Biological Functions And Molecular Mechanisms Of Mir-145 In Chondrogenic Differentiation Of Mesenchymal Stem Cells

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. Exploring the biology function of miRNAs in regulating chondrogenesis will provide a new insight on complicated regulation mechanisms of chondrogenesis.To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we designed a systemic scheme of experiments consisted of three parts: 1) Performing miRNAs microarray in MSCs at four different stages of transforming growth factor beta 3 (TGF-?3)-induced chondrogenic differentiation. 2) Screening the candidated miRNAs for regulating chondrogenic differentiation of MSCs and identifying their target genes by dual-luciferase reporter gene assay. 3) Validating the effect and mechanisms of the candidated miRNAs on controlling the chondrogenic differentiation of MSCs by gain-of-function and loss-of-function experiments.The following results have been obtained:1) Our microarray results revealed that eight miRNAs were significantly up-regulated and five miRNAs were down-regulated. Four of the thirteen miRNAs expression pattern were validated by qRT-PCR. Interestingly, we found two miRNAs clusters, miR-143/145 and miR-132/212, kept on down-regulation in the process. Using bioinformatics approaches, we analyzed the target genes of these differentially expressed miRNAs and found a series of them correlated with the process of chondrogenesis. Furthermore, the qRT-PCR results showed that the up-regulated (or down-regulated) expression of miRNAs were inversely associated with the expression of predicted target genes. Next, we identified that miR143 /145 cluster was decreased during TGF-?3-induced chondrogenic differentiation of murine MSCs. 2) Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3?-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. Unexpectedly, miR-143 failed to bind to its putative binding site in the 3?-UTR of RelA gene.3) In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for chondrogenic marker genes, such as type II collagen (Col2a1), type IX collagen (Col9a2), type XI collagen (Col11a1), aggrecan (Agc1) and cartilage oligomeric matrix protein (COMP) in C3H10T1/2 cells induced by TGF-?3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. The results of Alcian blue staining also showed that anti-miR-145 inhibitor promoted chondrogenic differentiation of MSCs by increasing production of GAGs. C/EBP?and C/EBP?are both Sox9 target genes, which are related to adipose differentiation of MSCs.The results of qRT-PCR did not showed that miR-145 effected the mRNA expression of C/EBP?and C/EBP?. Furthermore, miR-145 has no effect on the proliferation of MSCs. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9.In conclusion, our results first revealed the expression profiles of miRNAs and demonstrate that miR-145 is decreased during TGF-?3-induced chondrogenic differentiation of murine MSCs. The attenuation of miR-145 expression positively regulates its direct target gene Sox9 at protein level, and results in promoting early chondrogenic differentiation of mesenchymal stem cell line. Our findings indicate that the effect which miR-145 regulate chondrogenic differentiation of MSCs mediated by Sox9 in response to TGF-beta3 is a specific influence on genes associated with cartilage. The results may provide a novel mechanism in miRNA-mediated regulation of chondrogenic differentiation of MSCs.