ZEB介导microRNA-200家族对腹膜间皮细胞间质转分化的调控

Peritoneal fibrosis is a common complication of long-term peritoneal dialysis, and contributes to encapsulating peritoneal sclerosis and eventually peritoneal ultrafiltration failure, which restricts the wide application of peritoneal dialysis. Therefore, the prevention and treatment of peritoneal fibrosis is important to prolong peritoneal dialysis treatment. Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (MCs) is the key process in peritoneal fibrosis. However, the molecular regulation of EMT in peritoneum is not clear completely. MicroRNAs have been shown to regulate organ fibrosis and EMT, recently. The aim of our study is to prove that microRNA-200 family can regulate epithelial-mesenchymal transition of peritoneal mesothelial cells by targeting ZEB1/ZEB2. We use TGFβ-1 to induce EMT of MCs in vitro, and observe the expression of E-box-binding homeobox (ZEB)1 and ZEB2, which are E-cadherin transcriptional repressors. We construct ZEB1 and ZEB2 3'UTR luciferase reporters, and co-transfect miR-200s precursor or antimiR-200s into MCs to demonstrate ZEB was the target of miR-200s. TGF-β1 induced EMT MCs were transfected with miR-200s precursor or antimiR-200s, and then, western blot and real-time PCR were performed to detect the protein and mRNA level of ZEB1 ,ZEB2 and EMT markers. At last, we use miR-200s precursor or antimiR-200s to interfere high-glucose induced peritoneal fibrosis animal model to demonstrate that miR-200s can regulate peritoneal fibrosis in vivo.

腹膜纤维化是腹膜透析常见并发症，也是导致超滤衰竭和制约腹透发展最主要的原因。间皮细胞间质转分化（EMT）是腹膜纤维化发生和发展的关键环节。近年研究表明miRNAs参与了多种器官EMT和纤维化的过程，但其具体作用和机制仍不清楚。本项目用TGF-beta1诱导腹膜间皮细胞转分化，建立EMT体外模型，观察转分化关键转录因子bHLH、Snail、ZEB的表达。构建含ZEB1/2的3'-UTR序列荧光素酶报告基因质粒，与miR-200分子前体或抑制物共转染间皮细胞，以验证ZEB是其作用靶点。继而将miR-200分子前体或抑制物转染诱导转化的间皮细胞，观察其对ZEB1/2表达及EMT的影响。最后以miR-200分子前体或抑制物干预腹膜纤维化模型，体内证实miRNA对腹膜纤维化的调控。本研究拟证明miR-200通过调控ZEB1/ZEB2而影响EMT，为腹膜纤维化的治疗提出新思路。

腹膜透析相关腹膜纤维化是腹膜透析最常见的远期并发症，也是导致超滤衰竭及患者退出腹透最主要的原因。然而，其发生机制尚不明，仍无有效的治疗方法。microRNA是一组长度为22nt~的非编码RNA，参与了许多生理及病理过程的调控。本研究旨在探讨miR-200a在腹膜透析相关腹膜纤维化过程中的作用及机制。通过研究我们发现，在纤维化的大鼠腹膜组织中和TGF-beta1刺激的人腹膜间皮细胞内miR-200a的表达显著下降（2-3倍），同时间皮细胞表型蛋白E-cadherin的表达下降，肌成纤维细胞表型蛋白α-SMA及细胞外基质蛋白FN, ColⅠ的表达上调，结果提示miR-200a与腹膜纤维化及被认为是纤维化始动过程的EMT密切相关。于是我们通过在细胞内转染miR-200a mimics 和inhibitors上调及下调miR-200a的表达发现，上调miR-200a可抑制TGF-beta1诱导的腹膜间皮细胞EMT的发生，反之则促进该过程。由此确认了上述假设，即miR-200a在腹膜间皮细胞EMT的过程中发挥保护性作用。为验证ZEB1/2是否为miR-200a抑制腹膜间皮细胞EMT过程的下游靶基因，我们进行了荧光素酶报告实验，证实ZEB1/2的3’-UTR存在miR-200a的结合位点，miR-200a可显著调节ZEB1/2的表达。随后，我们在转染了miR-200a mimics的人腹膜间皮细胞中再次证实了miR-200a可下调ZEB1/2的表达，相反，抑制miR-200a的表达将上调ZEB1/2的表达。以上结果表明，miR-200a为腹膜间皮细胞EMT过程的保护性因子，它可通过作用其靶基因ZEB1/2发挥抑制或减弱TGF-beta1诱导的腹膜间皮细胞EMT的发生。提示miR-200a可能为治疗腹膜透析相关腹膜纤维化提供新的治疗思路和位点。