miR-122通过SELENBP1调控克罗恩病肠粘膜氧化应激及炎症反应

Crohn's disease, a subtype of inflammatory bowel disease with unknown origin, serious morbidity, high relapse rate, increased risk of gastrointestinal malignancy as well as difficulty for therapy, imposes huge healthy burden on individuals. Oxidative stress is one of the most important pathological processes. MiR-122 is involved in oxidative stress, but the exact mechanisms on modulation remain to be explored. Our previous studies have found that SELENBP1, a target of miR-122，participated in oxidative stress in intestinal epithelial cells in Crohn's disease. And it has been reported that SELENBP1 can act as a pro-oxidant. In this subject, we first observed the regulatory role of miR-122 on SELENBP1 expression by collecting mucosal biopsy specimens from patients with Crohn's disease and building oxidative stress model of human intestinal epithelial cells. And then, we analysed whether miR-122 played an important role in modulating oxidative stress and inflammation via SELENBP1 in oxidative stress model of human intestinal epithelial cells and TNBS-induced colitis mice model.we intervened miR-122 in the TNBS-induced colitis model to elucidate that miR-122 played a protective role in intestinal inflammation by modulating oxidative stress and inflammation through SELENBP1. Finally, we explored the mechanism of the abnormal levels of miR-122 in Crohn's disease samples and determined whether it was due to hypermethylation. This research will provide the theoretical and experimental basis to explore new therapeutic targets for Crohn's disease.

克罗恩病是炎症性肠病的一种亚型，目前病因未明，病情较重，有较高复发率，也有一定的癌变风险，治疗困难，严重危害人类健康。氧化应激在其发病机制中发挥重要作用，miR-122参与氧化应激，但其确切的调节机制尚不清楚。我们的前期研究已经发现miR-122的靶分子SELENBP1参与克罗恩病肠上皮细胞的氧化应激，也有文献证实SELENBP1作为亲氧化剂对氧化应激有促进作用。本课题拟通过收集克罗恩病病人肠粘膜活检组织，并构建人肠上皮细胞的氧化应激模型，研究miR-122对SELENBP1的调控机制；分析在肠上皮细胞的氧化应激模型和三硝基苯磺酸（TNBS）诱导的小鼠结肠炎模型中miR-122是否能通过SELENBP1调节氧化应激和炎症反应，从而在肠道炎症中发挥保护作用；研究miR-122在克罗恩病肠粘膜中的表达异常是否由其启动子甲基化引起。课题研究将为寻找克罗恩病的新的治疗靶点提供理论及实验基础。

克罗恩病是炎症性肠病的一种亚型，病因未明，其多发于青年人群，给社会、家庭及个人带来极大的负担。MicroRNA（miRNA）表达异常导致了多种疾病的发展和进展，包括克罗恩病（CD）。然而，CD中miRNA的作用及机制尚不清楚。我们的前期研究发现miR-122的靶分子硒结合蛋白1（SELENBP1，SBP1）SELENBP1参与CD肠上皮细胞的氧化应激。本文中应用正常志愿者和CD患者的结肠组织样本， 2,4,6-三硝基苯磺酸（TNBS）诱导的急性小鼠结肠炎模型和由H2O2诱导的HT-29细胞氧化应激模型来探讨氧化应激对miR-122和SELENBP1的表达，p65NF-κB信号转导和DNA甲基化水平的影响。通过实验，我们发现SBP1主要位于肠上皮细胞，并且在活动性CD患者中表达量显著增加。SBP1蛋白是miR-122的靶基因。在TNBS诱导的结肠炎或氧化应激刺激时，miR-122表达下调，而SBP1表达上调。 pre-miR-122或siRNA SBP1（si-SBP1）治疗可以缓解TNBS诱导的急性结肠炎和H2O2诱导的氧化应激。pre-miR-122和si-SBP1的共同作用可以使该治疗效果明显增强。此外，pre-miR-122和si-SBP1可以通过诱导IκBα的磷酸化来激活p65NF-κB信号通路，从而加重结肠炎症反应。miR-122的启动子区域中的CpG岛的亚硫酸氢盐测序显示在氧化应激状态下其启动子区域甲基化明显增加。用5'-AZA（DNA去甲基化剂）处理细胞可显著增加miR-122的表达。我们的研究结果表明，氧化应激诱导miR-122启动子区域的甲基化，进而使其靶蛋白SBP1的表达减少，激活p65NF-κB信号转导，加重了TNBS诱导的结肠炎，可能促进CD的进展。