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. 2014 Oct 30;5(10):e1499.
doi: 10.1038/cddis.2014.462.

microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells

D Hamam  1 , D Ali  1 , R Vishnubalaji  1 , R Hamam  1 , M Al-Nbaheen  1 , L Chen  2 , M Kassem  3 , A Aldahmash  3 , N M Alajez  1
Affiliations

microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells

D Hamam et al. Cell Death Dis. .

Abstract

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.

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Figures

Figure 1
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. ***P<0.0005 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. *P<0.05, ***P<0.0005
Figure 2
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 pre-miR-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, *P<0.05, **P<0.005, ***P<0.0005
Figure 3
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, *P<0.05, ***P<0.0005
Figure 4
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. *P<0. 05, **P<0.005, ***P<0.0005
Figure 5
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, *P<0.05, ***P<0.0005
Figure 6
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. *P<0.05. (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 pre-miR-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. **P<0.005. (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
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