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Profiling microRNA expression in bovine articular cartilage and implications for mechanotransduction.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 8, August 2009, pp 2333–2339
DOI 10.1002/art.24678
© 2009, American College of Rheumatology
Profiling MicroRNA Expression in Bovine Articular Cartilage
and Implications for Mechanotransduction
Walter Dunn, Grayson DuRaine, and A. Hari Reddi
Objective. Articular cartilage is an avascular tissue with precise polarity and organization comprising 3
distinct functional zones: the surface, middle, and deep
zones. Each zone has a different gene expression pattern
that plays a specific role in articular cartilage development and maintenance. MicroRNA (miRNA) are small
noncoding gene products that play an important regulatory role in determining cell differentiation and function. The purpose of this study was to test our hypothesis that miRNA expression profiles in the different
articular cartilage zones as well as between regions
subjected to different levels of weight-bearing stresses
are unique.
Methods. Using an miRNA microarray approach
in conjunction with quantitative reverse transcription–
polymerase chain reaction, we identified miRNA in
bovine articular cartilage that were differentially expressed in the different functional zones and in the
anterior weight-bearing and posterior non–weightbearing regions of the medial femoral condyle (M1 and
M4, respectively).
Results. We identified miRNA-221 and miR-222
as part of a subset of differentially expressed miRNA
that were up-regulated in articular cartilage in the
anterior, M1, greater weight-bearing location. Additionally, miR-126, miR-145, and miR-335 were down-
regulated in monolayers of tissue-cultured chondrocytes
as compared with levels determined directly from intact
native cartilage.
Conclusion. In conclusion, miR-222 expression
patterns in articular cartilage are higher in the weightbearing anterior medial condyle as compared with the
posterior non–weight-bearing medial condyle. Thus,
miR-222 might be a potential regulator of an articular
cartilage mechanotransduction pathway. These data
implicate miRNA in the maintenance of articular cartilage homeostasis and are therefore targets for articular
cartilage tissue engineering and regenerative medicine.
MicroRNA (miRNA) are a class of ⬃22-nucleotide noncoding RNAs that play an important role in
modulating gene expression during embryonic development and cell differentiation (1). These RNAs are
encoded within the genome as part of a predicted RNA
hairpin precursor. Generation of miRNA begins with a
long RNA transcript, termed primary miRNA, which is
initially processed into an ⬃60-nucleotide hairpin RNA
precursor (pre-miRNA) by the nuclear ribonuclease
Drosha (1). The resultant pre-miRNA is then processed
by Dicer to generate a ⬃22-bp duplex. Subsequently,
one strand of the duplex is incorporated into the RNAinduced silencing complex (RISC) of the RNA interference (RNAi) pathway and guides the RISC to the target
transcript that is complementary to the miRNA sequence (1). RISC binding results in either cleavage or
translation inhibition of the messenger RNA, depending
on the degree of complementarity between the miRNA
and the target transcript (1).
Although the function of most miRNA have yet
to be determined, the importance of miRNA-mediated
gene regulation in humans is underscored by the fact
that miRNA have been found in every human tissue type
examined thus far. Interestingly, subsets of these
miRNA are expressed in a tissue-specific manner and
Supported in part by NIH grant 1R01-AR-47345 and by the
Lawrence J. Ellison Endowed Chair in Musculoskeletal Molecular
Biology held by Dr. Reddi. Dr. Dunn’s work was supported by a
Medical Student Research Fellowship from the University of California, Davis School of Medicine.
Walter Dunn, PhD, Grayson DuRaine, BS, A. Hari Reddi,
PhD: University of California, Davis at Sacramento.
Address correspondence and reprint requests to A. Hari
Reddi, PhD, Lawrence Ellison Center for Tissue Regeneration and
Repair, Department of Orthopaedic Surgery, University of California,
Davis, 4635 Second Avenue, Research Building I, Room 2000, Sacramento, CA 95817. E-mail: ahreddi@ucdavis.edu.
Submitted for publication December 4, 2008; accepted in
revised form April 15, 2009.
2333
2334
sometimes only during certain developmental stages
(1–3). The large number of miRNA genes found in the
human and animal genomes, their unique distribution
and expression patterns, along with the findings of
functional studies in Caenorhabditis elegans and Drosophila melanogaster, suggest that miRNA represent a
general mechanism for the regulation of gene expression
in specific cell types and tissues (4).
An intriguing observation regarding miRNA expression and function is that their function depends on
the particular tissue type in which they are found and the
cellular environment in which they are expressed. For
example, although a specific miRNA species may be
expressed similarly in 2 differing tissue types, their
downstream function may be radically different. In one
tissue type, the miRNA may target a cellular replication
inhibitor, and thus, the expression of the miRNA may
lead to up-regulation of cellular replication. In another
tissue type, an miRNA may target an inducer of cell
proliferation, and thus, the expression of the miRNA
may suppress that inducer, resulting in inhibition of cell
proliferation. Thus, the role of miRNA is not only a
function of their own expression patterns, but also those
of their potential targets and other associated factors
(1,4).
The profiling of miRNA expression patterns continues to be an active area of research because there are
a multitude of tissue types and developmental stages yet
to be examined. Additionally, as more novel miRNA
sequences are identified, previously screened samples
need to be reexamined for the presence or absence of
the new miRNA. The investigation of miRNA expression in articular cartilage may provide opportunities for
the identification of targets in development and in
diseases such as osteoarthritis.
Articular cartilage in diarthrodial joints provides
a low-friction interface for skeletal movement during
locomotion. This unique tissue, although avascular and
recalcitrant to regenerative capacity, has the unique
capacity to resist significant mechanical loading stresses
during the lifetime of the organism. The surface, middle,
and deep zones within the articular cartilage are distinct
domains, and they exhibit differential gene expression
patterns and attendant functional roles. The tissue architecture and secretion of superficial zone protein is
unique to the surface of articular cartilage and functions
as a lubricant in friction-free movement (5). To date,
there have been no published reports on the miRNA
expression patterns in articular cartilage.
Using a bovine stifle joint articular cartilage
model (5), we examined the miRNA expression patterns
DUNN ET AL
across the regions of the femoral condyle in articular
cartilage with precise geometry and differing mechanical
loading conditions. Our results demonstrated a differential pattern of miRNA expression, suggesting that some
miRNA may be up-regulated in response to mechanical
stress. Furthermore, we examined the miRNA expression profiles of the different zones of articular cartilage,
interrogating the microRNA expression patterns in the
superficial zone versus the middle zone of the articular
cartilage.
MATERIALS AND METHODS
Cartilage procurement and RNA extraction. Cartilage
procurement was performed as described previously (5).
Briefly, stifle joints from 2–3-month-old calves were harvested
within 18 hours of slaughter. A 5-mm coring reamer (Fisher
Scientific, Fair Lawn, NJ) was used to extract cores of articular
cartilage down to the depth of the subchondral bone. The
medial condyle was divided into 4 regions, designated M1
through M4, with M1 representing the most anterior (weightbearing) region and with M4 representing the most posterior
(non–weight-bearing) region. Plugs of articular cartilage were
taken from each region and further sectioned into superficial,
middle, and deep zones. Each plug was placed in a custom
cutting jig and sectioned into a superficial zone section measuring 0.5 mm in thickness and a middle zone measuring 1.25
mm in thickness, with the remainder representing the deep
zone. Cartilage sections were then frozen in liquid nitrogen
and homogenized with a custom tissue homogenizer.
A modified RNA extraction procedure was used to
purify total RNA from the cartilage sections, as previously
described (5). After resuspension and centrifugation of homogenized cartilage in TRIzol reagent (Invitrogen, Carlsbad,
CA), the supernatant was removed, and total RNA was
extracted using a mirVana miRNA isolation kit (Ambion,
Austin, TX) according to the manufacturer’s total RNA extraction protocol. RNA species ⬍200 bp were preserved with
this protocol. RNA samples from 7 different animals were
pooled, the purity and quantity of the isolated total RNAs
were assessed using a spectrophotometer measurement, and
then the isolated total RNAs were submitted to LC Sciences
(Houston, TX) for miRNA microarray hybridization and analysis.
MicroRNA microarray analysis. Each customized microarray chip contained a probe set based on the miRBase
version 10.1 release of human/mouse/rat and bovine sequences
(online at http://microrna.sanger.ac.uk). The probe sets contained 1,193 unique miRNA sequences that were deposited in
triplicate on the array. The array also included perfectly
matched and single-basepair–mismatched control probes for a
20-mer RNA oligo spiked into the RNA samples before
labeling. The microarray experiments analyzing the M1 superficial zone versus the M1 middle zone and the M1 superficial
zone versus the M4 superficial zone consisted of 2-color
competition hybridization with a pair of samples labeled with
either Cy3 or Cy5. Subtraction of background values and
normalization were then performed. For each dual-sample
MicroRNA EXPRESSION PROFILES IN BOVINE ARTICULAR CARTILAGE
2335
Figure 1. Regions and zones of articular cartilage from the medial femoral condyle. A, Locations on the
articular surface of the bovine femoral condyle where the plugs of articular cartilage were extracted. The
medial femoral condyle was divided into 4 regions, with M1 representing the most anterior (weightbearing) region, which experiences the highest contact stresses across all the regions of the joint surface
(5), and M4 representing the most posterior (non–weight-bearing) region, which experiences significantly
less contact stress than the M1 site (5). B, Sectioning of the articular cartilage plugs into superficial, middle,
and deep zones.
experiment, a P value calculation was performed, and a list of
miRNA candidates was generated based on their differential
expression between the 2 samples.
Quantitative reverse transcription–polymerase chain
reaction (RT-PCR). Total RNA was isolated as described for
the microarray hybridization. RNA used for the RT-PCR
samples was pooled from 8 different animals collected on 2
separate harvest dates and did not include any RNA used for
the microarray hybridization. MicroRNA levels were measured in the M1 superficial, the M1 middle, and the M4
superficial cartilage zones and in chondrocytes from tissue
culture monolayers. Real-time quantitative PCR was performed on selected miRNA using TaqMan MicroRNA Assays
(Applied Biosystems, Foster City, CA) according to the manufacturer’s protocol and as previously described (6). TaqMan
miRNA primer sets designed for human miRNA were used to
quantify bovine miRNA after verifying that the human and
bovine miRNA sequences were identical. Each sample was run
in triplicate. GAPDH transcript levels were determined using
SYBR Green, and miRNA expression was normalized based
on GAPDH expression.
Tissue culture. Juvenile bovine femoral condyles were
obtained within 6 hours after slaughter, and the superficial
zone cartilage was manually sectioned off with a surgical
dermatome. Cartilage across the entire medial and lateral face
of the femoral condyle was removed in this manner. The
superficial zone cartilage was then placed in a collagenase
solution in a rotating incubator at 37°C for 5 hours. Chondrocytes from the surface zone were isolated and plated as a
monolayer in Dulbecco’s modified Eagle’s medium/F-12 medium (Invitrogen, Carlsbad, CA) containing 1% antibiotic
solution, 1% bovine serum albumin, and 50 ␮g/ml of ascorbic
acid–phosphate for 2 days before harvesting total RNA using a
mirVana miRNA isolation kit.
RESULTS
Findings of miRNA microarray studies. MicroRNA for the microarray studies were generated by
directly extracting total RNA from specific regions of
articular cartilage from freshly harvested bovine stifle
joint femoral condyles obtained from several animals.
The regions of interest were obtained by taking cores of
articular cartilage from the anterior and posterior regions of the medial femoral condyle and further sectioning them into superficial, middle, and deep zone slices
(Figure 1B). Previous studies have shown that cartilage
from the anterior regions of the femoral condyle exhibit
higher contact pressures than does cartilage from the
posterior regions (5). Thus, the medial condyle was
divided into 4 regions, designated M1–M4, with the M1
cartilage plug taken from the most anterior region and
the M4 plug taken from the most posterior region (5)
(Figure 1A).
Two-color competitive microarray experiments
were then performed to determine the differential expression pattern of known miRNA based on locations
within and across the articular cartilage obtained from
the femoral condyles. The first array experiment examined miRNA expression in cartilage from the anterior
weight-bearing M1 superficial zone, where contact pressures are maximal, compared with cartilage from the
posterior non–weight bearing M4 superficial zone,
2336
DUNN ET AL
Table 1. Hybridization values and ratios identified by microarray analyses of bovine articular cartilage*
Normalized hybridization value
miRNA analysis
M1 superficial zone vs. M1 middle zone
Cartilage zone analyzed
Probe ID
bta-miR-221
bta-miR-222
hsa-miR-34c-3p
hsa-miR-574-5p
hsa-miR-374b
hsa-miR-140-5p
hsa-miR-411
hsa-miR-130a
hsa-miR-155
hsa-miR-23a
M1 superficial zone vs. M4 superficial zone
Cartilage zone analyzed
Probe ID
bta-miR-221
bta-miR-222
hsa-miR-126
hsa-miR-143
hsa-miR-145
hsa-miR-34c-3p
hsa-miR-155
hsa-miR-495
hsa-miR-376a
hsa-miR-181a
Cartilage sample A
Cartilage sample B
Log2 ratio
M1 superficial
M1 middle
Superficial/middle
3,229.53
2,524.47
2,099.08
1,732.88
2,393.41
1,553.91
1,739.44
1,136.82
2,677.12
51,060.78
206.72
350.17
362.29
6,140.02
851.05
4,032.21
4,090.37
2,766.62
975.6
2,3748.42
4.04
2.85
2.55
⫺1.77
1.49
⫺1.37
⫺1.31
⫺1.28
1.24
1.23
M1 superficial
M4 superficial
M1/M4 superficial
5,097.17
4,256.4
15,539.59
4,090.81
10,018.33
1,173.87
4,381.34
984.6
1,250.4
3,229.99
452.1
494.34
2,739.21
803.25
1,972.54
299.99
1,132.04
3,550.88
4,065.49
1,164.43
3.42
3.08
2.60
2.35
2.34
2.07
1.95
⫺1.80
⫺1.70
1.54
* Values are from microarray experiments examining microRNA (miRNA) levels in the anterior medial femoral condyle (M1) superficial zone
versus the M1 middle zone, as well as levels in the M1 superficial zone versus the posterior medial femoral condyle (M4) superficial zone. The
normalized microarray hybridization values are given for each 2-color competition experiment (P ⬍ 0.01). For each dual-sample experiment, a P
value calculation was performed, and a list of miRNA candidates was generated based on their differential expression between the 2 samples. The
log2 ratios are based on the hybridization values for each condition. The top 10 miRNA with the greatest absolute log2 ratios, indicating the greatest
differential expression between the test conditions, are listed for each comparison. The miRNA candidates were also screened to include only those
for which the greater of the 2 compared hybridization values was at least ⬎1,000 (cutoff value).
where contact pressures are minimal (5). The second
experiment investigated miRNA expression in cartilage
from the M1 superficial zone compared with cartilage
from the M1 middle zone.
The LC Sciences miRNA microarray platform
was synthesized with all known human/mouse/rat and
bovine miRNA in version 10.1 of the miRBase database.
Non-bovine probes were included on the array, since the
database contained only a limited number of verified
bovine miRNA sequences. Based on our preliminary
bioinformatics analysis (Dunn W, et al: unpublished
observations) a number of verified human/mouse/rat
miRNA sequences were found in the bovine genome
sequence with perfect or significant sequence identity,
but which were not in the miRBase bovine miRNA
database. LC Sciences performed the labeling of the
miRNA, hybridization onto the array, and subsequent
analysis of the data, generating a list of differentially
expressed miRNA based on the log2 ratios between the
competing conditions. Table 1 shows a modified list of
those differentially expressed miRNA. The top 10 candidates with the greatest differential expression and for
which the hybridization value was ⬎1,000 (cutoff value)
for at least 1 of the 2 compared samples are shown. A
signal value of 1,000 on the array is the approximate
threshold for which miRNA can be detected by quantitative RT-PCR. The top 2 differentially expressed
miRNA on both arrays were miR-221 and miR-222.
Findings of quantitative RT-PCR analysis of
miRNA expression. We then selected miRNA-221 and
miR-222, along with several other miRNA, for further
quantification using quantitative RT-PCR techniques.
The same comparison conditions were used for the
quantitative RT-PCR analyses as for the microarray
experiments. We compared miRNA expression in cartilage from the M1 superficial zone versus the M4 superficial zone and from the M1 superficial zone versus the
M1 middle zone. The cartilage samples used for the
quantitative RT-PCR were taken from a separate harvest than those used for the array experiments, and total
MicroRNA EXPRESSION PROFILES IN BOVINE ARTICULAR CARTILAGE
2337
Figure 2. Quantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis of microRNA
(miRNA) expression and the average fold change. Quantitative RT-PCR was used to quantify selected
miRNA expressed in bovine articular cartilage chondrocytes. The anterior medial femoral condyle (M1)
and posterior medial femoral condyle (M4) values are from RNA that was extracted directly from sections
of the articular cartilage plugs. Tissue culture monolayer samples were extracted from superficial layer
chondrocytes that were plated as a monolayer in cell culture. The ‚Ct values are the threshold cycle values
normalized against GAPDH; ‚‚Ct values can be calculated from the ‚Ct values to obtain the fold
difference in miRNA expression under 2 different conditions. For example, the ‚Ct value for miR-221 in
the M1 superficial zone is 11.2, and the ‚Ct value for miR-221 in the M4 superficial zone is 9.0. The ‚‚Ct
value is calculated as 11.2 – 9.0 ⫽ 2.2. Therefore, there is a 22.2 ⫽ 4.59-fold greater amount of miR-221
in cartilage from the M1 superficial zone than in cartilage from the M4 superficial zone.
RNA from several knees were pooled for these analyses.
The quantitative RT-PCR results correlated well with
the array data, with miR-221 and miR-222 being the top
2 differentially expressed miRNA of the miRNA that
were analyzed. There was a 27-fold greater amount of
miR-221 and miR-222 in the M1 superficial zone cartilage as compared with the M1 middle zone cartilage.
The quantitative RT-PCR data also demonstrated that
there was a 4-fold greater amount of miR-221 and
miR-222 in the M1 superficial zone cartilage than in the
M4 superficial zone cartilage (Figure 2).
In addition to the miRNA that were up-regulated
in the anterior weight-bearing M1 region of cartilage,
quantitative RT-PCR identified miRNA that were
down-regulated. In particular, miR-148a was downregulated ⬃5-fold in cartilage from the M1 superficial
zone as compared with that from the M4 superficial
zone (Figure 2). The quantitative RT-PCR data for
miRNA levels in the superficial zone cartilage sections
were compared with data from superficial zone chondrocytes that had been extracted from their cartilage
matrix and plated as a monolayer in cell culture medium
for 2 days. Loss of the extracellular matrix environment
and growth in cell culture conditions significantly altered
the expression of several miRNA species. Levels of
miRNA-126 were 274-fold lower in the monolayer chon-
drocytes than in the M1 superficial zone intact cartilage
plug. It is noteworthy that miR-145 and miR-335 were
present at 111-fold and 55-fold lower levels in the
monolayer chondrocytes, respectively. Interestingly,
miR-221 and miR-222 levels were also decreased in the
monolayer chondrocytes, but not as dramatically as the
decreases observed for miR-126, miR-145, or miR-335.
Levels of miRNA-221 and miR-222 in the monolayer
chondrocytes were similar to those found in the M1
middle zone, which represented an ⬃27-fold decrease
from levels in the M1 superficial zone (Figure 2).
The mean fold difference in miRNA expression
levels between the M1 superficial zone of bovine articular cartilage and other zones or regions of articular
cartilage were calculated from the average ‚Ct values
shown in Figure 2. These additional data are available
upon request from the author.
DISCUSSION
Although the function of many miRNA have yet
to be elucidated, several identified in this study as being
differentially expressed in bovine articular cartilage have
previously been reported in other biologic contexts
(7–9). Both miRNA-221 and miR-222 have previously
been identified as playing a role in cancer cell prolifer-
2338
ation through their targeting of p27, a known cell cycle
inhibitor (8–10). Thus, these miRNA may play a role in
chondrocyte proliferation by possibly inhibiting p27 in
the chondrocytes found in the anterior higher weightbearing regions of articular cartilage. There is evidence
of increased cell division activity in the superficial zone
of cartilage as compared with the middle and deep
zones, as well as evidence of a stem cell progenitor
population in the superficial zone (11,12). The upregulation of miR-221 and miR-222 observed in this
study may be part of the stem cell population signature.
In addition, the findings in the anterior weightbearing area with maximum contact pressure (M1 superficial zone) as compared with the posterior non–weightbearing area with minimum contact pressure (M4
superficial zone) suggest that greater mechanical stress
may lead to greater chondrocyte proliferation, possibly
through a mechanism of suppressing inhibitors of proliferation via miRNA. This implies that these miRNA
may be examples of mechanical stress–induced miRNA
that are up-regulated in response to greater weight
bearing and maximum contact pressures. Previous studies in plants, such as the California poplar tree, have
identified stress-induced plant miRNA, but this would
be the first example of miRNA regulated in response to
mechanical stress in a mammalian system (13).
MicroRNA-148a, which was down-regulated in
the M1 superficial zone cartilage as compared with the
M4 superficial zone, has also been implicated in acute
myeloid leukemia. Its expression was found to be inversely correlated with the expression of the BAALC
gene, whose up-regulated expression is thought to be
associated with early markers of progenitor cell phenotypes (14). Thus, lower miR-148a expression may correlate with higher levels of early progenitor cell markers
that indicate a phenotype that favors cell proliferation
over differentiation. Again, this is consistent with our
observations, since lower miR-148a levels were found in
the M1 region as compared with the M4 region and since
the M1 cartilage bears comparatively more weight and,
thus, more cell proliferation is stimulated.
The present investigation on the differential expression of miRNA in articular cartilage yielded novel
findings of the differential expression of miRNA between the different zones and in the anterior weightbearing regions of articular cartilage. The juvenile calf
cartilage used in the current study is immature cartilage
and may not be directly comparable to adult human
articular cartilage. This investigation identified several
miRNA of interest that were differentially expressed
and perhaps related to regulation of chondrocyte prolif-
DUNN ET AL
eration. Furthermore, miR-221 and miR-222 were identified as mechanosensitive stress–induced miRNA, and
represent a novel discovery in a mammalian articular
cartilage. This insight into the role of miRNA in the
biology of articular cartilage will provide a basis for
further systematic investigation of articular cartilage in
health as well as in disease, such as in the osteoarthritis,
a degenerative disease associated with aging.
Since the completion of our analyses, Iliopoulous
et al (15) reported their finding of a 16-miRNA osteoarthritis gene signature from their studies comparing
osteoarthritic and nondiseased human cartilage. It is
noteworthy that the miRNA identified in bovine cartilage in our investigation are novel as compared with
those identified in human osteoarthritic cartilage. In
conclusion, miRNA-221 and miRNA-222 appear to be
expressed in cartilage of the anterior weight-bearing
superficial zone and may therefore represent targets of
mechanotransduction in articular cartilage.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Reddi had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Dunn, DuRaine, Reddi.
Acquisition of data. Dunn, DuRaine.
Analysis and interpretation of data. Dunn, Reddi.
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