miR-200b对炎症性肠病肠上皮屏障功能的调控及分子机制

Intestinal epithelial barrier injury is one of the most important mechanisms of inflammatory bowel disease ( IBD). Therefore, maintaining the intestinal barrier function has potential roles in improving pathophysiological and clinical outcomes of IBD. Through the prophase research, our group found that miR-200b decreased significantly in inflamed mucosa of IBD. With endogenous over expression of miR-200b, the proliferation of IEC-6 cells increased significantly. Meanwhile, epithelial-mesenchymal transition(EMT) induced by transforming growth factor-beta1(TGF-β1) in IEC-6 was inhibited. The results indicated that miR-200b could enhance intestinal epithelial barrier function by transcellular pathway. In order to further research the role of miR-200b on the intestinal epithelial barrier fuction mediated by paracellular pathway and relevant molecular mechanisms, follow-up study will be focused on the following issues: ①To investigate the expression of miR-200b , of the genes associated with tight junction as well as their correlation in IBD; ②To validate the protective role of miR-200b on the intestinal epithelial barrier function in vivo and in vitro; ③To explore the role of miR-200b-Myosin Light Chain Kinase(MLCK) signal pathway in the intestinal mucosal barrier injury in IBD. Above mentioned researches can help to clarify the molecular mechanism of intestinal barrier function injury in IBD and eventually provide theoretical basis and potential therapeutic targets for IBD.

肠上皮屏障损伤是炎症性肠病（IBD）的重要发病机制，保护肠上皮屏障功能可改善IBD预后。课题组前期工作发现，IBD受损肠黏膜组织中miR-200b表达下降，过表达miR-200b则可促进肠上皮细胞增殖，抑制转化生长因子（TGF-β1）诱导的上皮-间质转分化（EMT），提示miR-200b可通过跨细胞途径改善肠上皮屏障功能。为进一步研究miR-200b对细胞旁途径介导的IBD肠上皮屏障功能的调控作用及其分子机制，我们拟开展如下工作：①研究IBD时miR-200b与肠上皮细胞间紧密连接相关基因的表达及关联性；②在细胞与动物模型上验证miR-200b对IBD肠上皮屏障功能的改善作用；③探讨miR-200b-肌球蛋白轻链激酶（MLCK）信号通路在肠上皮屏障功能调控中的作用。项目完成可补充和完善IBD肠上皮屏障功能损伤的分子机制，为寻求可能的分子靶点奠定理论基础。

本课题研究发现，miR-200b在IBD肠上皮分化及屏障功能调控中发挥着不可或缺的作用，其机制与促进肠上皮细胞增殖、抑制肠上皮细胞EMT过程、调控靶基因MLCK表达、抑制紧密连接蛋白丢失有关，从而分别通过跨上皮途径和细胞旁途径影响肠道上皮通透性，改善肠上皮屏障功能。现总结如下：1.活动期IBD患者miR-200b表达明显下调，与肠上皮标志E钙黏蛋白明显下调和肠上皮细胞增殖凋亡失衡呈明显正相关；2. TGF-β诱导的肠上皮-间质转换细胞模型中，miR-200b表达受抑与E-钙粘蛋白表达下调明显相关，TGF-β刺激使上皮细胞之间连接松散甚至消失，而外源给予miR-200b可显著改善肠上皮紧密连接功能减轻屏障损伤；3.在TNF-α诱导的肠粘膜屏障损伤细胞模型中，过表达下调的miR-200b可明显改善TNF-α造成的跨膜电阻抗的下降，降低肠上皮细胞通透性，一方面靶向调控MLCK抑制MLCK/P-MLC信号通路来修复TNFα造成的肠上皮紧密连接损伤，另一方面靶向调控c-JUN抑制JNK、c-JUN/AP-1信号通路减少趋化因子IL-8的成熟和分泌；4.在以TNF-α模拟构建的IBD肠上皮屏障损伤细胞模型中，自噬降解过程受抑是导致紧密连接蛋白Claudin-2上调和肠屏障损伤的重要原因，该过程可能与溶酶体酸性环境破坏相关，而mTOR抑制剂PP242可激活自噬流，缓解上述Claudin-2和上皮屏障功能的改变，提示mTOR通路及下游自噬可能成为IBD治疗新靶点；5.在以LPS模拟构建的IBD肠上皮细胞损伤模型中，TLR4-MyD88-ERK MAPK信号通路的异常活化激活mTOR，并通过进一步活化NFκB通路联合抑制下游自噬进程，加剧IBD肠上皮细胞炎性细胞因子的分泌和氧化应激损伤；6.自噬相关蛋白Atg16L1缺陷可引起P62在肠上皮细胞中聚集，并通过P38 MAPK信号通路促进IL-1β诱导的炎性细胞因子IL-8的表达增加。以上研究结果从miRNA调控肠上皮细胞旁途径的新视角阐释了炎症性肠病肠屏障损伤的机理，拓展了对炎症性肠病肠上皮屏障损伤的机制认识，靶向miR-200b可为炎症性肠病临床干预策略提出新的见解。