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.
Highlights
IntroductionMicroRNAs (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.[11,12,13,14,15,16,17] 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]
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 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)
We have identified miR-320 family as novel regulator of bone marrow-derived human MSC (hMSC) differentiation into ADs
Summary
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.[11,12,13,14,15,16,17] 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]. We identified several novel pro-adipogenic miRNAs, and found that miR-320 to be an important regulator of adipocytic differentiation of hMSC
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