软骨形成过程中参与Hedgehog信号通路的microRNA的分析

Analysis of microRNA Involved in the Hedgehog Signaling Pathway During Chondrogenesis

间充质细胞增殖并聚集(condensation)是软骨形成的第一步,大量研究表明。在这一过程中,Hedgehog信号通路扮演着重要角色。但是,哪些Hedgehog信号通路相关的微小RNA(microRNA)参与这一过程目前还不清楚。本研究采用Hh信号通路特异性抑制剂(cyclopamine)和激活剂(purmorphamine)分别处理原代培养的胚胎期小鼠肢芽间充质细胞,经细胞微团培养(micromass culture),对间充质细胞聚集过程进行microRNA表达差异化芯片分析。研究找到了Hh信号通路相关的特异性microRNA:miR-494和miR-1906,并且我们发现在hedgehog信号被特异性激活后,miR-494和miR-1906的表达量显著下降,hedgehog信号被抑制后,miR-494和miR-1906的表达量则上升,呈现稳定的负相关变化趋势。通过microRNA数据库预测分析发现Gli3基因是miR-494潜在的靶基因。尽管初步体外验证试验没有发现miR-494对Gli3的直接调控,但是,这一发现为进一步探索microRNA在软骨发育过程中的功能和调控机制的研究提供了有价值的参考依据。

Proliferation and condensation of mesenchymal cells are the first step in chondrogenesis. Lots of studies have shown that Hedgehog signaling pathway plays an important role in the process of chondrogenesis. However, it remains unclear that which microRNAs that related to the Hedgehog signaling pathway are participating in this process. In this study, we treated the primary cultured mesenchymal cells from mouse embryonic limb bud individually with Hh signaling pathway specific inhibitor （Cyclopamine） and activator （Purmorphamine）, and then we analyzed the microRNA expression difference in the process of mesenchymal cell condensation by microarray after micromass cultivation. The study found the specific microRNAs that related to Hh signaling pathway which were miR-494 and miR-1906. We further observed that the expression levels of miR-494 and miR-1906 significantly decreased after hedgehog was activated by specificity, while the expression levels increased when hedgehog signal was inhibited, which showed a stable negative correlation trend. Based on the predictive analysis of microRNA database, we found that Gli3 was a potential target gene of miR-494. Even though we did not find miR-494 directly regulate Gli3 in preliminary vitro tests, this study provides valuable reference for further exploration on function and regulation mechanisms ofmicroRNA during chondrogenesis.