Yes相关蛋白通过Wnt/β-catenin信号通路调控软骨细胞Sox9表达的研究

Yes-associated protein regulates the expression of Sox9 in chondrocytes by Wnt/β-catenin signaling pathway

目的研究Yes相关蛋白(Yes-associated protein,YAP)通过Wnt/β-catenin信号通路调控软骨细胞Sox9表达的机制。方法用si RNA分别沉默YAP基因和Lats1基因来调控YAP的活性,通过Realtime PCR检测软骨特异性基因和Wnt/β-catenin信号通路下游基因的表达;通过Western Blot检测GSK3β的磷酸化水平、活化β-catenin的蛋白水平以及Sox9的蛋白水平;并通过阿利新蓝染色检测软骨细胞外基质的分泌情况。用si RNA沉默YAP基因后,再用氯化锂干预细胞,通过Western Blot检测Sox9的表达和GSK3β的磷酸化水平。结果沉默YAP基因后,磷酸化GSK3β和活化β-catenin的蛋白水平减少,c-Myc和Nanog基因表达减少,Sox9、Col2和Aggrecan基因表达升高,并且软骨细胞分泌基质增多;沉默Lats1基因后,磷酸化GSK3β和活化β-catenin的蛋白水平增加,c-Myc和Nanog基因表达上调,Sox9、Col2和Aggrecan基因表达减少,并且软骨细胞分泌基质减少。此外,氯化锂可以阻断沉默YAP引起的Sox9表达上调。结论 YAP通过Wnt/β-catenin信号通路调控软骨细胞Sox9的表达。

Objective To investigate the mechanisms of Sox9 regulated by Yes- associated protein（Yap） in chondrocytes via Wnt signaling pathway. Methods Yap or Lats1 was knocked down with small inter-fering RNA to regulate the activity of Yap. Genes related to Wnt/β-catenin signaling pathway and cartilage-spe-cific genes were detected by Real-time PCR. Wnt/β-catenin signaling was determined by detection of p-GSK3βand active β-catenin via Western blotting and Sox9 was also detected by Western blotting. The cartilage-specif-ic extracellular matrix was assessed by alcian blue staining. Chondrocytes with Yap knockdown were treatedwith lithium chloride, then p-GSK3β and Sox9 were detected by Western blotting. Results After knockdownof Yap, Wnt signaling pathway was inhibited, leading to increased expression of Sox9, Col2 and Aggrecan andenhanced secretion of the cartilage-specific extracellular matrix. After Lats1 was knocked down, the obtained re-sult was opposite completely. Moreover, lithium chloride could block the up-regulation of Sox9 expression in-duced by knockdown of Yap. Conclusion Yap regulates the expression of Sox9 in chondrocytes by Wnt signaling pathway.