Mirna-140 Inhibits C3h10t1/2 Mesenchymal Stem Cell Proliferation By Targeting Cxcl12 During Transforming Growth Factor-Beta 3-Induced Chondrogenic Differentiation

The aim of the present study was to investigate the role of microRNA (miRNA or miR)-140 in C3H10T1/2 mesenchymal stem cells (MSCs). Cluster analysis was used to evaluate the miRNA expression profile. The expression level of miRNA-140 was validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). TargetScan and microRNA.org databases were used to predict target miRNAs and cartilage-associated target genes. Binding sites between miR-140 and the target gene were predicted by bioinformatics software. A dual-luciferase reporter assay was performed to determine whether miR-140 could target C-X-C motif chemokine ligand 12 (CXCL12). Following the promotion/inhibition of miR-140, 1, 7 and 14 days following transforming growth factor-beta 3 (TGF-beta 3)-induction, western blotting was utilized to evaluate CXCL12 protein levels. MTT assays and alcian blue staining were applied to assess C3H10T1/2 MSC viability and chondrogenic differentiation, respectively. In the TGF-beta 3-induced group, RT-qPCR verified that the mRNA level of Mus musculus (mmu)-miR-140 was significantly elevated when compared with the control group. miR-140 was predicted to recognize and interact with CXCL12-3'UTR and the dual luciferase reporter assay further validated that miR-140 targeted the predicted region of CXCL12. CXCL12 was markedly decreased following miR-140 overexpression and visibly increased following miR-140 inhibition. In addition, the level of CXCL12 expression declined as the duration of induction increased. Following the promotion/inhibition of miR-140, at 1 and 7 days following TGF-beta 3-induction, C3H10T1/2 MSCs inhibited or promoted cell viability, respectively, when compared with the control groups. In addition, in pellets achieved by chondrogenic differentiation following the induction of C3H10T1/2 MSCs for 7 days, alcian blue staining revealed no significant difference in characteristic extracellular matrix glycosaminoglycans between the miR-140 up and downregulated groups, and their respective control groups. The present study concludes that miRNA-140 inhibition promoted C3H10T1/2 MSC viability however, not C3H10T1/2 MSC differentiation by targeting and reducing CXCL12 protein levels during the process of TGF-beta 3-induced chondrogenic differentiation. In conclusion, the present study provided a potential target for the treatment of cartilage defection.