miR-21负向调控PTEN影响肠上皮-间质转化在克罗恩病肠纤维化发病中的作用及机制

Stricture caused by intestinal fibrosis is a serious problem in the clinical practice in crohn's disease, as there is no effective diagnostic and therapeutic method.Studies have shown that EMT involved in the formation of the fibrosis, miR-21 participate in the fibrosis process,PTEN is its target gene,MiR-21 and PTEN were involved in regulation of EMT.Our previous studies had shown that miR-21 and PTEN were associated with the pathogenesis of CD,Inhibition of PTEN downstream of PI3K/Akt/mTOR signaling pathway can reduce intestinal fibrosis.So we assume that:miR-21 up-regulate in CD intestinal fibrosis tissue, which combined with PTEN to inhibit PTEN expression,and the PI3K/Akt/mTOR signaling pathway signal was increased, thereby promoting the EMT,and the intestinal fibrosis promoted. To confirm this hypothesis,we will clarify miR-21 regulates EMT and participate in CD intestinal fibrosis formation by inhibiting PTEN including both in vitro and vivo tests. It would provide a new theoretical basis for the molecular mechanism of regulation of intestinal fibrosis by miRNAs.

肠纤维性狭窄是克罗恩病（CD）临床工作中的难点，目前缺乏有效诊治措施。研究显示EMT参与纤维化形成，miR-21参与纤维化的调控，PTEN是miR-21靶基因，miR-21、PTEN参与EMT的调控。我们前期研究发现，miR-21、PTEN与CD的发病相关，抑制PTEN下游PI3K/Akt/mTOR信号通路可减轻肠道纤维化。因此我们假设：CD患者miR-21表达上调，与PTEN结合抑制PTEN表达，上调PI3K/Akt/mTOR信号通路，继而促进EMT发生，从而促进肠道纤维化形成，最终导致肠狭窄。本研究拟结合体内、外实验，采用Real-time PCR、Western blotting、siRNA、Luciferase报告基因技术等技术，进一步证实miR-21可通过抑制PTEN，调控EMT，参与CD肠道纤维化形成，期望通过本研究为miRNAs调控肠道纤维化的分子机制提供新的理论依据。

背景：克罗恩病肠纤维化的形成是一种复杂、动态的病理生理过程，最终导致肠梗阻（CD 的严重并发症）。先前的研究表明miR-21调节器官的上皮-间质转化（EMT）和纤维化。本研究旨在探讨miR-21在克罗恩病（CD）相关肠纤维化EMT过程中的作用及机制。.方法：取CD患者和非CD患者的纤维化和非纤维化小肠的组织活检；用TNBS建立慢性肠纤维化模型，用miR-21拮抗剂治疗；用miR-21模拟物或抑制剂转染人肠上皮细胞NCM460。检测PTEN和mTOR、EMT相关标志物的表达以及结肠炎和纤维化的严重程度。.结果：与对照组相比，miR-21在纤维狭窄的CD患者肠组织中显著上调，随后PTEN表达降低，EMT标志物和mTOR表达增加，MMP9（基质金属蛋白酶9）/TIMP1（基质金属蛋白酶组织抑制剂1）比例失衡。miR-21下调NCM460细胞中PTEN的表达，上调mTOR信号。此外，细胞转染下调miR-21在体外降低了EMT。动物实验用拮抗剂抑制miR-21，通过抑制EMT和平衡MMPs/TIMPs，在体内逆转TNBS诱导的肠纤维化。.结论：miR-21通过靶向PTEN和mTOR参与肠纤维化过程中的EMT，抑制miR-21通过调节细胞外基质（ECM）重塑减轻肠纤维化。我们的研究结果表明，miR-21是治疗CD纤维化并发症的潜在新靶点。