OPEN
Citation: Cell Death and Disease (2014) 5, e1499; doi:10.1038/cddis.2014.462
& 2014 Macmillan Publishers Limited All rights reserved 2041-4889/14
www.nature.com/cddis
microRNA-320/RUNX2 axis regulates adipocytic
differentiation of human mesenchymal (skeletal)
stem cells
D Hamam1,3, D Ali1,3, R Vishnubalaji1, R Hamam1, M Al-Nbaheen1, L Chen2, M Kassem1,2, A Aldahmash1,2 and NM Alajez*,1
The molecular mechanisms promoting lineage-specific commitment of human mesenchymal (skeletal or stromal) stem cells
(hMSCs) into adipocytes (ADs) are not fully understood. Thus, we performed global microRNA (miRNA) and gene expression
profiling during adipocytic differentiation of hMSC, and utilized bioinformatics as well as functional and biochemical assays, and
identified several novel miRNAs differentially expressed during adipogenesis. Among these, miR-320 family (miR-320a, 320b, 320c,
320d and 320e) were ~ 2.2–3.0-fold upregulated. Overexpression of miR-320c in hMSC enhanced adipocytic differentiation and
accelerated formation of mature ADs in ex vivo cultures. Integrated analysis of bioinformatics and global gene expression profiling
in miR-320c overexpressing cells and during adipocytic differentiation of hMSC identified several biologically relevant gene targets
for miR-320c including RUNX2, MIB1 (mindbomb E3 ubiquitin protein ligase 1), PAX6 (paired box 6), YWHAH and ZWILCH.
siRNA-mediated silencing of those genes enhanced adipocytic differentiation of hMSC, thus corroborating an important role for
those genes in miR-320c-mediated adipogenesis. Concordant with that, lentiviral-mediated stable expression of miR-320c at
physiological levels (~1.5-fold) promoted adipocytic and suppressed osteogenic differentiation of hMSC. Luciferase assay
validated RUNX2 (Runt-related transcription factor 2) as a bona fide target for miR-320 family. Therefore, our data suggest miR-320
family as possible molecular switch promoting adipocytic differentiation of hMSC. Targeting miR-320 may have therapeutic
potential in vivo through regulation of bone marrow adipogenesis.
Cell Death and Disease (2014) 5, e1499; doi:10.1038/cddis.2014.462; published online 30 October 2014
Bone marrow fat is increasingly recognized as an important
component of the bone marrow microenvironment with
potential role in regulating bone formation, hematopoiesis
and the whole body’s energy metabolism.1,2 During aging and
in a number of skeletal diseases, an inverse relationship
between bone marrow trabecular bone mass and fat mass has
been reported, suggesting a common regulatory genetic
program.3–6 Based on a large number of in vitro studies, bone
marrow adipocytes (ADs) and osteoblasts originate from a
common progenitor cells within the bone marrow stroma
known as mesenchymal (skeletal or stromal) stem cells
(MSCs).7 It is thus envisaged that controlling MSC fate into
osteoblasts or AD can be a target for intervention with the aim
of enhancing bone formation in bone loss disorders.8 To
achieve this goal, molecular mechanisms controlling MSC
commitment to ADs versus osteoblasts need to be identified.
MicroRNAs (miRNAs) are double-stranded noncoding RNA
molecules of ~ 22 nucleotides that function as posttranscriptional regulators of gene expression and are found
in a wide variety of organisms, from plants, insects to
humans.9,10 miRNAs have been identified to affect multiple
biological functions including stem cell differentiation, neurogenesis, hematopoiesis, immune response, skeletal and
cardiac muscle development.11–17 Several previous studies
have identified a number of miRNAs as important regulators of
MSC differentiation into osteoblasts (for review, see Taipaleenmaki et al.,18 Eskildsen et al.19 and Zeng et al.20) and
chondrocytes.21 On the other hand, few studies have
examined miRNA regulation of MSC differentiation into
ADs.22,23 In the present study, we carried out a comprehensive
analysis of miRNA expression profiling and bioinformatics
analyses of human MSC (hMSC) during in vitro AD
differentiation. We identified several novel pro-adipogenic
miRNAs, and found that miR-320 to be an important regulator
of adipocytic differentiation of hMSC.
Results
Identification of differentially expressed miRNAs during
adipocytic differentiation of hMSCs. Using standard ADinduction medium (AIM), hMSC differentiated readily into
1
Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia and 2KMEB, Department of Endocrinology, University of Southern
Denmark, Odense, Denmark
*Corresponding author: NM Alajez, Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia. Tel: +966 1 4679421;
Fax: +966 1 4671498; E-mail: nalajez@ksu.edu.sa
3
These authors contributed equally to this work.
Abbreviations: 3′UTR, 3′-Untranslated region; C/EBP, CCAAT-enhancer-binding protein; DMEM, Dulbecco’s modified Eagle’s medium; FACS, Fluorescence-activated
cell scan; FBS, Fetal bovine serum; HEK-293, Human embryonic kidney 293 cells; hMSCs, Human mesenchymal stem cells; hTERT, Human telomerase reverse
transcriptase; miRNAs, MicroRNAs; MSCs, Mesenchymal stem cells; PBS, Phosphate-buffered saline; PPARγ, Peroxisome proliferator-activated receptor-g; qRT PCR,
Quantitative real-time reverse transcription PCR; RUNX2, Runt-related transcription factor 2; MIB1, Mindbomb E3 ubiquitin protein ligase 1; PAX6, Paired box 6
Received 28.6.14; revised 03.9.14; accepted 17.9.14; Edited by G Raschella'
miRNA-320/RUNX2 axis regulates adipocytic differentiation
D Hamam et al
2
mature lipid-filled ADs as demonstrated by positive staining
for Oil Red O (Figure 1a) and increased expression of several
AD-specific genes (Figure 1b). Global miRNA expression
profiling carried out on AD-differentiated hMSC revealed 38
miRNAs to be differentially expressed on day 13 compared
with day 0 (Po0.05; Figure 1c and Table 1). Eleven miRNAs
were found to be differentially expressed on day 7 compared
with day 0 (Po0.05; Figure 1d and Supplementary Table 1).
The expression levels of selected group of miRNAs identified
in the microarray experiment, miR − 374 − 5p, − 30b, − 222,
− 320c, − 186, − 320a, − 320e and − 29c, were validated
using quantitative real-time reverse transcription PCR (qRTPCR) that confirmed the microarray results and showed
upregulation of miR-30b, miR-320 family (320a/320c/ 320e)
on day 7 post-AD differentiation induction and further
increase in expression levels of the same miRNAs in addition
to miR-186 on day 13 (Figure 1e). miR-222 was found to be
downregulated on day 13 (Figure 1e). Among the identified
miRNAs, several members of the miR-320 (miR-320a, 320b,
320c, 320d and 320e) family were differentially expressed
and were chosen for further investigation, as they have not
previously been implicated in regulating the adipocytic
differentiation of MSCs.
Overexpression of miR-320c and miR-30b promote
adipocytic differentiation of hMSCs. To examine for the
potential role of selected miRNAs, miR-320c and -30b in
regulating the adipocytic differentiation of hMSC, cells were
transfected with pre-miR-320c, pre-miR-30b or pre-miRnegative control and subsequently were exposed to AIM.
qRT-PCR revealed significant increase in miRNA expression
in transfected cells (data nor shown). As shown in Figure 2a,
cell transfected with pre-miR-320c and -30b exhibited
enhanced formation of lipid-filled mature ADs. Concordant
with those data, Nile red staining and fluorescence-activated
cell scan (FACS) analysis revealed increased number of Nile
Red High population in hMSC cultures transfected with premiR-320c and -30b compared with the controls (Figures 2b
and c). As miR-320 family was the most novel family of
miRNAs identified in current study as a possible regulator of
adipocytic differentiation of hMSCs, all subsequent experiments focused on miR-320c member. In order to confirm that
the enhanced adipocytic differentiation mediated via
miR-320c was specific and not because of nonspecific effect
as a result of transfection, we generated hMSCs stably
expressing miR-320c using lentiviral-mediated transduction.
As shown in Figures 2d and e, stable expression of miR-320c
indeed led to enhanced adipocytic differentiation of hMSCs
compared with cells transduced with control lentivirus.
Representative images of Oil Red O staining are shown in
Figure 2e, while quantification of Oil Red O staining
demonstrated enhanced adipogenesis in LV miR-320c cells
compared with control cells (Figure 2f). Similarly Nile red
staining and quantification also demonstrated enhanced lipid
droplet accumulation in miR-320c compared with control cells
(Figures 2g and h). We observed no significant difference in
cell viability on day 7 post-AD differentiation induction
between LV miR-320c and LV control cells (Figure 2i),
therefore the difference in Nile red staining is not due to
difference in cells numbers. Concordant with that, the
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expression of AD-specific genes was higher in LV miR-320c
cells compared with control cells (Figure 2j). Taken together,
our data indicated enhanced adipocytic differentiation of
hMSCs overexpressing miR-320c.
Identification of bona fide mRNA targets for miR-320c. In
order to identify possible gene targets of miR-320c that
regulate MSC differentiation into ADs, we used the following
approach. hMSC transfected with miR-320c or control miRNA
were cultured in presence or absence of AIM, total RNA was
extracted from baseline and following 7 days of AD induction
and subjected to microarray analysis. As shown in Figures 3a
and b, hMSC overexpressing miR-320c did cluster together
compared with cells transfected with miRNA control.
Supplementary Tables 2 and 3 contain a list of genes that
were downregulated (−1.3-fold, Po0.05) in hMSCs transfected with miR-320c compared with control cells at 72 h post
transfection and in day 7 adipocytic induced hMSC,
respectively. In addition to these genes that were experimentally determined, a third list of predicted miR-320c gene
targets were curated from TargetScan database. The intersection in Venn diagram between three lists, identified 210
common genes, that is, genes that were downregulated upon
miR-320c overexpression, downregulated during adipocytic
differentiation of hMSCs and that were predicted to be
targeted by miR-320c in silico. Gene ontology and pathway
analysis of the identified 210 common genes showed strong
enrichment for genes involved in regulating the cell cycle and
cell differentiation (Figure 3d and Supplementary Table 4).
Concordant with those data, we observed that un-induced
hMSC stably expressing miR-320c had slower proliferation
rate compared with control cells (Figure 3e). Several genes
involved in cell cycle regulation and cell differentiation that
were common to the three lists (MIB1, PAX6, ZWILCH,
YWHAH and SEMA5A) or genes that were common to Exp.
determined and in silico (TGFBR1, TGFBR2, NRP1, RASA1,
ULK1, RUNX2 (Runt-related transcription factor 2), BMPR1A
and KITLG) were chosen for further investigation. As
confirmation of the microarray data, qRT-PCR showed good
concordance between microarray and qRT-PCR except for
one gene KITLG (Figure 3f).
Validating the role of selected miR-320c targets in
regulating adipocytic differentiation of hMSC. To confirm
whether the identified miR-320c targets are indeed involved
in regulating adipocytic differentiation of hMSCs, we used
siRNA approach to knock down the expression of selected
genes. As shown in Figure 4a, siRNA-mediated silencing of
MIB1, PAX6, RUNX2, YWHAH and ZWILCH led to increased
number of adipocytic cells differentiated from hMSC. Concordant with that, qRT-PCR indicated upregulation of several
adipocytic markers, AdipoQ, PPARγ (peroxisome proliferatoractivated receptor-λ) and FABP4, in cells transfected with
YWHAH, MIB1, RUNX2 and ZWILCH siRNAs, suggesting a
plausible role for these genes in miR-320-mediated effects on
adipocytic differentiation of hMSC.
miR-320c suppresses osteogenic differentiation of
hMSCs. Interestingly, RUNX2, which is a key transcription
factor (TF) involved in osteogenesis, was among the novel
miRNA-320/RUNX2 axis regulates adipocytic differentiation
D Hamam et al
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Figure 1 miRNA expression profiling during adipocyte (AD) differentiation of hMSC. hMSC-TERT cells were induced to AD differentiation. (a) Oil Red O staining of lipid-filled
mature ADs on days 7 and 13. (b) qRT-PCR analysis of AD marker genes (peroxisome proliferator-activated receptor-γ, PPARγ; AD protein 2, AP2; leptin, LEP; adiponectin,
AdipoQ). Gene expression was normalized to GAPDH and β-actin. Data are presented as mean ± S.E. of fold changes compared with non-induced controls, n = 6 from two
independent experiments. ***Po0.0005 between non-induced and induced samples. hMSC were induced to AD differentiation and on days 0, 7 and 13. miRNA expression
profiling was done using the miRCURY LNA miRNA Array (6th GEN). (c) Heat map and unsupervised hierarchical clustering were performed on the top 30 miRNAs differentially
expressed on AD day 13 (D13) versus AD D0; the color scale illustrates the relative expression level of miRNAs (log2). Red color represents an expression level below the
reference channel, and green color represents expression higher than the reference. (d) Venn diagram depicting the overlap in miRNAs that were differentially expressed on AD
D13 and AD D7. (e) Validation of selected miRNAs identified in c using Taqman miRNA qRT-PCR. Data are presented as mean ± S.E., n = 6. *Po0.05, ***Po0.0005
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miRNA-320/RUNX2 axis regulates adipocytic differentiation
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Table 1 Significantly differentially expressed miRNAs on AD day 13 versus AD day 0
No.
Annotation
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
hsa-miR-320c
hsa-miR-30b
hsa-miR-320e
hsa-miR-320a
hsa-miR-320b
hsa-miR-29c
hsa-miR-10b
hsa-miR-19a
hsa-miR-101
hsa-miR-320d
hsa-miR-148b
hsa-miR-29a
hsa-miR-193b
hsa-miR-151-5p
hsa-miR-129*
hsa-miR-193b*
hsa-miR-30c
hsa-miR-374b
hsa-miR-365
hsa-miR-103a
hsa-miR-3647-5p
hsa-miR-30e
hsa-miR-3647-3p
hsa-miR-190
hsa-miR-222
hsa-miR-152
hsa-miR-374a
hsa-miR-26b
hsa-miR-28-5p
hsa-miR-186
hsa-miR-191
hsa-miR-26a
hsa-miR-30a
hsa-miR-16
hsa-miR-15a
hsa-miR-1285
hsa-miR-151-3p
hsa-miR-30d
logFC
AvgExpr
AvgHy3
P-value
Adj. P-value
1.200823
1.615031
1.206532
1.182796
1.248647
2.620266
1.383596
0.646275
2.644133
1.178505
0.803186
1.228473
1.192514
0.701698
1.434831
0.392681
1.44055
1.202467
1.24017
0.989911
0.715019
1.047744
0.872259
1.532023
− 1.15414
0.717162
1.285674
1.519303
0.686888
1.166416
1.222257
1.611705
1.400624
0.831693
0.969761
− 1.24066
0.491623
1.343111
0.234397
0.310867
0.222737
0.204731
0.139694
0.310883
0.156777
0.285073
-0.05242
0.28873
0.294123
0.347
0.18976
0.079185
0.064513
0.04686
0.202695
0.155924
0.331153
0.22999
− 0.17482
0.480218
0.087001
0.435949
-0.14702
0.380272
0.256238
0.156895
0.012992
0.40042
0.357161
0.16245
0.282938
0.543501
0.440476
− 0.31694
0.195363
0.511484
8.424051
8.365077
8.432005
8.699699
8.734353
8.044084
8.122331
8.533174
8.501706
8.23224
6.764786
12.13131
8.324068
7.519235
6.293654
5.884442
8.307646
7.191527
8.881532
8.47661
6.152502
7.490472
8.168902
6.418949
10.71594
7.531555
7.48288
8.870564
5.912515
6.430794
7.655313
7.403967
7.650121
10.61485
9.501428
7.180472
6.432898
7.202426
6.39E − 07
3.12E − 06
5.03E − 06
5.64E − 06
8.83E − 06
1.32E − 05
1.37E − 05
1.47E − 05
1.63E − 05
1.71E − 05
2.19E − 05
2.29E − 05
2.44E − 05
2.51E − 05
3.11E − 05
3.59E − 05
3.74E − 05
3.76E − 05
4.11E − 05
4.64E − 05
4.66E − 05
5.47E − 05
5.85E − 05
5.96E − 05
6.03E − 05
6.32E − 05
6.46E − 05
6.49E − 05
6.82E − 05
7.04E − 05
7.42E − 05
7.54E − 05
8.02E − 05
8.99E − 05
9.15E − 05
9.24E − 05
9.56E −05
0.000103
0.00026
0.0013
0.0021
0.0023
0.0037
0.0055
0.0057
0.0061
0.0068
0.0072
0.0092
0.0096
0.010
0.010
0.013
0.015
0.015
0.015
0.017
0.019
0.019
0.023
0.024
0.025
0.025
0.026
0.027
0.027
0.028
0.029
0.031
0.031
0.033
0.037
0.038
0.038
0.040
0.043
gene targets identified for miR-320c family in current study.
Therefore, we sought to assess the effect of miR-320c
expression on osteogenic differentiation of hMSC. Data
presented in Figure 5a showed lower ALP staining in LV
miR-320c compared with LV control cell, as well as
decreased expression of osteoblast marker genes, whereas
the most reduction was seen for RUNX2 expression
(Figure 5b). Similarly, ALP quantification revealed lower
ALP activity on day 10 post-OB differentiation induction in
LV miR-320c cells compared LV controls (Figure 5c). There
was no difference in cell viability of OB-differentiated LV
miR-320c and LV control hMSC (Figure 5d).
Identification of RUNX2 as bone fide target for miR-320c
during adipogenesis. Among the miR-320c-identified gene
targets, RUNX2 was the most prominent. In addition to
TargetScan, another database (HOCTAR, http://hoctar.tigem.
it/), which examines for an inverse relationship between the
expression of miRNA host gene and a potential target, was
also used. We found that miR-320 family to be among the top
10% miRNAs predicted to regulate RUNX2, which further
supports a role for miR-320 family in regulating RUNX2
expression during AD differentiation (Supplementary Table
5). Interestingly, RUNX2 had four predicted miR-320 family
binding sites on it 3′-untranslated region (3′UTR) located
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between nucleotides 1175 and 3142 (Figure 6a). Overexpression of pre-miR-320c led to significant reduction in
RUNX2 expression in hMSC (Figure 6b). To confirm that
RUNX2 is indeed a direct target for miR-320 family, we
constructed reporter vector carrying the predicted binding site
(s) of RUNX2 downstream of a firefly luciferase gene in the
pMIR-REPORTER miRNA Expression Reporter vector
(Figure 6c).24 A mutant version of RUNX2 UTR reporter
vector with mutations in the predicted miR-320 seed region(s)
in the 3′UTRs was also generated using the primer
combination listed in Table 2. The pRL-SV40 (encoding for
renilla luciferase) was used for normalization. Co-transfection
experiments in HEK-293 (human embryonic kidney 293) cells
using two different RUNX2 UTR reporter clones demonstrated significant regulation of the RUNX2 reporter by
miR-320c miRNA (~50%; Figure 6d). The regulation of
RUNX2 UTR by miR-320c was specific, as mutating the
seed region completely abrogated this effect.
Discussion
In the present study, we have identified miR-320 family as
novel regulator of bone marrow-derived hMSC differentiation
into ADs. Our data corroborate an increasing number of
studies demonstrating the role of miRNAs in regulation of
miRNA-320/RUNX2 axis regulates adipocytic differentiation
D Hamam et al
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Figure 2 Forced expression of miR-320c- and miR-30b-enhanced AD differentiation of hMSCs. hMSCs were transfected with 30 nM of pre-miR-320c, pre-miR-30b and premiR-Neg, then were subjected to AD differentiation. (a) AD differentiation was assessed on day 7 using Oil Red O staining. (b) The percentage of Nile redhigh cells was
enumerated using flow cytometry. (c) Quantitative presentation of the data obtained in b. Data are presented as mean ± S.E, n = 3. (d) Stable expression of miR-320c in hMSC
cells using lentiviral expression vector led to significant increase in miR-320c expression. (e) Oil Red O quantification in LV miR-320c and LV control cells after 7 days of adipocytic
differentiation. (g) Nile red staining of LV miR-320c and LV control cells on day 7 adipocytic induction. The level of Nile red staining was quantified using molecular devices M5
microplate reader using fluorescence well-scan mode (h). Data are representative of three independent experiments, n = 36. Cell numbers was quantified using the alamarBlue
assay on LV miR-320c and LV control cells (i), n = 9. (j) Expression of adipo-specific markers in LV miR-320c or LV control cells after 7 days of adipocytic differentiation, n = 6,
*Po0.05, **Po0.005, ***Po0.0005
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miRNA-320/RUNX2 axis regulates adipocytic differentiation
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Figure 3 Identification of miR-320c bona fide gene targets during adipogenic differentiation of hMSCs. (a) Hierarchical clustering of hMSC transfected with miR-320c or
control miRNA 72 h post transfection based on mRNA expression levels. Each column represents one replica. Expression level of each gene in a single sample is depicted
according to the color scale. (b) Hierarchical clustering of control MSCs or MSCs differentiated into ADs (day 7) based on mRNA expression levels, where each column
represents one replica. Expression level of each gene in a single sample is depicted according to the color scale. (c) Venn diagram demonstrating the overlap between
experimentally determined miR-320c targets at baseline or following adipocytic differentiation of hMSC, and the in silico-predicted miR-320c targets based on TargetScan
database. (d) Pie chart illustrating the distribution of the top 20 GO categories for the 210 predicted miR-320c gene targets. The pie section size corresponds to fold enrichment.
(e) Quantification of cell viability of control hMSC or hMSC stably transduced with miR-320c LV on day 4 using alamarBlue assay. (f) qRT-PCR validation (blue) of selected
miR-320c gene targets identified from microarray (red) (c). Data are presented as mean ± S.E., n = 6 from two experiments, *Po0.05, ***Po0.0005
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miRNA-320/RUNX2 axis regulates adipocytic differentiation
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Figure 4 Functional validation of the identified miR-320c targets in regulating adipocytic differentiation of MSCs. hMSC were transfected with the indicated siRNA or control
siRNA and were subjected to adipocytic differentiation induction for 7 days. (a) Oil Red O staining of mature lipid-filled ADs on day 7. (b) qRT-PCR analysis of AD marker genes
(AdipoQ, PPARγ and FABP4). Gene expression was normalized to GAPDH and β-actin. Data are presented as mean fold change compared with cells transfected with control
siRNA ± S.E., n = 6 from two independent experiments. *Po0. 05, **Po0.005, ***Po0.0005
hMSC cell lineage fate as well as increasing number of other
types of stem cells.17,25 Although several miRNA candidates
that control osteoblastic differentiation of MSC have been
described, only few miRNA have been reported to regulate
their adipocytic differentiation. Among the reported miRNAs
are miR-143, miR-138 and miR-637 that were implicated in
regulating AD differentiation via modulation of ERK5, EID-1
and Osterix, respectively.26–28
In current study, we used an integrated analysis of miRNA
expression profiling combined with bioinformatics analyses.
Interestingly, several of the identified differentially regulated
miRNAs during AD differentiation of hMSC have previously
been reported to regulate hMSC differentiation (e.g., miR-222,
miR-138 and miR-30 family23,27,29,30), indicating the importance of the regulatory network controlled by miRNAs as they
are preserved across different cellular models of MSC.
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miRNA-320/RUNX2 axis regulates adipocytic differentiation
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Figure 5 Overexpression of miR-320c suppressed ALP activity in hMSC. (a) Representative hMSC were stably transduced with a lentivirus containing miR-320c or control
miRNA and were subjected to osteoblast differentiation induction for 10 days. ALP staining for control hMSC (LV control) or hMSC stably expressing miR-320c (LV miR-320c) is
shown. (b) qRT-PCR analysis of osteoblast gene markers (RUNX2 and osteonectin). (c) Quantification of ALP activity on cells from a. Data is presented as relative ALP activity
compared to cells transduced with LV control. Data are presented as mean ± S.E. from five independent experiments, n = 50 (d) Quantification of cell viability of LV control or LV
miR-320c cells on day 10 post-osteogenic induction showing no significant difference between the two groups, *Po0.05, ***Po0.0005
We identified miR-320 family as the most prominent novel
regulator of hMSC differentiation into ADs. Using Tri-Pronged
approach combined with functional and biochemical assays,
we identified several novel gene targets for miR-320 family
during adipocytic differentiation of hMSC. Among these MIB1,
PAX6, YWHAH, ZWILCH and RUNX2 were most relevant to
adipogenesis. Interestingly, several of the identified genes are
known to have a role in regulating cell proliferation and stem
cell differentiation. For example, MIB1 and PAX6 were
implicated in regulating neural stem cell differentiation.31,32
YWHAH has recently been implicated in regulating cell
division during meiosis,33 while ZWILCH has been shown to
be essential for kinetochore functions during cell division.34
However, our data revealed an additional role of these proteins
in bone marrow adipogenesis. RUNX2 was one target
identified in this study that is known for its being a master
TF for inducing osteoblast differentiation. Therefore, it is
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plausible that miR-320 family promote adipogenesis via
blocking other MSC differentiation pathways (i.e., osteoblast;
Figure 6d).
Bioinformatics analysis revealed that RUNX2 3′ UTR
harbors four potential binding sites for miR-320 family.
Regulation of RUNX2 expression by miR-320 was subsequently confirmed using qRT-PCR and luciferase assay. The
interaction between miR-320 and RUNX2 3′ UTR was found to
be specific, as mutating miR-320 seed region in the 3′UTR of
RUNX2 completely abrogated its regulatory effects. RUNX2 is
osteoblast-specific TF that has an important role in MSC
differentiation to osteoblasts.35–37 Previous studies have
demonstrated that adipocytic differentiation of MSC is
suppressed by RUNX2 and RUNX2− / − calvarial cells exhibited an enhanced AD differentiation.38 Our data corroborate
that osteoblast differentiation is the primary default differentiation pathway for bone marrow-derived hMSC and thus
miRNA-320/RUNX2 axis regulates adipocytic differentiation
D Hamam et al
9
Figure 6 Direct regulation of RUNX2 by miR-320c. (a) Schematic presentation showing the alignment of miR-320c mature sequence and the putative binding sites within the
3′UTR region of the RUNX2 mRNA using TargetScan database. The exact positions of the interaction between RUNX2 3′UTR and miR-320c seed region are indicated.
(b) Overexpression of miR-320c was associated with significant decrease in RUNX2 mRNA and protein. *Po0.05. (c) An illustration of the construction of luciferase reporter
vector carrying the predicted RUNX2-miR-320c binding sites downstream of the firefly luciferase gene in the pMIR-REPORT vector. The number of predicted miR-320c binding
sites within the 3′UTR region of RUNX2 is shown as black bars. (d) The indicated wild-type or mutant reporter vector was co-transfected with a pre-miR control (100 nM) or premiR-320c (100 nM) in HEK-293 cells, and luciferase activity was measured 24 h following transfection. Renilla luciferase activity was used for normalization. Data are presented
as mean ± S.E, n = 6. **Po0.005. (e) A working model depicting the possible mechanisms by which miR-320c promotes adipocytic differentiation of hMSCs through targeting
genes involved in multiple genetic pathways
inhibition of RUNX2 activity is needed to promote AD
differentiation. Interestingly, previous studies have also
identified RUNX2 as a bona fide target for pro-adipocytic
miRNAs such as miR-30a, 30d and miR-204/211. Therefore,
RUNX2 appears to be a key negative regulator of adipogenesis that seems to be targeted by several miRNA families,
including the miR-320 family in our study. This is not surprising
as RUNX2 has relatively large 3′UTR (3.777 kb) that makes it
a likely target for several groups of miRNAs. Interestingly,
lentiviral-medicated stable expression of miR-320c at physiological levels (~1.5-fold; Figure 2d) also promoted adipocytic
differentiation of hMSC, thus further supporting that the
observed effects have physiological relevance.
MSC commitment to specific lineage, AD or osteoblast, was
shown to be regulated by the expression of different TFs,
which are involved in different cellular pathways. MSCs
express several adipogenic TFs, for example, CCAATenhancer-binding protein (C/EBP) and PPARγ, as well as
osteoblastic TF, for example, RUNX2, MSX2, DLX5 and
Osterix.39–46 It is plausible that the undifferentiated state of
MSC is maintained by suppression of lineage-specific TFs.
Most of the studies focused on the ability of miRNA to induce
Cell Death and Disease
miRNA-320/RUNX2 axis regulates adipocytic differentiation
D Hamam et al
10
Table 2 Primer Sequences used for cloning and qRT-PCR
No. Name
Sequence
A Cloning primers
1 RUNX2 wt F 5′GTTGTTACTAGTTCTTTGAATGCCTCTAA
UTR
CACAGCTTTGCCTTTACCCAAGGCC CCA
CTGGCAGCTTTCCACATATCAGAGTTCCAGA
R 5′GTTGTTAAGCTTTCCTTAAAGCTGTACA
CACATCTCCTCAAACCAAAGCTGTGGTAC
CTGTTCTGGAACTCTGATATGTGGAAAGCTG
2 RUNX2 mut F 5′GTTGTTACTAGTTCTTTGAATGCCTCTAA
UTR
CAagaagggGCCTTTACCCAAGGCCCCACT
GGagaagggCCACATATCAGAGTTCCAGA
R 5′GTTGTTAAGCTTTCCTTcccttctTACACAC
ATCTCCTCAAACCcccttctTGGTACCTGTTC
TGGAACTCTGATATGTGGcccttct
B qRT-PCR primers
1 AP2
F
R
2 PPARγ
F
R
3 AdipoQ
F
R
4 LEP
F
R
5 GAPDH
F
R
6 ALPL
F
R
7 Osteonectin F
R
5′ TGGTTGATTTTCCATCCCAT
5′ GCCAGGAATTTGACGAAGTC
5′ GCTTCTGGATTTCACTATGG
5′ AAACCTGATGGCATTATGAG
5′ GCAGTCTGTGGTTCTGATTCCATAC
5′ GCCCTTGAGTCGTGGTTTCC
5′ CAGCGGTTGCAAGGCCCAAGA
5′ GGCCAAAGCCACAAGAATCCGC
5′ CTGGTAAAGTGGATATTGTTGCCAT
5′ TGGAATCATATTGGAACATGTAAACC
5′ GACGGACCCTCGCCAGTGCT
5′ AATCGACGTGGGTGGGAGGGG
5′ GAGGAAACCGAAGAGGAGG
5′ GGGGTGTTGTTCTCATCCAG
Lower case letter indicate sites of mutations in seed regions
lineage-specific differentiation through induction of lineagespecific TFs. Our study suggests that suppressing of the TFs
belonging to an alternative differentiation lineage is an
important mechanism controlling MSC lineage fate choice.
miRNA can thus be a target for pharmacological intervention
to control lineage fate of MSC.
Materials and Methods
Cell culture. As a model for primary human bone marrow-derived MSC
(hMSC), we used a Telomerized hMSC line that has been created through
overexpression of human telomerase reverse transcriptase gene (hTERT)
transduction (hMSC-TERT).47 The hMSC-TERT expresses all known markers of
primary hMSCs48,49 and exhibit ‘stemmness’ characteristics by being able to form
bone and bone marrow microenvironment when implanted in vivo.19 The hMSCTERT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with D-glucose 4500 mg/l, 4 mM L-glutamine and 110 mg/l sodium
pyruvate, 10% fetal bovine serum (FBS), 1% penicillin–streptomycin (Pen–Strep)
and non-essential amino acids 1. For transfection studies, HEK-293 cells were used
and the cells were cultured in the same culture medium as above. All reagents were
provided from Gibco–Invitrogen (Carlsbad, CA, USA). All cells were incubated in a
humidified atmosphere containing 95% air and 5% CO2 at 37 °C, medium was
replaced once a week or as needed.
Adipogenic differentiation of hMSCs. hMSC-TERT cells were cultured in
basal medium in 24-well tissue culture plates. When cells reached 80–90%
confluence, medium was replaced with AIM (DMEM medium supplemented with
10% FBS, 10% horse serum (Sigma, St Louis, MO, USA), 1% Pen–Strep, 100 nM
dexamethasone, 0.45 mM isobutyl methyl xanthine (Sigma), 3 μg/ml insulin (Sigma)
and 1 μM Rosiglitazone (BRL49653). The AIM was replaced every 3 days. Control
cells were cultured in parallel in normal DMEM medium. Cells were assessed for
adipogenic differentiation on days 7 and 13 post differentiation.
Cell Death and Disease
Osteogenic differentiation of hMSCs. For osteogenic differentiation of
hMSCs, cells were cultured as above then were exposed to osteogenic induction
medium (DMEM containing 10% FBS, 1% Pen–Strep,50 μg/ml L-ascorbic acid
(Wako Chemicals GmbH, Neuss, Germany), 10 mM β-glycerophosphate (Sigma)
and 10 nM calcitriol (1α,25-dihydroxy vitamin D3; Sigma) and 10 nM Dexamethasone (Sigma)).
Lentiviral transduction. Lentiviral particles encoding for has-miR-320c-1
(LP-HmiR0470-MR03-0200-S) or control lentiviral particles (LP-MCHR-LV105-0200)
were purchased from Genecopoeia (Genecopoeia Inc., Rockville, MD, USA).
Hundred thousand hMSCs were seeded in complete DMEM in 24-well plate. Fortyeight hours later (~80 confluency), media was removed and then 20 μl of crude
lentiviral particles in 500 μl of DMEM+5% heat-inactivated serum (Invitrogen) and
1% Pen–Strep supplemented with polybrene (8 μg/ml; Sigma) was added to the
cells. Seventy-two hours later, media was removed and transduced cells were
selected with puromycin (1 μg/ml, Sigma) for 1 week until stably transduced cells
were generated.
Total RNA isolation and quantification of miRNA and mRNA
expression. Total RNA containing the small RNA fraction were isolated from
hMSC-TERT cells using Total RNA Purification Kit (Norgen-Biotek Corp., Thorold,
ON, Canada) according to the manufacturer’s instructions. The concentrations of
total RNA were measured using NanoDrop 2000 (Thermo Scientific, Wilmington,
DE, USA). qRT-PCR analysis was performed as previously described50 to assess
the expression levels of miRNAs using TaqMan miRNA Assays (Applied Biosystems
Inc., Foster City, CA, USA). In brief, 10 ng of total RNA was subjected to reverse
transcription using miRNA-specific primers supplied by ABI. Subsequently, second
set of miRNA-specific primers was used to amplify each miRNA according to the
manufacturer’s recommendations. Expression levels of adipogenic-related genes,
PPARγ, LEP, AP2 and AdipoQ, were assessed using qRT-PCR. Reverse
transcription was performed on 500 ng of total RNA using High Capacity Reverse
Transcriptase Kit (Applied Biosystems Inc.) according to manufacturer’s specifications. qRT-PCR was done using FAST-SYBR Green Master Mix (Applied
Biosystems Inc.) and the StepOne Plus Real-Time PCR Detection System (Applied
Biosystems Inc.). Primers used for gene expression analysis are listed in Table 1
and were either previously published or were designed using NCBI Primer-BLAST
(http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The 2∆CT value method was used
to calculate relative expression of miRNAs and mRNAs.51
miRNA expression profiling. All miRNA microarray experiments and
analyses were conducted by Exiqon (Copenhagen, Denmark). hMSC-TERT cells
were differentiated into ADs, and on days 0, 7 and 13 RNA was extracted as
described above.19 The samples were labeled using the miRCURY LNA microRNA
Hi-Power Labelling Kit, Hy3/Hy5, and hybridized on the miRCURY LNA microRNA
Array (6th GEN) as outlined in Supplementary Figure 1. Samples that did not meet
quality requirements were excluded from the data analyses. Data were normalized
using the global Lowess regression algorithm, then were subjected to unsupervised
as well as supervised data analysis. P-values were corrected for multiple testing
using the Bonferroni adjustment method. Subsequently, miRNAs found to be
significantly regulated by the one-way ANOVA test were subjected to the Tukey's
‘Honest Significant Difference' test to determine which groups contribute most to the
significant difference. All data analyses were done using R/bioconductor software.
Gene ontology analyses were conducted using DAVID Bioinformatics Database
functional-annotation tools, as we have previously described.52 miRNA expression
data sets were deposited to the Gene Expression Omnibus (GEO), accession
number GSE59684.
miRNA and siRNA transfection experiments. To investigate the role of
selected miRNAs in regulating hMSC differentiation into ADs, hMSC cells were
transfected with the indicated miRNA precursors (pre-miR-Neg, pre-miR-320c and
pre-miR30b, Ambion, Foster City, CA, USA) or siRNAs (Ambion) using reverse
transfection protocol and Lipofectamine 2000 (Invitrogen) as we previously
described.52 In brief, 0.05 × 106 cells were reverse-transfected with 30 nM of the
indicated pre-miRs or siRNAs complexed with 1.5 μl of Lipofectamine 2000 in a
24-well tissue culture plate. Transfection cocktail was subsequently replaced after
4 h with normal DMEM without antibiotics. On day 3, medium was replaced with
DMEM–AIM as described above and fresh induction medium was replaced every
3 days.
miRNA-320/RUNX2 axis regulates adipocytic differentiation
D Hamam et al
11
Cloning of RUNX2 3′UTR and luciferase assay. A reporter vector
(pMir-Report, ABI) carrying the predicted miR-320 binding site(s) from RUNX2 3′
UTR was constructed using partially complementary primer pairs listed in Table 1.
Amplification was conducted as we previously described24 and using Amplitaq gold
DNA polymerase (Applied Biosystems Inc.). As positive control, we constructed a
vector carrying the full-length complementary sequence to Let-7b miRNA. A mutant
version of RUNX2 3′UTR reporter plasmid was generated by mutating the seed
region for the miR-320 miRNA family using the indicated primers in Table 1. All
regions were subsequently cloned into the SpeI and HindIII sites downstream of the
firefly luciferase gene in the pMIR-REPORT vector (Applied Biosystems Inc.). To
assess the direct interaction between miR-320 miRNA family and the 3′UTR from
RUNX2, HEK-293 cells were co-transfected with 100 nM of pre-miR-Neg or premiR-320c and 100 ng of pMIR-REPORT carrying either wt or mutant 3′UTR
sequences, along with 20 ng of pRL-SV40 vector (Promega, Madison, WI, USA)
carrying the Renilla luciferase gene. Transfection experiments were conducted using
Lipofectamine 2000 (Invitrogen). At 48 h post transfection, luciferase activity was
measured using the Dual-Glo luciferase assay system (Promega). Firefly luciferase
activity was then normalized to that of Renilla luciferase.
ALP activity quantification. To quantify ALP activity in control and
differentiated hMSC, we used the BioVision ALP activity colorimetric assay kit
(BioVision, Inc., Milpitas, CA, USA) with some modifications. Cells were cultured in
96-well plates under normal or osteogenic induction conditions, then on day 10,
wells were rinsed once with PBS and were fixed using 3.7% formaldehyde in 90%
ethanol for 30 s at room temperature. Subsequently, fixative was removed and 50 μl
of pNPP solution was added to each well and incubated for 1 h in the dark at room
temperature. Reaction was subsequently stopped by adding 20 μl stop solution and
gently shaking the plate. O.D. was then measured at 405 nm using SpectraMax/M5
fluorescence spectrophotometer plate reader.
Gene expression microarray. hMSC were differentiated into ADs as
described above. On day 7, total RNA was extracted using Total RNA Purification Kit
(Norgen-Biotek Corp.) according to the manufacturer’s instructions. For miR-320ctransfected cells, MSCs were transfected with either pre-miR-control or
pre-miR-320c. Seventy-two hours later, total RNA was isolated as described
above. The concentrations of total RNA were measured using NanoDrop 2000
(Thermo Scientific). Extracted RNA was labeled and then hybridized to the Agilent
Human SurePrint G3 Human GE 8 × 60 k microarray chip (Agilent Technologies,
Santa Clara, CA, USA). All microarray experiments were conducted at the
Microarray Core Facility (Stem Cell Unit, King Saud University College of Medicine,
Riyadh, Saudi Arabia). Data analyses were conducted using GeneSpring 12.0
software (Agilent Technologies) and DAVID bioinformatic tool as described
before.52,53 Percentile Shift was used for data normalization while Benjamini–
Hochberg false discovery rate method was used for multiple testing corrections. The
gene expression profiling in hMSCs transfected with miR-320c and the gene
expression profiling during adipogenic differentiation of hMSCs data sets were
deposited to the GEO, accession numbers GSE59458 and GSE59450, respectively.
AlamarBlue cell viability assay. Cell viability was measured using
alamarBlue assay according to the manufacturer’s recommendations (AbD Serotec,
Raleigh, NC, USA). In brief, we cultured cells in 96-well plates in 100 μl of the
appropriate medium and at the indicated time point, and 10 μl of alamarBlue
substrate was added and plates were incubated in the dark at 37 °C for 1h. Reading
was subsequently taken using fluorescent mode (Ex 530 nm/Em 590 nm) using
BioTek Synergy II microplate reader (BioTek Inc., Winooski, VT, USA).
Oil Red O staining for ADs. At the indicated time points, adipogenic
differentiation was determined by Oil Red O staining for lipid-filled mature ADs. Cells
were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min and were incubated with a newly made and filtered (0.45 μM) Oil
Red O staining solution (Sigma; 0.05 g in 60% isopropanol) for 1 h at room
temperature. Photomicrographs were acquired using inverted Zeiss microscope
(Thornwood, NY, USA).
AD enumeration by flow cytometry. Nile Red Staining was performed as
we described previously.54 In brief, following trypsinization, the cells were washed
with calcium and magnesium-free PBS. Subsequently, Nile Red dye (N3013; Sigma)
was added at a final concentration of l00 ng/ml. Following 5 min incubation at 4 °C,
the cells were washed in PBS, centrifuged and re-suspended in 500 μl PBS and
were analyzed using BD FACSCalibur flow cytometer (BD Biosciences, Franklin
Lakes, NJ, USA). Staining was detected in the green fluorescence channel (FL1);
the gating strategy is presented in Figure 2. Data were analyzed using FlowJo
software (Tree Star, Ashland, OR, USA).
Nile red fluorescence determination and quantification of
adipogenesis using microplate reader. Stock solution of Nile red
(1 mg/ml) in DMSO was prepared and stored at − 20 °C protected from light.
Staining was performed on unfixed cells. Cultured undifferentiated and differentiated
cells (were grown in Corning polystyrene flat bottom 96-well TC-treated black
microplates, Corning, NY, USA) were washed once with PBS. The dye was then
added directly to the cells (5 μg/ml in PBS), and the preparation was incubated for
10 min at room temperature then washed twice with PBS. Fluorescent signal was
measured using SpectraMax/M5 fluorescence spectrophotometer plate reader
(Molecular Devices Co., Sunnyvale, CA, USA) using bottom well-scan mode where
nine readings were taken per well using Ex (485 nm) and Em (572 nm) spectra.
Furthermore, fluorescence images were taken using FLoid cell imaging station (Life
Technologies Inc., Grand Island, CA, USA).
RUNX2 quantification. For quantification of RUNX2 protein, hMSC were
transfected with pre-miR-Neg or pre-miR-320c (30 nM), and 72 h later cells were
collected and washed with PBS. Cells were lysed in 100 μl PBS containing protease
inhibitors using five freeze-thaw cycles. Cell lysate was subsequently spun down at
maximum speed for 10 min, and supernatant was collected and stored at − 80°C.
Subsequently, RUNX2 was quantified using the RUNX2 ELISA kit according to the
manufacturer’s recommendation (Uscn Life Science Inc., Wuhan, PRC).
Statistics. Statistical analyses and graphing were performed using Microsoft
excel 2010 and GraphPad Prism 6.0 software (Graphpad software, San Diego, CA,
USA). P-values were calculated using the two-tailed t-test.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. This work was supported by the National Science
Technology and Innovation Plan strategic technologies program, grant number
(11-BIO-1941-02) in Saudi Arabia.
1. Naveiras O, Nardi V, Wenzel PL, Hauschka PV, Fahey F, Daley GQ. Bone-marrow
adipocytes as negative regulators of the haematopoietic microenvironment. Nature 2009;
460: 259–263.
2. Gimble JM, Robinson CE, Wu X, Kelly KA. The function of adipocytes in the bone marrow
stroma: an update. Bone 1996; 19: 421–428.
3. Menagh PJ, Turner RT, Jump DB, Wong CP, Lowry MB, Yakar S et al. Growth hormone
regulates the balance between bone formation and bone marrow adiposity. J Bone Miner
Res 2010; 25: 757–768.
4. Syed FA, Oursler MJ, Hefferanm TE, Peterson JM, Riggs BL, Khosla S. Effects of estrogen
therapy on bone marrow adipocytes in postmenopausal osteoporotic women. Osteoporos Int
2008; 19: 1323–1330.
5. Justesen J, Stenderup K, Ebbesen EN, Mosekilde L, Steiniche T, Kassem M. Adipocyte
tissue volume in bone marrow is increased with aging and in patients with osteoporosis.
Biogerontology 2001; 2: 165–171.
6. Fazeli PK, Horowitz MC, MacDougald OA, Scheller EL, Rodeheffer MS, Rosen CJ et al.
Marrow fat and bone–new perspectives. J Clin Endocrinol Metab 2013; 98: 935–945.
7. Aldahmash A, Zaher W, Al-Nbaheen M, Kassem M. Human stromal (mesenchymal) stem
cells: basic biology and current clinical use for tissue regeneration. Ann Saudi Med 2012; 32:
68–77.
8. Gimble JM, Zvonic S, Floyd ZE, Kassem M, Nuttall ME. Playing with bone and fat. J Cell
Biochem 2006; 98: 251–266.
9. Perera RJ, Ray A. MicroRNAs in the search for understanding human diseases. BioDrugs
2007; 21: 97–104.
10. Lakshmipathy U, Hart RP. Concise review: microRNA expression in multipotent
mesenchymal stromal cells. Stem Cells 2008; 26: 356–363.
11. Krichevsky AM, Sonntag KC, Isacson O, Kosik KS. Specific microRNAs modulate embryonic
stem cell-derived neurogenesis. Stem Cells 2006; 24: 857–864.
12. Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM et al. The role of
microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat
Genet 2006; 38: 228–233.
13. Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific
microRNA that targets Hand2 during cardiogenesis. Nature 2005; 436: 214–220.
Cell Death and Disease
miRNA-320/RUNX2 axis regulates adipocytic differentiation
D Hamam et al
12
14. Pedersen I, David M. MicroRNAs in the immune response. Cytokine 2008; 43: 391–394.
15. Alajez NM. Cancer stem cells. From characterization to therapeutic implications. Saudi Med
J 2011; 32: 1229–1234.
16. Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH. Targeted inhibition of
miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet
development. PLoS Biol 2007; 5: e203.
17. Tay YM, Tam WL, Ang YS, Gaughwin PM, Yang H, Wang W et al. MicroRNA-134 modulates
the differentiation of mouse embryonic stem cells, where it causes post-transcriptional
attenuation of Nanog and LRH1. Stem Cells 2008; 26: 17–29.
18. Taipaleenmaki H, Bjerre Hokland L, Chen L, Kauppinen S, Kassem M. Mechanisms in
endocrinology: micro-RNAs: targets for enhancing osteoblast differentiation and bone
formation. Eur J Endocrinol 2012; 166: 359–371.
19. Eskildsen T, Taipaleenmaki H, Stenvang J, Abdallah BM, Ditzel N, Nossent AY et al.
MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem
cells in vivo. Proc Natl Acad Sci USA 2011; 108: 6139–6144.
20. Zeng Y, Qu X, Li H, Huang S, Wang S, Xu Q et al. MicroRNA-100 regulates osteogenic
differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2.
FEBS Lett 2012; 586: 2375–2381.
21. Tuddenham L, Wheeler G, Ntounia-Fousara S, Waters J, Hajihosseini MK, Clark I et al. The
cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett
2006; 580: 4214–4217.
22. Laine SK, Alm JJ, Virtanen SP, Aro HT, Laitala-Leinonen TK. MicroRNAs miR-96, miR-124,
and miR-199a regulate gene expression in human bone marrow-derived mesenchymal
stem cells. J Cell Biochem 2012; 113: 2687–2695.
23. Skarn M, Namlos HM, Noordhuis P, Wang MY, Meza-Zepeda LA, Myklebost O. Adipocyte
differentiation of human bone marrow-derived stromal cells is modulated by microRNA-155,
microRNA-221, and microRNA-222. Stem Cells Dev 2012; 21: 873–883.
24. Alajez NM, Shi W, Wong D, Lenarduzzi M, Waldron J, Weinreb I et al. Lin28b promotes head
and neck cancer progression via modulation of the insulin-like growth factor survival
pathway. Oncotarget 2012; 3: 1641–1652.
25. Tome M, Lopez-Romero P, Albo C, Sepulveda JC, Fernandez-Gutierrez B, Dopazo A et al.
miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal
stem cells. Cell Death Differ 2011; 18: 985–995.
26. Esau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV et al.
MicroRNA-143 regulates adipocyte differentiation. J Biol Chem 2004; 279: 52361–52365.
27. Yang Z, Bian C, Zhou H, Huang S, Wang S, Liao L et al. MicroRNA hsa-miR-138 inhibits
adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells through
adenovirus EID-1. Stem Cells Dev 2011; 20: 259–267.
28. Zhang JF, Fu WM, He ML, Wang H, Wang WM, Yu SC et al. MiR-637 maintains the balance
between adipocytes and osteoblasts by directly targeting Osterix. Mol Biol Cell 2011; 22:
3955–3961.
29. Zaragosi LE, Wdziekonski B, Brigand KL, Villageois P, Mari B, Waldmann R et al. Small RNA
sequencing reveals miR-642a-3p as a novel adipocyte-specific microRNA and miR-30 as a
key regulator of human adipogenesis. Genome Biol 2011; 12: R64.
30. Karbiener M, Neuhold C, Opriessnig P, Prokesch A, Bogner-Strauss JG, Scheideler M.
MicroRNA-30c promotes human adipocyte differentiation and co-represses PAI-1 and ALK2.
RNA Biol 2011; 8: 850–860.
31. Barsi JC, Rajendra R, Wu JI, Artzt K. Mind bomb1 is a ubiquitin ligase essential for mouse
embryonic development and Notch signaling. Mech Dev 2005; 122: 1106–1117.
32. St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss P. Pax6 is required for
differentiation of glucagon-producing alpha-cells in mouse pancreas. Nature 1997; 387:
406–409.
33. De S, Kline D. Evidence for the requirement of 14-3-3eta (YWHAH) in meiotic spindle
assembly during mouse oocyte maturation. BMC Dev Biol 2013; 13: 10.
34. Williams BC, Li Z, Liu S, Williams EV, Leung G, Yen TJ et al. Zwilch, a new component of the
ZW10/ROD complex required for kinetochore functions. Mol Biol Cell 2003; 14: 1379–1391.
35. Bialek P, Kern B, Yang X, Schrock M, Sosic D, Hong N et al. A twist code determines the
onset of osteoblast differentiation. Dev Cell 2004; 6: 423–435.
36. Shen R, Wang X, Drissi H, Liu F, O'Keefe RJ, Chen D. Cyclin D1-cdk4 induce runx2
ubiquitination and degradation. J Biol Chem 2006; 281: 16347–16353.
37. Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrow-derived
mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci
2009; 66: 236–253.
38. Kobayashi H, Gao Y, Ueta C, Yamaguchi A, Komori T. Multilineage differentiation of
Cbfa1-deficient calvarial cells in vitro. Biochem Biophys Res Commun 2000; 273: 630–636.
39. Bennett CN, Longo KA, Wright WS, Suva LJ, Lane TF, Hankenson KD et al. Regulation of
osteoblastogenesis and bone mass by Wnt10b. Proc Natl Acad Sci USA 2005; 102:
3324–3329.
40. Clement-Lacroix P, Ai M, Morvan F, Roman-Roman S, Vayssiere B, Belleville C et al.
Lrp5-independent activation of Wnt signaling by lithium chloride increases bone formation
and bone mass in mice. Proc Natl Acad Sci USA 2005; 102: 17406–17411.
41. Taylor-Jones JM, McGehee RE, Rando TA, Lecka-Czernik B, Lipschitz DA, Peterson CA.
Activation of an adipogenic program in adult myoblasts with age. Mech Ageing Dev 2002;
123: 649–661.
42. Arango NA, Szotek PP, Manganaro TF, Oliva E, Donahoe PK, Teixeira J. Conditional
deletion of beta-catenin in the mesenchyme of the developing mouse uterus results in a
switch to adipogenesis in the myometrium. Dev Biol 2005; 288: 276–283.
43. Hooper JE, Scott MP. Communicating with Hedgehogs. Nat Rev Mol Cell Biol 2005; 6:
306–317.
44. Spinella-Jaegle S, Rawadi G, Kawai S, Gallea S, Faucheu C, Mollat P et al. Sonic hedgehog
increases the commitment of pluripotent mesenchymal cells into the osteoblastic lineage and
abolishes adipocytic differentiation. J Cell Sci 2001; 114: 2085–2094.
45. Zehentner BK, Leser U, Burtscher H. BMP-2 and sonic hedgehog have contrary
effects on adipocyte-like differentiation of C3H10T1/2 cells. DNA Cell Biol 2000; 19:
275–281.
46. Suh JM, Gao X, McKay J, McKay R, Salo Z, Graff JM. Hedgehog signaling plays a conserved
role in inhibiting fat formation. Cell Metab 2006; 3: 25–34.
47. Simonsen JL, Rosada C, Serakinci N, Justesen J, Stenderup K, Rattan SI et al. Telomerase
expression extends the proliferative life-span and maintains the osteogenic potential of
human bone marrow stromal cells. Nat Biotechnol 2002; 20: 592–596.
48. Abdallah BM, Haack-Sorensen M, Burns JS, Elsnab B, Jakob F, Hokland P et al.
Maintenance of differentiation potential of human bone marrow mesenchymal stem cells
immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive
proliferation. Biochem Biophys Res Commun 2005; 326: 527–538.
49. Al-Nbaheen M, Vishnubalaji R, Ali D, Bouslimi A, Al-Jassir F, Megges M et al. Human
stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit
differences in molecular phenotype and differentiation potential. Stem Cell Rev 2013; 9:
32–43.
50. Hui AB, Shi W, Boutros PC, Miller N, Pintilie M, Fyles T et al. Robust global micro-RNA
profiling with formalin-fixed paraffin-embedded breast cancer tissues. Lab Invest 2009; 89:
597–606.
51. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402–408.
52. Alajez NM, Shi W, Hui AB, Yue S, Ng R, Lo KW et al. Targeted depletion of BMI1 sensitizes
tumor cells to P53-mediated apoptosis in response to radiation therapy. Cell Death Differ
2009; 16: 1469–1479.
53. Al-toub M, Almusa A, Almajed M, Al-Nbaheen M, Kassem M, Aldahmash A et al. Pleiotropic
effects of cancer cells’ secreted factors on human Stromal (mesenchymal) stem cell. Stem
Cell Res Ther 2013; 4: 114.
54. Vishnubalaji R, Manikandan M, Al-Nbaheen M, Kadalmani B, Aldahmash A, Alajez NM. In
vitro differentiation of human skin-derived multipotent stromal cells into putative endotheliallike cells. BMC Dev Biol 2012; 12: 7.
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