of protecting human brain neurons
from A.beta.-induced neurotoxicity in Alzheimer's diseases,
comprising: (a) at least a vector capable of expressing said
hairpin-like pre-miRNA mimics through a eukaryotic promoter,
wherein said vector is pLenti-EF1alpha/CMV-RGFP/GFP-miR302; and (b)
at least a transcription inducer capable of delivering and inducing
the expression of said hairpin-like pre-miRNA mimics in treated
neurons, wherein the expression of said hairpin-like pre-miRNA
mimics is induced by mixing (a) and (b) in a cell substrate
containing the treated neurons.
[0087] Alternatively, the present invention is a method of
protecting human brain neurons from A.beta.-induced neurotoxicity
in Alzheimer's diseases with hairpin-like RNA mimics of microRNA
precursors (hairpin-like pre-miRNA mimics), comprising: (a)
treating at least one neuron with a vector, wherein the vector
contains SEQ. ID. NO.2 and is capable of expressing at least one
hairpin-like pre-miRNA mimic through a eukaryotic promoter; and (b)
inducing an expression of said at least one hairpin-like pre-miRNA
mimic in the treated neurons with an administration of at least one
transcription inducer.
[0088] In principle, the present invention provides a novel
composition design and its applicable strategy for inducing
adaptation of prokaryotes to use eukaryotic promoters for producing
a large amount of hairpin-like RNAs as drugs for treating
Alzheimer's diseases in human brain neurons. Preferably, said
hairpin-like pre-miRNA mimics are miR-302 precursors (pre-miR-302)
in a structural conformation selected from the group consisting of
microRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA
(siRNA), their precursors and homologs, and a combination
thereof.
[0089] Preferably, said prokaryote is a bacterial cells in
particular, Escherichia coli (E. coli), and said transcription
inducer is 3-morpholinopropane-l-sulfonic acid (MOPS), ethanol or
glycerin, or a combination thereof. Also preferably, said
eukaryotic RNA promoter is either a eukaryotic pol-2 promoter, such
as EF 1 alpha, or a pol-2 compatible viral promoter, such as
cytomegaloviral (CMV) promoter or retroviral long terminal repeat
(LTR) promoter. The gene mediated by said eukaryotic RNA promoter
is coded for either a non-coding or a protein-coding RNA
transcript, or both, selected from the group consisted of microRNA
(miRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA),
messenger RNA (mRNA), their precursors and homologs, and a
combination thereof.
DESCRIPTION OF THE DRAWINGS AND TABLES
[0090] The patent application or application filed contains at
least one drawing executed in color. Copies of this patent or
patent application publication with color drawings will be provided
by the Office upon request and payment of the necessary fee.
[0091] Referring particularly to the drawings for the purpose of
illustration only and not limitation, there is illustrated:
[0092] FIGS. 1A and 1B show the basic design of a eukaryotic
promoter-driven hairpin RNA expression composition (A) and its
related RNA processing and translation mechanisms (B). Individual
components of the eukaryotic promoter-driven hairpin RNA expression
composition (i.e. the pLenti-EF lalpha-RGFP-miR302 plasmid vector
which may carry both EF lalpha and CMV promoters) can be re-located
in different places of the vector or even deleted for providing
more compact and effective delivery into targeted cells. According
to the disclosed mechanisms in (B), it is possible for an ordinary
skill in the art to use any microRNA/shRNA in place miR-302 or any
mRNA/protein in place of RGFP as taught in the present invention.
Black arrows indicate the pathways for protein/peptide production,
while white arrows indicate the steps for hairpin RNA
generation.
[0093] FIG. 2 depicts the results of E. coli culture broths treated
with (left) or without (right) the mixture of about 0.1% (v/v) MOPS
and about 0.05% (v/v) glycerin. The E. coli bacteria were
transformed by pLenti-EF1alpha-RGFP-miR302 before treatments.
[0094] FIG. 3 shows the results of different bacterial pellets
after treated with about 0.1% (v/v) MOPS. The E. coli bacteria were
transformed by either pLVX-GFP-miR302+367 (green) or pLenti-EF 1
alpha-RGFP-miR302 (red) vector, respectively, before the MOPS
treatment.
[0095] FIG. 4 shows the inducibility of different chemical inducers
for stimulating EF1alpha and/or CMV promoter-driven gene expression
in competent E. coli cells. Among all chemicals tested in the
present invention, the top three most potent transcription inducers
are MOPS, glycerin and ethanol. The inducer concentrations used can
be ranged from about 0.001% to about 10%, most preferably, from
about 0.05 to about 4%.
[0096] FIG. 5 shows the Western blotting results of red RGFP
protein expression induced by MOPS, glycerin, and ethanol,
respectively. Bacterial RuvB protein is used as a house-keeping
standard to compare the levels of induced RGFP expression. Proteins
and RNAs extracted from original E. coli cells without any vector
transformation serve as negative controls.
[0097] FIG. 6 shows the Northern blotting results of miR-302 and
its pre-miRNA/pri-miRNA cluster expression induced by MOPS,
glycerin, and ethanol, respectively. RNAs extracted from original
E. coli cells without any vector transformation serve as negative
controls.
[0098] FIG. 7 shows iPS cell (iPSC) generation using miR-302 and
pre-miR-302 isolated from bacterial extracts (BE), of which the
miR-302/pre-miRNA expression has been confirmed by Northern blot
analysis as shown in FIG. 6. As previously reported (Lin 2008,
2010, 2011), the miR-302-reprogrammed iPS cells (mirPSCs) form
sphere-like cell colonies and express strong ESC marker Oct4
proteins (labeled by Oct4-promoter-driven green fluorescent protein
expression).
[0099] FIG. 8 shows the global DNA demethylation of Oct4 and Sox2
gene promoters induced by miR-302 and pre-miR-302 isolated from
bacterial extracts (BE), of which the miR-302/pre-miRNA expression
has been confirmed by Northern blot analysis as shown in FIG. 6. As
reported by Simonsson and Gurdon (Nat Cell Biol. 6, 984-990, 2004),
both events of global DNA demethylation and Oct4 expression are
required for somatic cell reprogramming to form iPSCs.
[0100] FIGS. 9A and 9B show comparison of the healing results
between untreated (9A) and miR-302-treated (9B) wounds in vivo. The
isolated miR-302 molecules (20.about.400 .mu.g/mL) were formulated
with glycylglycerin and antibiotic ointment to form candidate drugs
for testing topic treatments of large 2cm.times.2cm open wounds on
pig back skins in vivo (n=6 for each group). After about two-week
treatments (one treatment per day), the healed wounds were
dissected and further made into tissue sections for histological
examination under a microscope. The data showed that no or very
little scar could be seen in the miR-302-treated wounds (9B top,
n=6/6), whereas almost all untreated (treated with only antibiotic
ointment) wounds contained large scars (9A top, n=5/6). Also, a
significantly large amount of CD34-positive adult stem cell
clusters (labeled by green fluorescent antibodies) were found in
the miR-302-treated wounds (9B bottom, n=6/6), but not in untreated
control wounds (9A bottom, n=0/6).
[0101] FIGS. 10A and 10B show the results of HPLC purification and
analysis using a synthetic standard uDNA (made by Sigma-Genosys)
and freshly extracted miR-302s/pre-miR-302s (or called
pro-miR-3025) isolated from pLenti-EF1alpha-RGFP-miR302-transformed
E. coli cells. The standard uDNA was designed to mimic a natural
pre-miR-302a as: 5'-CCACCACUUA AACGUGGAUG UACUUGCUUU GAAACUAAAG
AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3' (SEQ. ID. NO.3).
[0102] FIGS. 11A and 11B show the results of microRNA (miRNA)
microarray analyses using small RNAs extracted from either blank E.
coli competent cells or pLenti-EF1alpha-RGFP-miR302
(RGFP-miR302)-transformed/transfected cells. The extracted small
RNAs were further purified by HPLC as shown in the green-labeled
area of FIG. 10B. FIG. 11A shows that RNAs from blank E. coli cells
present almost no microRNA (green dots mean non-statistically
significant whereas red dots indicate positive results). This is
because prokaryotes lack several essential enzymes required for
microRNA expression and processing, such as Pol-2, Drosha and RNase
III Dicer. Also, prokaryotic RNA polymerases do not transcribe
small RNAs with high secondary structures, such as hairpin-like
pre-miRNAs and shRNAs, which resemble intrinsic transcription
termination signals in prokaryotes. As a result, only using the
present invention, we can stimulate the expression of specific
microRNAs, such as miR-302a, a*, b, b*, c, c*, d and d* as shown in
FIG. 11B, in prokaryotic cells. Since prokaryotes do not possess
Dicer, most microRNAs so obtained remain in their hairpin-like
precursor conformations, such as pri-miRNA (4-hairpin cluster)
and/or pre-miRNA (1 hairpin precursors).
[0103] FIG. 12 shows the lists of expressed microRNAs extracted
from either blank E. coli cells (Group 1 as shown in FIG. 11A) or
pLenti-EF1alpha-RGFP-miR302-transformed/transfected cells (Group 2
as shown in FIG. 11B). Signals less than 500 are not statistically
significant (as shown in green in FIGS. 11A and 11B), which may be
caused by either low copy number expression or high background.
[0104] FIGS. 13A and 13B show the sequencing results of the miR-302
familial cluster (13A) and the individual pro-miR-302a,
pro-miR-302b, pro-miR-302c, and pro-miR-302d sequences (13B). The
result of the whole miR-302 familial cluster transcript
(=pri-miR-302) is
TABLE-US-00001 (SEQ.ID.NO. 2) 5'- AAUUUUUUUC UUCUAAAGUU AUGCCAUUUU
GUUUUCUUUC UCCUCAGCUC UAAAUACUCU GAAGUCCAAA GAAGUUGUAU GUUGGGUGGG
CUCCCUUCAA CUUUAACAUG GAAGUGCUUU CUGUGACUUU AAAAGUAAGU GCUUCCAUGU
UUUAGUAGGA GUGAAUCCAA UUUACUUCUC CAAAAUAGAA CACGCUAACC UCAUUUGAAG
GGAUCCCCUU UGCUUUAACA UGGGGGUACC UGCUGUGUGA AACAAAAGUA AGUGCUUCCA
UGUUUCAGUG GAGGUGUCUC CAAGCCAGCA CACCUUUUGU UACAAAAUUU UUUUGUUAUU
GUGUUUUAAG GUUACUAAGC UUGUUACAGG UUAAAGGAUU CUAACUUUUU CCAAGACUGG
GCUCCCCACC ACUUAAACGU GGAUGUACUU GCUUUGAAAC UAAAGAAGUA AGUGCUUCCA
UGUUUUGGUG AUGGUAAGUC UUCUUUUUAC AUUUUUAUUA UUUUUUUAGA AAAUAACUUU
AUUGUAUUGA CCGCAGCUCA UAUAUUUAAG CUUUAUUUUG UAUUUUUACA UCUGUUAAGG
GGCCCCCUCU ACUUUAACAU GGAGGCACUU GCUGUGACAU GACAAAAAUA AGUGCUUCCA
UGUUUGAGUG UGGUGGUUCC UACCUAAUCA GCAAUUGAGU UAACGCCCAC ACUGUGUGCA
GUUCUUGGCU ACAGGCCAUU ACUGUUGCUA-3',
while the individual sequences of pro-miR-302a, pro-miR-302b,
pro-miR-302c, and pro-miR-302d are as follows: 5'-CCACCACUUA
AACGUGGAUG UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG-3'
(SEQ. ID. NO.3), 5'-GCUCCCUUCA ACUUUAACAU GGAAGUGCUU UCUGUGACUU
UAAAAGUAAG UGCUUCCAUG UUUUAGUAGG AGU-3' (SEQ. ID. NO.4),
5'-CCUUUGCUUU AACAUGGGGG UACCUGCUGU GUGAAACAAA AGUAAGUGCU
UCCAUGUUUC AGUGGAGG-3' (SEQ. ID. NO.5), and 5'-CCUCUACUUU
AACAUGGAGG CACUUGCUGU GACAUGACAA AAAUAAGUGC UUCCAUGUUU GAGUGUGG-3'
(SEQ. ID. NO.6), respectively.
[0105] FIGS. 14A-14F show that treatments of miR-302 inhibit
A.beta.-induced apoptosis in human SK-N-MC neuronal cells. (14A)
Transfection of SK-N-MC cells with either the pLVX-Grn-miR302
vector (black bar, to form miR-302-overexpresed cells) or an empty
vector (white bar, to serve as control cells), using a
lipofectamine 2000 reagent. Positively transfected cells were
detected by co-expression of a green fluorescent protein (AcGFP)
under an inverted fluorescent microscope. (14B) RT-qPCR analyses of
miR-302 expression using total RNA samples extracted from
miR-302-transfected (black bar) or control (white bar) cells,
respectively. The detected miR-302 expression levels in transfected
cells were normalized with the levels of control cells (n=3,
p<0.01). (14C) Cell viability was determined by MTT assays.
Cells were seeded in 24-welled plates overnight and then treated
with 2.5 .mu.M A.beta. for 24 hours. The results of cell viability
were normalized using the level of control cells, showing that
ectopic miR-302 expression significantly reduced A.beta.-induced
cell death. (14D) Morphological changes of nuclear chromatins
during apoptosis were observed under fluorescent microscopy with
DAPI staining. Cells were cultivated on coated slides and treated
with 2.5 .mu.M A.beta. for 24 hours. The nuclei fragmentation was
labeled (white arrow) and was quantified by counting four random
fields per condition (14E). (14F) A.beta.-induced cell apoptosis
was determined by western blotting of Caspase 3 and PARP cleavage
after A.beta. treatment (2.5 .mu.M A.beta. for 24 hours). The
results were normalized with the density levels of control cells,
showing markedly attenuated A.beta.-induced cell apoptosis in
miR-302-transfected cells (n=3, p<0.01). (A.beta.,
amyloid-.beta.; +, with treatment; -, without treatment. All values
were presented as mean .+-.S.E.M. Significant differences were
determined by multiple comparisons using Dunnett's post-hoc trest
for * p <0.05 and **p<0.01.)
[0106] FIGS. 15A-15D show that ectopic miR-302 expression activates
Akt signaling and hence diminishes A.beta.-induced cytotoxicity.
(15A) Western blot analyses of pSer307-IRS-1, pTyr-IRS-1, and
pSer473-Akt expressions 24 hours after A.beta. treatment (2.5
.mu.M), showing marked elevation of pSer307-IRS-1 (n=3, p<0.01)
as well as reduction of both pTyr-IRS-1 and pSer473-Akt levels
(n=3, p<0.05) in control groups compared to those of
miR-302-transfected cells. (15B) Western blot analysis of
pSer473-Akt levels after treatments of 2.5 .mu.M A.beta. or 20
.mu.M LY294002, or both for 24 hours. (15C) Cell viability in
response to the treatments of (15B), as determined by MTT assays.
(15D) Western blot measurement of pSer9-GSK3.beta., and pThr231-tau
levels in response to the treatments of miR-302s (15B), showing
that miR-302 could stimulate Akt signaling to counteract
A.beta.-mediated cytotoxicity, resulting in a marked increase of
GSK3.beta. Ser9 phosphorylation and decrease of tau-Thr231
phosphorylation (n=3, p<0.05). Yet, further co-treatment of
A.beta. (2.5 .mu.M) and LY294002 (20 .mu.M) abolished all these
protective effects of Akt signaling in miR-302-transfected cells
(n=3, p<0.05). (A.beta., amyloid-.beta.; +, with treatment; -,
without treatment. All values were presented as mean .+-.S.E.M.
Significant differences were determined by multiple comparisons
using Dunnett's post-hoc trest for *p<0.05 and **p<0.01.)
[0107] FIGS. 16A-16E show that miR302-induced Akt signaling
activation attenuates A.beta.-induced oxidative stress. (16A)
Intracellular superoxide radical anions stained with DHE were
detected by fluorescence microscopy. Cells were treated with 2.5
.mu.M A.beta. or 1 .mu.M insulin, or both, for 2 hours and then
analyzed with DHE staining. The intensity of red fluorescent dye
was normalized with the level of control cells before comparison.
(16B) After 24-hour A.beta. treatment (2.5 .mu.M), western blot
analyses showed that the expression of Nrf2 and HO-1 were decreased
in control cells compared to those of miR-302-transfected cells
(n=3, p<0.05). (16C) Cells were treated with 2.5 .mu.M A.beta.
in the presence of 1 .mu.M insulin or 20 .mu.M LY294002, or both,
and then analyzed with western blotting for Nrf2. As shown,
co-treatment of A.beta. and LY294002 inhibited Nrf2 expression
(n=3, p<0.05), whereas further treatment with insulin (1 .mu.M)
prevented this inhibitory effect on Nrf2 expression (n=3,
p<0.05). (16D) Cells of (16C) were further stained with JC-1 dye
and observed under an inverted fluorescent microscope, showing that
A.beta. treatment reduced the intensity of JC-1 green fluorescence
in miR-302-transfected cells (n=3, p<0.05), while further
treatment of LY294002 (20 .mu.M) prevented this effect. (16E)
Western blotting analyses showing that a significant increase of
tBid and decrease of Bcl-2 were observed in control cells compared
to miR-302-transfected cells after 24-hour A.beta. treatment (2.5
.mu.M). (A.beta., amyloid-.beta.; +, with treatment; -, without
treatment. For fluorescent density quantification, the levels of
tested cells were normalized with that of control cells before
comparison. Values were presented as mean .+-.S.E.M. Significant
differences were determined by multiple comparisons using Dunnett's
post-hoc trest for *p<0.05 and **p<0.01.)
[0108] FIGS. 17A-17F show that miR-302 targets PTEN and upregulates
Nanog through Akt signaling. (17A) Alignment of predicted miR-302
binding sites within human PTEN 3'UTR was shown. (17B and 17C)
Cells lysates were obtained from untreated control cells and
miR-302-transfected cells, respectively, and further analyzed with
western blotting for PTEN and Nanog, showing the downregulation of
PTEN and upregulation of Nanog in miR-302-transfected cells (n=3,
p<0.05). (17D) Western blot analyses of PTEN, pSer473 Akt, and
Nanog expressions after 24-hour A.beta. treatment (2.5 .mu.M),
showing an increase of PTEN (p<0.05) and decreases of pSer473
Akt (p<0.05) and Nanog in control cells (n=3, p<0.01)
compared to those of miR-302-transfected cells (n=3, p<0.05).
(17E) Western blot analyses of pSer473 Akt and Nanog expressions 24
hours after treatment of A.beta. (2.5 .mu.M) or LY294002 (20
.mu.M), or both, showing that both pSer473 Akt and Nanog were
significantly decreased in miR-302-transfected cells treated with
both A.beta. and LY294002 (n=3, p<0.05). (17F) The
miR-302-transfected cells were transiently transfected with
shRNA-Nanog, and then treated with A.beta. (2.5 .mu.M) for 24
hours. shRNA-directed knockdown of Nanog markedly elevated
pSer307-IRS-1 and reduced the levels of pTyr-IRS-1, pSer473-Akt and
pSer9-GSK3.beta. expressions in miR-302-transfected cells compared
to those of control cells treated with A.beta. alone. (A.beta.,
amyloid-.beta.; shRNA-Nanog, shRNA gene silencer directed against
human Nanog. +, with treatment; -, without treatment. The results
of density quantification were normalized with the level of control
cells. Values were presented as mean .+-.S.E.M. Significant
differences were determined by multiple comparisons using Dunnett's
post-hoc trest for *p<0.05 and **p<0.01.)
[0109] FIGS. 18A-18D show that Comparison of the expression levels
of Naong and LARP7 mRNAs in vitro and in vivo after miR-302
treatments. (18A) After 24-hour A.beta. treatment (2.5 .mu.M), the
expression of Nanog mRNA was markedly decreased in control cells in
vitro (n=3, p<0.05). (18B) Both AD patients' (n=7) and normal
age-matched individual's (n=6) blood samples were collected,
separately, and total RNAs were then extracted and used for RT-qPCR
analyses. The results showed that AD patients' PBMCs express
significantly lower Nanog mRNAs than that of normal individuals
(p<0.05). (18C) Following 24-hour A.beta. treatment (2.5 .mu.M),
the expression of LARP7 mRNA was markedly reduced in control cells
compared to that of miR-302-transfected cells in vitro (n=3,
p<0.05). (18D) AD patients' PBMCs expressed significantly lower
LARP7 mRNA levels than that of normal individuals (p<0.05). (AB,
amyloid-.beta.; AD, Alzheimer diseases. Levels of mRNA expression
were normalized with the levels of control cells or normal healthy
individuals. Values were presented as mean .+-.S.E.M. Significant
differences were determined by using multiple comparisons of
Dunnett's post-hoc trest for *p<0.05 and **p<0.01.)
[0110] FIG. 19 shows a proposed scheme for the protective effects
of miR-302 against A.beta.-induced neurotoxicity. Upregulation of
miR-302 can silence PTEN to activate Akt signaling, which
subsequent (i) stimulates Nrf2/HO-1 elevation and hence attenuates
A.beta.-induced oxidative stress and apoptosis, and (ii) stimulates
Nanog expression to suppress p-Ser307 IRS-1 expression, resulting
in a significant increase of insulin/IRS-1/Akt signaling, so as to
inhibit GSK3.beta.-mediated tau hyperphosphorylation.
[0111] FIG. 20 shows Table 1, which shows data of AD patients and
age-matched healthy individuals included in this trial study of
miR-302 treatments for AD therapy. The table presents gender, age,
MMSE and CASI scores for AD patients and healthy individual
controls, respectively.
EXAMPLES
[0112] Referring particularly to the Examples provided for the
purpose of practical demonstration only and not limitation.
1. Bacterial Cell Culture and Chemical Treatments
[0113] Competent cells of E. coli DH5alpha strain were acquired
from the z-competent E. coli transformation kit (Zymo Research,
Irvine, Calif.) and transformed by mixing with about 1-10 .mu.g of
a desired plasmid vector such as pLVX-Grn-miR302+367 and/or
pLenti-EF1alpha-RGFP-miR302 vectors. Non-transformed bacterial
cells were normally grown in Luria-Bertani (LB) broth supplemented
with 10 mM MgSO.sub.4 and 0.2 mM glucose at 37.degree. C. with
frequent agitation at 170 rpm, whereas the transformed bacterial
cells were cultivated under the same condition with further
addition of 100 .mu.g/mL ampicillin. For chemical induction, about
0.1.about.10 mL of MOPS, glycerin, and/or ethanol, respectively or
in combination, was added into per litter of LB broth supplemented
with 10 mM MgSO.sub.4 and 0.2 mM glucose in the presence of 100
.mu.g/mL ampicillin. As negative controls, the transformed cells
were cultivated in the same ampicillin-added LB broth but without
addition of any chemical inducer.
2. Human Cell Culture and MicroRNA Transfection
[0114] For inducing stem cell derivation with miR-302, human
epidermal skin cells (hpESCs) were isolated and dissociated from a
minimum of 2 cubic mm by 4 mg/mL collagenase I digestion at
37.degree. C. for 35 min in fresh RPMI 1640 medium supplemented
with 20% FBS. For culturing keratinocytes, the isolated cells were
cultivated in EpiLife serum-free cell culture medium supplemented
with human keratinocyte growth supplements (HKGS, Invitrogen,
Carlsbad, Calif.) in the absence of antibiotics at 37.degree. C.
under 5% CO.sub.2. Culture cells were passaged at 50%-60%
confluency by exposing cells to trypsin/EDTA solution for 1 min and
rinsing once with phenol red-free DMEM medium (Invitrogen), and the
detached cells are replated at 1:10 dilution in fresh EpiLife
medium with HKGS supplements. Human cancer/tumor cell lines MCF7,
HepG2 and Tera-2 were obtained from the American Type Culture
Collection (ATCC, Rockville, MD) and maintained according to
manufacturer's suggestions. For microRNA/shRNA transfection, 15
.mu.g of isolated miR-302 and/or precursor thereof was dissolved in
1 mL of fresh EpiLife medium and mixed with 50 .mu.L of X-tremeGENE
HP DNA transfection reagent. After 10 min incubation, the mixture
was added into a 100-mm cell culture dish containing 50%-60%
confluency of hpESCs or the cancer/tumor cells, respectively. The
medium was replaced by fresh EpiLife medium with HKGS supplements
or the conditioned medium suggested by ATCC 12 to 18 hours later.
This transfection procedure could be repeated 3 to 4 times every
three-four days to increase transfection efficiency. After cell
morphology became sphere-like, the cells (mirPSCs) were grown and
passaged in knockout DMEM/F-12 medium (Invitrogen, CA) supplemented
with 20% knockout serum, 1% MEM nonessential amino acids, 100 .mu.M
B-mercaptoethanol, 1 mM GlutaMax, 1 mM sodium pyruvate, 10 ng/mL
bFGF, 10 ng/mL FGF-4, 5 ng/mL LIF, 100 IU/ml penicillin/100
.mu.g/mL streptomycin, 0.1 .mu.M A83-01, and 0.1 valproic acid
(Stemgent, San Diego, Calif.), at 37.degree. C. under 5%
CO.sub.2.
[0115] In the tests for treating AD with miR-302, human
neuroblastoma SK-N-MC cells were obtained from the American Type
Culture Collection (ATCC, Bethesda, Md., USA). Cells were
maintained in Minimal Eagle's medium (MEM, Gibco), supplemented
with 10% fetal bovine serum, 100 units/mL penicillin, 100 .mu.g/mL
streptomycin, and 2 mM L-glutamine at 37.degree. C., 5% CO.sub.2.
For inducing miR-302 expression, a pLVX-Grn-miR-302 vector was
applied to transfect the SK-N-MC cells, using a lipofectamine 2000
reagent (Invitrogen) following the manufacturer's instructions, so
as to form miR-302-transfected cells. The miR-302-transfected cells
were identified by the presence of a co-expressed AcGFP green
fluorescent protein. For silencing Nanog expression, another shRNA
gene silencer vector directed against human Nanog mRNAs, called
shRNA-Nanog, was obtained from Academia Sinica in Taiwan. In some
experiments, we further transfected the shRNA-Nanog vector into the
miR-302-transfected cells with the lipofectamine 2000 reagent.
3. Protein Extraction and Western Blot Analysis
[0116] Cells (10.sup.6) are lysed with a CelLytic-M
lysis/extraction reagent (Sigma) supplemented with protease
inhibitors, Leupeptin, TLCK, TAME and PMSF, following the
manufacturer's suggestion. Lysates are centrifuged at 12,000 rpm
for 20 min at 4.degree. C. and the supernatant is recovered.
Protein concentrations are measured using an improved SOFTmax
protein assay package on an E-max microplate reader (Molecular
Devices, CA). Each 30 .mu.g of cell lysate are added to SDS-PAGE
sample buffer under reducing (+50 mM DTT) and non-reducing (no DTT)
conditions, and boiled for 3 min before loading onto a 6-8%
polyacylamide gel. Proteins are resolved by SDS-polyacrylamide gel
electrophoresis (PAGE), electroblotted onto a nitrocellulose
membrane and incubated in Odyssey blocking reagent (Li-Cor
Biosciences, Lincoln, NB) for 2 hours at room temperature. Then, a
primary antibody is applied to the reagent and incubated the
mixture at 4.degree. C. Primary antibodies include Oct3/4 (Santa
Cruz Biotechnology, Santa Cruz, Calif.), Sox2 (Santa Cruz), Nanog
(Santa Cruz), CDK2 (Santa Cruz), cyclin D1 (Santa Cruz), cyclin D2
(Abcam), BMI-1 (Santa Cruz), keratin 16 (Abcam), .beta.-actin
(Chemicon, Temecula, Calif.), RuvB (Santa Cruz) and RGFP
(Clontech). After overnight, the membrane is rinsed three times
with TBS-T and then exposed to goat anti-mouse IgG conjugated
secondary antibody to Alexa Fluor 680 reactive dye (1:2,000;
Invitrogen-Molecular Probes), for 1 hour at the room temperature.
After three additional TBS-T rinses, fluorescent scanning of the
immunoblot and image analysis are conducted using Li-Cor Odyssey
Infrared Imager and Odyssey Software v.10 (Li-Cor).
4. RNA Extraction and Northern Blot Analysis
[0117] Total RNAs (10 .mu.g) are isolated with a mirVana.TM. miRNA
isolation kit (Ambion, Austin, Tex.), fractionated by either 15%
TBE-urea polyacrylamide gel or 3.5% low melting point agarose gel
electrophoresis, and electroblotted onto a nylon membrane.
Detection of miR-302 and/or pre-miR-302 is performed with a
[LNA]-DNA probe (5'-[TCACTGAAAC] ATGGAAGCAC TTA-3') (SEQ. ID. NO.1)
probe. The probe has been purified by high-performance liquid
chromatography (HPLC) and tail-labeled with terminal transferase
(20 units) for 20 min in the presence of [.sup.32P]-dATP (>3000
Ci/mM, Amersham International, Arlington Heights,Ill.).
5. Plasmid Amplification and Plasmid DNA/Total RNA Extraction
[0118] Competent E. coli DH5alpha cells treated with plasmid
transformation (from Example 1) are cultivated overnight in LB
broth supplemented with 10 mM MgSO.sub.4 and 0.2 mM glucose at
37.degree. C. with frequent agitation at 170 rpm. For inducing
eukaryotic promoter-driven RNA and/or protein production, 0.5 to 2
ml of MOPS, glycerin, and/or ethanol is added into every 1 litter
of LB broth for the above bacterial cultivation and amplification.
All amplified plasmid DNAs and expressed mRNAs/microRNAs are
isolated together using a HiSpeed plasmid purification kit (Qiagen,
Valencia, Calif.), following the manufacturer's protocol but with a
minor modification that RNase A is not added into the P1 buffer.
The final extracted products containing both plasmids and
mRNAs/microRNAs are dissolved in DEPC-treated ddH.sub.2O and stored
at -80.degree. C. before use. For purifying only the amplified
plasmid vectors, RNase A is added into the P1 buffer and the
extraction procedure is performed following the manufacturer's
protocol.
6. MicroRNA and mRNA Isolation/Purification
[0119] Total RNAs isolated from the above Example 5 are further
purified using a mirVana.TM. miRNA isolation kit (Ambion, Austin,
Tex.), following the manufacturer's protocol. The final products
are dissolved in DEPC-treated ddH.sub.2O and stored at -80.degree.
C. before use. Because bacterial RNAs are degraded very fast (a few
hours) in nature while eukaryotic poly-A RNAs (mRNAs) and
hairpin-like microRNA precursors (pre-miRNA or pri-miRNA) remain
relatively stable at 4.degree. C. (half-life up to 3-4 days), we
can use this difference to acquire pure mRNAs and/or pre-miRNAs for
further applications. For example, RGFP mRNA can be used to
identify the transfected cells, while pre-miR-3025 are used to
reprogram somatic cells to ESC-like iPS cells. The purified
pre-miR-302s can also be added into stem cell culture medium to
facilitate and maintain the reprogramming process.
7. Immunostaining Assay
[0120] Embedding, sectioning and immunostaining tissue samples are
performed as reported (Lin et al., RNA 2008). Primary antibodies
include Oct4 (Santa Cruz), Sox2 (Santa Cruz), Nanog (Santa Cruz),
and RGFP (Clontech). Fluorescent dye-labeled goat anti-rabbit or
horse anti-mouse antibody is used as the secondary antibody
(Invitrogen-Molecular Probes). Positive results are examined and
analyzed at 100.times. or 200.times. magnification under a
fluorescent 80i microscopic quantitation system with a Metamorph
imaging program (Nikon).
8. Bisulfite DNA Sequencing
[0121] Genomic DNAs are isolated from about two million cells using
a DNA isolation kit (Roche, Indianapolis, Iowa) and 1 .mu.g of the
isolated DNAs are further treated with bisulfite (CpGenome DNA
modification kit, Chemicon, Temecula, Calif.), according to the
manufacturers' suggestions. The treatment with bisulfite converts
all unmethylated cytosine to uracil, while methylated cytosine
remains as cytosine. For bisulfite DNA sequencing analyses, we
amplify the promoter regions of Oct4 and Nanog with PCR. Primers
include 5'-GAGGCTGGAG CAGAAGGATT GCTTTGG-3'(SEQ. ID. NO.2) and
5'-CCCTCCTGAC CCATCACCTC CACCACC-3'(SEQ. ID. NO.3) for Oct4, and
5'-TGGTTAGGTT GGTTTTAAAT TTTTG-3' (SEQ. ID. NO.4) and 5'-AACCCACCCT
TATAAATTCT CAATTA-3'(SEQ. ID. NO.5) for Nanog. The
bisulfite-modified DNAs (50 ng) are first mixed with the primers
(total 100 pmole) in 1.times. PCR buffer, heated to 94.degree. C.
for 2 min, and immediately cooled on ice. Next, 25 cycles of PCR
are performed as follows: 94.degree. C. for 1 min and 70.degree. C.
for 3 min, using an Expand High Fidelity PCR kit (Roche). The
amplified DNA product with a correct size is further fractionized
by 3% agarose gel electrophoresis, purified with a gel extraction
filter (Qiagen), and then used in DNA sequencing. A detailed
profile of the DNA methylation sites is then generated by comparing
the unchanged cytosine in the converted DNA sequence to the
unconverted one.
9. Materials and Preparations
[0122] 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), 4- ,6-diamidino-2-phenylindole (DAPI), dihydroethidium
(DHE), and JC-1 were purchased from Sigma (Munchen, Germany).
Amyloid-.beta. (A.beta.) 1-42 was acquired from AnaSpec Inc. (San
Jose, Calif., USA), and solutions were prepared according to our
previous report (Li et al, 2015). Antibodies used were directed
against Akt, p-Akt, GSK3.beta., p-GSK3.beta., IRS-1, Nrf2, HO-1,
tBid, Bc1-2, Caspase 3, poly (ADP-ribose) polymerase (PARP) (from
Santa Cruz, Calif., USA), p-tyrosine, p-Tau, Tau (from Merck
Millipore, Darmstadt, Germany), (.beta.-actin (from Novus
Biologicals, Littleton, Colo., USA), p-IRS-1, Nanog, and PTEN (from
Cell Signaling Technology, Danvers, Ma., USA), respectively.
10. Cell Viability Assays
[0123] Cells were seeded in 24-well plates overnight and then
treated as indicated. After 24 hours, the tetrazolium salt MTT was
added to the medium following the manufacturer's instructions. Only
viable cells could metabolize MTT into a purple formazan product,
of which the color density (OD) was further quantified by a Bio-Rad
spectrophotometer at 550 nm. Cell viability was determined by the
percentage of OD from treated cells or transfected cells divided by
OD from control cells.
11. Examination of Nucleus Morphology
[0124] Cells were cultivated on coated slides at 60% confluency and
then treated with drugs for 24 hours. Thereafter, changes in cell
nucleus morphology, in particular characteristics of apoptosis,
were examined, using a fluorescence microscope. The cells were
fixed in 4% paraformaldehyde after 24 hours of treatment with the
indicated compounds, permeabilized in ice-cold methanol, incubated
with 1 ng/mL of DAPI stain for 15 min at room temperature, and then
observed under a fluorescence microscope (DP80/BX53, Olympus).
Apoptotic cells were quantified by counting four random fields per
condition of treatment.
12. Analysis of Mitochondrial Membrane Potential (MMP)
[0125] MMP was investigated using a vital mitochondrial cationic
dye JC-1, which accumulates in mitochondria in a
potential-dependent matter. Cells were treated with 1 .mu.M of JC-1
in fresh medium and incubated at 37.degree. C. for 30 min. Cell
morphology was then observed and photographed using an inverted
fluorescence microscope (DP72/CKX41, Olympus). In normal cells,
JC-1 remained as red fluorescent aggregations, whereas during the
induction of apoptosis the mitochondrial potential collapsed and
hence JC-1 formed monomers producing green fluorescence. MMP was
quantified by fluorescent intensity using Image J software (NIH,
Bethesda, Md.). Then, the normalized fluorescence intensity levels
from control cells were set as 100% for comparing the relative
expression levels of the fluorescent intensities in tested
groups.
13. Detection of ROS by Dihydroethidium (DHE) Staining
[0126] DHE is a fluorogenic reagent used for detecting
intracellular superoxide radical anion. Cells were treated in fresh
medium containing 10 .mu.M DHE and incubated for 30 min in the dark
at room temperature. After 30-min incubation, the staining medium
was discarded and the cells were washed twice with PBS and then
observed and photographed under an inverted fluorescence microscope
(DP72/CKX41). ROS levels were determined by oxidized DHE
fluorescence intensity using Image J software (NIH, Bethesda, Md.).
Then, the normalized fluorescence intensity levels from control
cells were set as 100% for comparing the relative expression levels
of the fluorescent intensities in tested groups.
14. Study Population and Blood Sample
[0127] Blood sampling from AD patients (n=7) and age-matched
healthy individuals (n=6) was performed according to standardized
procedures approved by the Institutional Review Board (IRB) of
Chung Shan Medical University Hospital (CSMUH No: CS 13233) (Table
1). Clinical AD diagnosis was determined by the Diagnostic and
Statistical Manual of Mental Disorders IV (DSM-IV) criteria and
completed with a Mini-Mental State Examination (MMSE) and cognitive
abilities screening instrument (CASI) test. MMSE scores were used
as a rough measurement of cognitive function. CASI scores ranged
from 1 to 100 were used for quantitative assessment on attention,
concentration, orientation, short-term memory, long-term memory,
language abilities, visual construction, list-generating fluency,
abstraction, and judgment. A detailed overview of AD patients (n=7,
mean age 80.0.+-.4.9 years, range 74-86 years) and age-matched
healthy individuals (n=6, mean age 80.0.+-.5.9 years, range 72-86
years) was summarized in Table 1. A number of AD patients (n=7,
Female/Male=4/3) had moderate dementia under MMSE (mean
scale=19.3.+-.2.6, range 16-23) and CASI (mean scale=65.1.+-.10.3,
range 49-79) measurement scales, showing most differences between
AD patients and age-matched healthy controls (n=6, Female/Male=3/3)
(Table. 1). Age-matched healthy individuals were recruited by local
advertisement at the Aging Research Unit, Chung Shan Medical
University, Taichung, Taiwan. Neither cognitive impairment nor any
dementia disorder was detected in all tested healthy individuals.
Both AD patients and age-matched healthy individuals were
volunteers with written informed consents had been obtained from
all participants and/or their closest relatives according to the
Declaration of Helsinki and the IRB-approved protocols.
Approximately 20 mL of venous peripheral blood mononucleated cells
(PBMCs) were obtained from each tested subject and then total RNAs
were isolated from each blood sample with an Qiagen RNeasy Kit
(Qiagen, Germantown, Md., USA) and further used for
spectrophotometric quantification following the manufacturer's
instructions.
15. Reverse transcription (RT) and quantitative PCR (qPCR)
[0128] Total RNAs were extracted from patients' PBMCs and cells,
respectively, using a Qiagen RNeasy Kit (Qiagen) and further
quantified spectrophotometrically. RT-qPCR was carried out using 1
.mu.g of total RNAs and following the protocols of an ABI
High-Capacity cDNA Archive Kit (ABI). Then, we diluted the
resulting cDNA into ten folds and used only 5 .mu.l of the diluted
cDNA in each of triplicate qPCRs run on a Applied Biosystems 7300
Real Time PCR System with Maxima SYBR Green qPCR Master Mix
(2.times.), ROX solution provided (Thermo), according to the
manufacturer's instructions. Levels of relative mRNA or miRNA
expression were acquired with the SDS software version 1.2.3
(Sequence Detection Systems 1.2.3-7300 Real Time PCR System,
Applied Biosystems) and then further normalized with the level of
housekeeping GAPDH expression in the same sample. The normalized
mRNA levels from control cells or normal healthy individuals were
set as 100% for comparing the relative expression levels of the
mRNA expression in tested groups.
16. Statistic Analysis
[0129] Each experiment was repeated at least for three times
(n>3). All data were presented as means .+-.standard error of
mean (S.E.M). For cell viability tests, the average population
number of control cells was set as 100% for comparing the survival
rates of other tested cells. For western blotting, the protein
level measured in each blot was first normalized with the
expression level of a housekeeping .beta.-actin protein, and then
compared to the normalized level of the protein expressed in
control cells, of which the control protein level was then set as
100% for further comparison. For RT-qPCR, the measured values of
mRNA expression were first normalized with the expression level of
housekeeping GAPDH, and then compared to the normalized mRNA levels
from control cells or normal healthy individuals, of which the
control mRNA levels were set as 100% for comparing the relative
expression levels of the mRNA in tested groups. For measuring
fluorescence intensity, the normalized fluorescence intensity
levels from control cells were set as 100% for comparing the
relative expression levels of the fluorescent intensities in tested
groups. Statistical significance of differences between compared
groups was determined by one-way analysis of variance (ANOVA)
following Dunnett's post-hoc test for multiple comparisons with a
SPSS statistical software (SPSS, Inc., Chicago, Ill., USA) as well
as the two-tailed Student's t-test. A probability value of <0.05
or <0.01 was taken to indicate statistical significance and
hence the significant levels were set at *p<0.05 or **p<0.01,
respectively, depending on individual experiments. Probability
values of p<0.05 is considered significant. All p values are
determined from two-tailed tests.
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TABLE-US-00002 [0164] SEQUENCE LISTING (1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 6 (2) INFORMATION FOR SEQ ID NO: 1: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: RNA (A) DESCRIPTION: /desc = ''natural'' (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO: 1: UAAGUGCUUC CAUGUUU 17 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 720 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: multiple
hairpins (ii) MOLECULE TYPE: RNA (A) DESCRIPTION: /desc =
''recombinant'' (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 2: AAUUUUUUUC UUCUAAAGUU
AUGCCAUUUU GUUUUCUUUC UCCUCAGCUC UAAAUACUCU GAAGUCCAAA GAAGUUGUAU
GUUGGGUGGG CUCCCUUCAA CUUUAACAUG GAAGUGCUUU CUGUGACUUU AAAAGUAAGU
GCUUCCAUGU UUUAGUAGGA GUGAAUCCAA UUUACUUCUC CAAAAUAGAA CACGCUAACC
UCAUUUGAAG GGAUCCCCUU UGCUUUAACA UGGGGGUACC UGCUGUGUGA AACAAAAGUA
AGUGCUUCCA UGUUUCAGUG GAGGUGUCUC CAAGCCAGCA CACCUUUUGU UACAAAAUUU
UUUUGUUAUU GUGUUUUAAG GUUACUAAGC UUGUUACAGG UUAAAGGAUU CUAACUUUUU
CCAAGACUGG GCUCCCCACC ACUUAAACGU GGAUGUACUU GCUUUGAAAC UAAAGAAGUA
AGUGCUUCCA UGUUUUGGUG AUGGUAAGUC UUCUUUUUAC AUUUUUAUUA UUUUUUUAGA
AAAUAACUUU AUUGUAUUGA CCGCAGCUCA UAUAUUUAAG CUUUAUUUUG UAUUUUUACA
UCUGUUAAGG GGCCCCCUCU ACUUUAACAU GGAGGCACUU GCUGUGACAU GACAAAAAUA
AGUGCUUCCA UGUUUGAGUG UGGUGGUUCC UACCUAAUCA GCAAUUGAGU UAACGCCCAC
ACUGUGUGCA GUUCUUGGCU ACAGGCCAUU ACUGUUGCUA 720 (2) INFORMATION FOR
SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 69 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
hairpin (ii) MOLECULE TYPE: RNA (A) DESCRIPTION: /desc =
''recombinant'' (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCACCACUUA AACGUGGAUG
UACUUGCUUU GAAACUAAAG AAGUAAGUGC UUCCAUGUUU UGGUGAUGG 69 (2)
INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 73 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: hairpin (ii) MOLECULE TYPE: RNA (A)
DESCRIPTION: /desc = ''recombinant'' (iii) HYPOTHETICAL: YES (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCUCCCUUCA
ACUUUAACAU GGAAGUGCUU UCUGUGACUU UAAAAGUAAG UGCUUCCAUG UUUUAGUAGG
AGU 73 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 68 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: hairpin (ii) MOLECULE TYPE:
RNA (A) DESCRIPTION: /desc = ''recombinant'' (iii) HYPOTHETICAL:
YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CCUUUGCUUU AACAUGGGGG UACCUGCUGU GUGAAACAAA AGUAAGUGCU UCCAUGUUUC
AGUGGAGG 68 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 68 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: hairpin (ii) MOLECULE TYPE:
RNA (A) DESCRIPTION: /desc = ''recombinant'' (iii) HYPOTHETICAL:
YES (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CCUCUACUUU AACAUGGAGG CACUUGCUGU GACAUGACAA AAAUAAGUGC UUCCAUGUUU
GAGUGUGG 68
Sequence CWU 1
1
6117RNAArtificial Sequencechemical synthesis 1uaagugcuuc cauguuu
172720RNAArtificial Sequencechemical synthesis 2aauuuuuuuc
uucuaaaguu augccauuuu guuuucuuuc uccucagcuc uaaauacucu 60gaaguccaaa
gaaguuguau guuggguggg cucccuucaa cuuuaacaug gaagugcuuu
120cugugacuuu aaaaguaagu gcuuccaugu uuuaguagga gugaauccaa
uuuacuucuc 180caaaauagaa cacgcuaacc ucauuugaag ggauccccuu
ugcuuuaaca uggggguacc 240ugcuguguga aacaaaagua agugcuucca
uguuucagug gaggugucuc caagccagca 300caccuuuugu uacaaaauuu
uuuuguuauu guguuuuaag guuacuaagc uuguuacagg 360uuaaaggauu
cuaacuuuuu ccaagacugg gcuccccacc acuuaaacgu ggauguacuu
420gcuuugaaac uaaagaagua agugcuucca uguuuuggug augguaaguc
uucuuuuuac 480auuuuuauua uuuuuuuaga aaauaacuuu auuguauuga
ccgcagcuca uauauuuaag 540cuuuauuuug uauuuuuaca ucuguuaagg
ggcccccucu acuuuaacau ggaggcacuu 600gcugugacau gacaaaaaua
agugcuucca uguuugagug uggugguucc uaccuaauca 660gcaauugagu
uaacgcccac acugugugca guucuuggcu acaggccauu acuguugcua
720369RNAArtificial Sequencechemical synthesis 3ccaccacuua
aacguggaug uacuugcuuu gaaacuaaag aaguaagugc uuccauguuu 60uggugaugg
69473RNAArtificial Sequencechemical synthesis 4gcucccuuca
acuuuaacau ggaagugcuu ucugugacuu uaaaaguaag ugcuuccaug 60uuuuaguagg
agu 73568RNAArtificial Sequencechemical synthesis 5ccuuugcuuu
aacauggggg uaccugcugu gugaaacaaa aguaagugcu uccauguuuc 60aguggagg
68668RNAArtificial Sequencechemical synthesis 6ccucuacuuu
aacauggagg cacuugcugu gacaugacaa aaauaagugc uuccauguuu 60gagugugg
68
* * * * *
References