Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing, People's Republic of China.
PLoS One. 2011;6(7):e21679. doi: 10.1371/journal.pone.0021679. Epub 2011 Jul 20.
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. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. 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. 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 three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation.
软骨细胞分化的间充质干细胞（MSCs）是由必要的转录因子和信号级联准确调控。然而，这一过程中涉及的精确机制仍有待确定。microRNAs（miRNAs）通过结合靶mRNA来衰减蛋白质合成，从而调节各种生物过程。为了研究miRNAs 介导的软骨分化调节的机制，我们发现 miR-145 在转化生长因子β3（TGF-β3）诱导的鼠 MSCs 软骨分化过程中降低。随后，双荧光素酶报告基因检测数据表明，miR-145 靶定 Sox9 基因 3'UTR 中的一个假定结合位点，Sox9 基因是软骨形成的关键转录因子。此外，miR-145 的过表达仅在蛋白水平上降低 Sox9 的表达，而 miR-145 抑制则显著提高 Sox9 蛋白水平。此外，miR-145 的过表达降低了 TGF-β3 诱导的 C3H10T1/2 细胞中三种软骨标志物基因（II 型胶原（Col2a1）、聚集蛋白（Agc1）、软骨寡聚基质蛋白（COMP）、IX 型胶原（Col9a2）和 XI 型胶原（Col11a1）的 mRNA 水平，而抗-miR-145 抑制剂则增加了这些软骨标志物基因的表达。因此，我们的研究表明，miR-145 通过在软骨分化早期直接靶向 Sox9，是软骨分化的关键负调控因子。
