HuR和miR-222 竞争性调控E-cadherin表达和肠黏膜屏障功能的分子机制研究

Intestinal mucosal barrier prevents the diffusion of toxins, allergens, and pathogen from the lumen into the tissue. Disruption of this barrier occurs commonly in various pathological conditions. Epithelial cells of the intestine undergo a rapid cell-renewal and require sufficient and rapid supply of junctional proteins. Posttranscriptional regulation is one of the most rapid and efficient mechanisms on regulating the expression of target proteins, but its effect on junctional proteins remain to be elucidated. The integrity and normal function of the epithelial barrier depend on apical junctional complexes (AJCs) composing of different intercellular junctions including tight junctions (TJs) and adherens junctions (AJs). E-cadherin, the main component of AJs, is also found to be required for formation of other junctions such as TJs and desmosome. However, the mechanisms underlying its posttranscriptional regulation remain to be elucidated. There are several computationally predicted hits of the HuR and miR-222 motif in the E-cadherin mRNA, suggesting that the E-cadherin mRNA maybe a direct target of HuR and miR-222. Our preliminary experiments showed that both HuR and miR-222 bound to E-cadherin mRNA and modulated its expression. Based on these findings, we hypothesize that HuR promotes, and miR-222 inhibits E-cadherin expression, thus modulate the integrity of intestinal barrier. In order to test this hypothesis, firstly we will elucidate the molecular mechanisms of HuR and miR-222 on modulating the expression of E-cadherin. Secondly, we will examine the effect of HuR and miR-222 on the barrier function using monolayer epithelial cells model. Finally, we will determine whether in vivo expression of E-cadherin is modulated by miR-222 and HuR using a mouse model of inflammation. The successful completion of this project will lead to a better understanding of the regulatory mechanisms of the intestinal barrier function, and help to improve therapeutic approarches for the epithelial barrier dysfunction related disease.

快速更新的肠上皮需要大量构成肠黏膜屏障的连接蛋白，转录后调控可高效地调节蛋白表达，但对连接蛋白作用尚待阐明。我们曾报道RNA结合蛋白HuR和CUGBP1竞争性调控紧密连接蛋白Occludin。顶端连接复合体(AJCs)是肠粘膜屏障的结构基础，中间连接蛋白E-cadherin介导AJCs形成。序列分析提示E-cadherin mRNA上有多个潜在的RNA结合蛋白和微小RNA结合位点。预实验发现HuR和miR-222与E-cadherin mRNA结合并调控其表达。推测HuR促进/miR-222抑制E-cadherin 表达从而影响AJCs形成和肠黏膜通透性。为此，首先阐明HuR和miR-222调节E-cadherin蛋白表达的分子机制，以及它们之间的相互关系；然后分别在细胞和整体水平评估HuR和miR-222对肠黏膜屏障功能的调控及规律，阐明临床相关性，为预防和治疗肠屏障相关疾病提供新思路。

背景：肠屏障的调控机制是目前基础与临床研究的关键问题之一。鉴于肠上皮细胞间顶端连接复合体对于肠屏障功能最为重要，RNA 结合蛋白和microRNA如何相互协作，精准高效地调节连接蛋白的表达值得深入研究。.方法：体外利用Caco-2单层细胞模型研究HuR和miR-222对E-cadherin表达、顶端连接复合体形成及单层细胞通透性的影响的影响。利用炎症因子处理Caco-2细胞模拟炎症损伤肠道屏障，探究炎症因子对HuR和miR-222表达的影响。采用动物炎症模型来证实在体内发生炎症的情况下， HuR、miR-222及E-cadherin mRNA三者表达的变化以及对肠屏障通透性的影响。.结果： HuR与E-cadherin mRNA3’UTR3679-4300片段处存在物理性结合，结合后可通过促进翻译的方式在转录后水平上调E-cadherin表达；miR-222同样与E-cadherin mRNA存在物理性结合，结合位点位于3"UTR 3577-3597片断处，MS2实验结果表明miR-222可通过招募E-cadherin mRNA进入P-bodies的方式对其进行快速靶向降解，导致后者的翻译受阻表达降低；炎症因子TGF-β和IFN-γ对HuR表达具有抑制作用，造成HuR与E-cadherin mRNA的结合显著减少而miR-222与E-cadherin mRNA的结合显著增加，这一平衡的打破造成E-cadherin mRNA在翻译水平受阻，肠屏障通透性增高。在体内水平我们通过盲肠结扎合并穿孔模型诱发炎症，发现HuR表达降低而miR-222表达升高，两者表达的变化直接导致E-cadherin mRNA与HuR和miR-222的结合失衡，致使E-cadherin表达量显著降低。通过抑制miR-222可见E-cadherin表达抑制的情况显著减轻，表明miR-222确实是介导炎症因子损伤肠屏障的致病靶点之一；通过过表达HuR同样可部分恢复E-cadherin表达抑制的情况并纠正肠屏障损伤，表明HuR确实也是炎症因子损伤肠屏障的有效干预靶点之一； .结论：HuR 与miR-222竞争性调控 E-cadherin表达并影响顶端连接复合体的形成。炎症因子通过调控E-cadherin 表达改变肠屏障通透性的分子机制与HuR 和miR-222密切相关。