miRNA-200s的转录调控在结肠癌EMT-MET可塑性调节中的作用机制研究

The plasticity between epithelial-mysenchymal transition(EMT) and mysenchymal-epithelial transition(MET) is involved in embryogenesis, wound healing and cancer progression and metastasis. The miRNAs are one of the regulating networks determining the celllular features besides transcription factors, but how these miRNAs are regulated in their expression is largely unknown and leaves an emerging field becomes a mojor research hot in RNA biology. The focused on the identification of transcriptional control of transcription factors predicted in the proximal promoters of miRNA-200 family on miRNA-200b-a-429 and miRNA-200c-141 clusters using siRNA techniques to target those predicted TF(s) in the intestinal epithelial cells. Using chromatin immuno-precipitation experiment(Chip) to determine whether these predicted TFs are bound to their consensus sequences located in the proximal promoters of miRNA-200s, and confirm the effects from their transcriptional regulation due to the knockdown of these TFs, including the changes of precursors of miR-200s and their mature counterpart, finally, exploring the exploring the effects on the target proteins determining the epithelial or myesenchymal features regulated by miR-200s due to the knockdown of these TFs and the changes of cellular behavior. The study will provide the new knowledge in the regulation of the plasticity of EMT-MET, and might provide the new target and theoretical insights for the injury of intestinal epithelial cells and their healing, provide the new clues about the homeostasis of intestinal mocosa.

细胞在上皮间充质转变（EMT）-间充质上皮转变（MET）之间的可塑性涉及胚胎发育、创伤愈合和肿瘤发生，其可塑性的机制认识有助于拓展新的治疗方法。miR-200s是EMT-MET可塑性的重要调控因子，其本身在转录水平是如何被调控表达缺乏深入的研究。本研究拟在预测的与miR-200s近端启动子（-2000~-1）的相应结合位点所结合的转录因子的基础上，通过对其RNA干扰或基因过表达，及定量PCR分析鉴定miR-200s水平，揭示miR-200s近段启动子区潜在结合的转录因子对其转录调节效应；通过Chip、双萤光素酶报告基因和EMSA实验揭示它们对miR-200s的转录调控机制；及其通过miR-200s调节EMT靶基因的作用和细胞生物学行为的变化。获得与miR-200s近段启动子区潜在结合的转录因子通过转录调控miR-200s的表达并调节EMT-MET可塑性的分子机制，为结肠癌治疗提供新的靶点。

细胞在上皮间充质转变（EMT）-间充质上皮转变（MET）之间的可塑性涉及胚胎发育、创伤愈合和肿瘤发生，其可塑性的机制认识有助于拓展新的治疗方法。miR-200s是EMT-MET可塑性的重要调控因子，其本身在转录水平是如何被调控表达缺乏深入的研究。本课题通过研究Ascl2、nanog、HIF-1α及黏蛋白Core 3 O-型糖链对miR-200s家族的调控进而调节结肠癌细胞EMT-MET可塑性的机制，发现（1）miR-200s家族在50例结肠癌组织中的表达高于正常组织，且和Ascl2的表达呈明显负相关；（2）Ascl2促进结肠癌细胞EMT进程；（3）Ascl2通过与miR-200b/a/429启动子区域的E-box元件结合调控 miR-200 / ZEB轴进而调节上皮和间充质结肠癌细胞的可塑性；（4）结肠癌细胞的干性基因Nanog与miR-200b和miR-200c的表达呈显著负相关；(5)Nanog通过与miR-200s家族启动子结合抑制miR-200b和miR-200c的转录，进一步促进结肠癌EMT发生；（6）缺氧诱导因子HIF-1α在50例结肠癌组织中的表达与Ascl2呈正相关，与miR-200b呈负相关；（7）HIF-1α与Ascl2启动子区域的缺氧反应元件HRE结合进而促进Ascl2的转录及通过Ascl2间接抑制miR-200b启动子转录活性；（8）miR-200b能够负反馈抑制HIF-1α mRNA表达水平从而形成HIF-1α/Ascl2/miR-200b负反馈环，通过此反馈环维持结肠癌细胞EMT表型特征。（9）Core 3合成酶影响结肠癌预后和诱导MET发生；（10）再表达Core 3合酶调节O-型糖链的结构、MUC1糖基化和MUC1-C核转位并进一步增强p53的表达；（11）Core 3合酶通过MUC1/p53/miR-200c调节结肠癌细胞EMT-MET可塑性。