miR-34c在胃肠运动功能障碍性疾病发生机制中的作用研究

Interstitial cells of Cajal (ICCs) serve as the pace maker of spontaneous rhythmic activities in gastrointestinal tract. The quantitative decrease and structural abnormality of ICCs are involved in the genesis and development of gastrointestinal motility disorders. It has been confirmed that the proliferation and survival of ICCs closely depend on the KIT/SCF signal molecules. Our group found that SCF expression in dilated colon of mice was significantly reduced. MicroRNA array revealed an up-regulation of miR-34c which is related to the SCF mRNA expression. However, the molecular mechanism of the regulation of miR-34c on SCF mRNA has been not well known yet. Therefore, we will use specimen of gastrointestinal motility disorder patients, cell culture and Pax6m/- mice to: ① identify the hyper-expression of miR-34c and hypo-expression of SCF in gastrointestinal motility disorder patients; ② up- and down-regulate the miR-34c so as to reveal the regulation of miR-34c on SCF expession in cultured smooth muscle cells; ③ construct a luciferase report to figure out the mediating mechanism of miR-34c on SCF mRNA; ④ observe the effect of LNA-antimiR on the SCF expression and gastrointestinal motility of mice with ectocolon. This proposal aims to provide new evidence on the mechanism of gastrointestinal motility disorders and the treatment of LNA-antimiR with gastrointestinal motility disorders.

已知Cajal间质细胞（ICCs）的数量减少和细胞网络稀疏与许多胃肠运动功能障碍性疾病密切相关，ICCs的增殖和存活受KIT/SCF信号分子的调控。本课题组发现：扩张肠管平滑肌表达SCF明显降低，芯片分析表明miR-34c明显上调，但miR-34c是否能够调节SCF表达尚不清楚。因此，本项目拟利用临床胃肠运动障碍性疾病患者的术后标本及细胞培养和动物实验：①明确胃肠运动障碍性疾病患者是否存在miR-34c的高表达和SCF的低表达；②培养平滑肌细胞，通过上调和下调miR-34c证明其对SCF表达的调控作用；③构建荧光素酶报告基因系统，明确miR-34c调控SCF mRNA的分子机制；④利用结肠扩张小鼠观察LNA-antimiR能否增加SCF的表达并改善胃肠运动功能障碍。本项目期望为深入理解胃肠运动障碍性疾病的发生机制提供新的实验依据，并为临床应用LNA-antimiR治疗相关疾病开辟新的方法。

胃肠运动功能障碍性疾病（Gastrointestinal motility disorders, GMDs），如糖尿病胃轻瘫、慢传输性便秘、先天性巨结肠病和假性肠梗阻等，为消化系统常见病和多发病。现已证实胃肠运动起搏细胞——Cajal间质细胞（Interstitial cell of Cajal, ICC）的减少和细胞网络的破坏是GMDs发生发展的重要原因之一，ICC的增殖、存活、表型和功能维持依赖于SCF/KIT信号的活化，所以，任何原因引起的SCF或KIT的减少均可导致ICC的减少和功能异常。大量研究表明，胃肠扩张是GMDs的常见病理改变，本课题组亦发现在糖尿病胃轻瘫小鼠模型中，扩张的胃壁内ICC减少之前，SCF的表达已发生明显地降低，据此我们推测持续胃肠扩张可能通过抑制SCF的表达引起ICCs的减少，最终导致GMDs的发生。本研究利用慢性结肠扩张小鼠模型、糖尿病胃肠轻瘫小鼠模型、先天性巨结肠患儿术后标本，以及离体结肠平滑肌细胞牵拉模型，深入研究持续扩张（牵张）所致胃肠平滑肌SCF表达下降和ICC减少的分子机制。结果表明：持续牵拉可使平滑肌细胞的钙离子通道开放，引起细胞内钙超载，进而通过激活ERK-c-Jun信号分子轴上调miR-34c的表达，致使miR-34c的靶基因SCF表达降低和ICC减少，最终引起胃肠运动功能障碍，上述结果可能将为临床GMDs提供新的诊断参考指标和治疗靶点。