miR-7/miR-375在炎症性肠病中对TFF3的调控及其对肠上皮屏障的影响

The research is focused on the role of miR-7/miR-375 in regulating trefoil factor family 3 (TFF3) on intestinal epithelial barrier during inflammatory bowel disease（IBD）. miR-7/miR-375, abnormally over-expressed in IBD,are predicted to be the goal miRNAs to regulate TFF3 assistanted by bioinformatics. Using the methods of Luciferase assay, western blot, gene transfection, SiRNA, quantitative PCR, miRNA inhibitor and other molecular biology techniques，adopting the intestinal epithelial barrier model established by Caco-2 cell in vitro and model of IBD mice induced by DSS，the aims are:1）To definite the binding of miR-7/miR-375 and TFF3; 2) To study the post-transcriptional control of miR-7/miR-375 on TFF3; 3) To investigate the effect of miR-7/miR-375 on intestinal epithelial barrier through regulating TFF3; 4) To observe the protection of TFF3 on mucosal barrier if inhibiting miR-7/miR-375. The study will prove that miR-7/miR-375 is the upstream regulating foctor of TFF3.Inhibiting the over-expression of miR-7/miR-375 will promote the intestinal mucosal barrier protect function of TFF3, then to offer a new proposal for IBD.

研究miR-7/miR-375对炎症性肠病（IBD）中屏障保护因子-肠三叶因子（TFF3）的调控作用。前期实验预测到IBD中高表达的miR-7/miR-375可能是调控TFF3的目标miRNAs，应用荧光素酶实验、免疫印迹、基因转染、SiRNA、定量PCR、miRNA inhibitor等技术，采用Caco-2体外肠屏障模型及IBD小鼠模型，研究：1）确定miR-7/miR-375与TFF3结合;2）探讨miR-7/miR-375对TFF3的转录后调控作用;3）研究miR-7/miR-375调控TFF3表达进而影响肠屏障功能的机制;4）抑制miR-7/miR-375的表达观察TFF3对IBD模型的屏障保护作用。试图证实miR-7 /miR-375是TFF3的上游调控因子,抑制miR-7 /miR-375的高表达可以发挥TFF3的肠黏膜屏障修复功能，为IBD的靶向治疗提供新的思路。

背景：肠三叶因子（TFF3）在炎症性肠病（IBD）中肠屏障保护中起到重要作用，本文旨在找出TFF3的上游调控microRNA，为IBD的靶向治疗提供新的思路。.方法：应用荧光素酶实验、免疫印迹、基因转染、SiRNA、定量PCR、miRNAinhibitor等技术，在LA174T细胞及IBD小鼠模型中评估miR-7对TFF3的调控作用。 结果：前期实验预测到IBD中高表达的miR-7/miR-375可能是调控TFF3的目标miRNAs，经过荧光素酶报告基因检测，我们最终确定miR-7为TFF3的靶向调控microRNA。经过在LS174T细胞中转染miR-7 mimic，进行western blot检测证实miR-7可以在转录后调控水平上影响TFF3蛋白的表达。同时我们检测了miR-7调控TFF3后对LS174T 细胞增殖和迁移能力的影响，均显示miR-7可以通过抑制TFF3的表达进而抑制细胞的增殖和迁移能力，且该过程中涉及到对PI3K/Akt通路的调控。在动物模型及IBD患者肠组织中我们均检测到miR-7及TFF3存在差异性表达。在动物模型中通过观察动物存活率、肠道形态改变、HE染色下病理改变、miR-7及TFF3基因表达情况、免疫组化和Western Blot 检测TFF3蛋白表达，结果显示在miR-7antagomir抑制miR-7基因表达的同时可以促进TFF3基因及蛋白的表达，miR-7antagomir可以修复TNBS对结肠炎小鼠的肠道损伤。结论：miR-7对TFF3有靶向调控作用，抑制miR-7的表达可以进一步发挥TFF3在IBD疾病中屏障保护作用。