shh通过miR-92b转录后调控KLF-4在血管平滑肌细胞去分化中的作用机制研究

VSMC由静止型去分化为增殖型而后发生无序增殖是血管狭窄性疾病发生发展的必要前提。我们在以往研究中证实shh信号通路与VSMC的生物学行为密切相关，在本申请课题的前期研究中我们又发现shh可诱导VSMC发生去分化，且通过基因芯片筛选分析发现此过程与miR-92b相关，KLF-4亦参与其中，但具体机制还有待进一步研究。以往研究显示shh可直接促进miR-92b的转录，且理论预测miR-92b与KLF4-mRNA-3'UTR有关键结合位点，因此本申请课题拟在以上实验及理论基础上通过荧光素酶报告基因表达分析和绿色荧光蛋白报告基因分析，确定miR-92b是否与KLF-4mRNA直接结合而抑制其表达；并分别在不同表型VSMC中进行shh，miR-92b的阻断及过表达研究，明确shh是否通过miR-92b对KLF-4进行转录后调控，从而促进VSMC的去分化。本课题将是我们前期研究结果的有益补充和完善。

Dedifferentiation of VSMC from nascence into proliferation state had been solidly confirmed to be essential for vascular stenotic diseases progression. We have proved that shh signaling pathway was related to the biological behavior of VSMC closely.In this protocol,our previous studies demonstrated that Shh signaling pathway could induce VSMC dedifferentiation, mediated by miR-92b and KLF-4 which was discovered by micRNA micro-array analysis.But the precise mechanism involving it need to be further investigated. One possible mechanism arising from is that miR-92b which activated by Shh, targeting its theoretical binding site on KLF4-3'UTR directly to inhibit expression of KLF-4 ,a differentiation oriented transcripitional factor. Next, we intend to clarify physical interaction between miR-92b and KLF-4 mRNA-3'UTR in vitro by luciferase reportor and GFP reportor analysis;Then we are going to repress or activate miR-92b or Shh in VSMC of different phenotype stages, to elucidate whether the dedifferentiation of VSMC induced by shh is through post-transcriptional suppression of KLF-4 by miR-92b.This protocol will be a intensive complement and improvement for our previous results.