转录因子SRF通过启动miR-199a/214基因簇促进腹膜间皮细胞转分化的分子机制研究

Epithelial-to-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) is the early stage in peritoneal fibrosis. Therefore, the identification of related molecular and pathway becomes a critical issue for reversing EMT of HPMCs.In our study, selecting by active TFs arrays, we found that SRF might be an important active TF in EMT of HPMCs,which were also verified in EMT in HPMCs cell lines, PD animal model and HPMCs from PD patients. Moreover, SRF inhibitor could reverse EMT of fibroblast-like HPMCs. All these show that SRF can be the target of peritoneal fibrosis. However,the mechanism of SRF in HPMCs EMT is still unclear.We then scan the downstream targe miRNAs and genes of SRF by miRNA and gene array respectively.Significant down-regulation of miR-199a/214 cluster and up-regulation of CDH1/CLDN2 was found after down-regulating SRF in HPMCs. And we also find that CDH1/CLDN2 may be the target of miR-199a/214 cluster predicting by miRbase.All this reveals that SRF may promote the expression of miR-199a/214 cluster directly, then silence the expression of E-cadherin and claudin-2 proteins by binding to CDH1/CLDN2 mRNA, and finally enhance EMT of HPMCs. In this study, we will investigate SRF-miR-199a/214-CDH1/CLDN2 pathway in HPMCs in vivo and in vitro. It will be helpful to find the effect target for reversing EMT of peritoneal fibrosis.

腹膜间皮细胞（HPMCs）转分化（EMT）是腹膜纤维化的重要原因，其调控机制尚未完全清楚。我们前期用转录因子（TF）活性谱芯片筛选不同时间腹透患者脱落HPMCs中活性差异的TF，并经细胞及动物实验证实：SRF在EMT的HPMCs中活性增加最显著，而SRF抑制剂可部分逆转EMT，以上提示：SRF可能为抑制腹膜EMT的有效靶点，但其具体作用及机制如何？进一步经miRNA及基因芯片筛选发现抑制SRF表达可引起miR-199a/214簇及CDH1/CLDN2表达显著改变，而CDH1/CLDN2恰为miR-199a/214预测靶分子。由此推测SRF可能通过启动miR-199a/214,抑制E-cadherin(CDH1)及claudin(CLDN2)表达，进而促HPMCs的EMT。本研究将进一步探讨HPMCs中SRF-miR-199a/214-CDH1/CLDN2通路，为探寻腹膜纤维化靶点提供依据。

腹膜间皮细胞（HPMCs）发生EMT是腹膜纤维化的起始环节和可逆阶段。课题组前期采用转录因子活性谱芯片分析技术筛选出SRF；下调EMT的HPMCs中SRF的表达，miRNA芯片筛选出下游靶分子miR-199a/214 基因簇。实验主要研究SRF/miR-199a/214在腹膜纤维化过程中发挥的作用。高糖（60 mmol/L）刺激HPMCs后， ChIP结果显示SRF可通过SRE位点直接结合miR-199a/214 基因簇；启动子报告基因结果表明SRF可直接结合并正向调控miR-199a/214簇的表达。高糖组（60 mmol/L）、对照组（5.6 mmol/L）培养永生化HPMCs，高糖刺激3天后，细胞免疫荧光显示E-cadherin和Claudin2表达降低，α-SMA和vimentin表达升高；高糖刺激1 d、3 d、5 d和7 d后，WB显示E-cadherin和Claudin2表达降低，α-SMA、CTGF和FN表达升高，同时RT-qPCR结果显示与对照组相比，miR-199a/214簇的表达与时间呈正相关。构建实验组（4.25%透析液）和对照组（生理盐水）SD大鼠模型，6 W后实验组注射SRF-inhibitor。Masson结果显示，实验组与对照组相比腹膜增厚，注射SRF-inhibitor后，实验组腹膜变薄；原位杂交证实miR-199a/214簇在纤维化的腹膜中高表达，E-cadherin和Claudin2的表达减弱。miR-199a/214mimic/inhibitor转染HPMCs，通过细胞免疫荧光、WB和RT-qPCR方法检测，显示上调miR-199a/214 簇后， E-cadherin和 Claudin2表达减弱，α-SMA和vimentin表达增强；下调后E-cadherin和 Claudin2表达增强，α-SMA和vimentin减弱。双荧光素酶报告基因检测表明CDH1和CLDN2是miR-199a/214簇的靶基因，miR-199a/214簇与CDH1和CLDN2的mRNA结合，抑制转录后水平，导致E-cadherin和Claudin2蛋白表达下降。因此，miR-199a/214簇通过CDH1/CLDN2通路促进腹膜间皮细胞发生间充质转分化，下调 SRF或者miR-199a/214簇可以部分逆转间皮细胞转分化和腹膜纤维化的发生。