TM6SF2基因E167K变异与非酒精性脂肪肝相关性及其机制研究

Based on our previous liver eQTL study, we found that TM6SF2 gene E167K variant was associated with Non-alcoholic Fatty Liver Disease (NAFLD). In the further experiment, we found that in E167K variant cells, TM6SF2 protein was reduced, cholesterol was accumulated while the secretion of VLDL was blocked. After analysis on protein structure and the latest research progress, we speculated that E167K variant could change the protein structure and promote degradation of TM6SF2, reduce the cholesterol transport and block the synthesis of VLDL, so as to affect the transport of lipid outwards the liver cells and induce to NAFLD. There is a signal pathway: E167K variant→TM6SF2 degradation→cholesterol transport disorder→reduction of VLDL secretion→NAFLD. Pirola found that TM6SF2 was associated with the content of VLDL in peripheral blood, which implied the correctness of our speculation. This study aims to study the degradation mechanism of TM6SF2 through NMR and DXMS, to clarify the signal pathway through which cholesterol transport disorder affects VLDL secretion with protein spectrum and GST Pull-down, and to explore the feasibility of Crispr correcting E167K and improving NAFLD. It is expected to elucidate the molecular mechanism of E167K variant inducing NAFLD, and to provide potential targets for disease warning and treatment.

在课题组肝脏eQTL研究基础上，申请人发现TM6SF2基因E167K变异人群非酒精性脂肪肝（NAFLD）发病率升高。进一步实验发现E167K变异细胞TM6SF2蛋白含量下降，胆固醇蓄积伴VLDL分泌减少。结合生物信息学分析，推测E167K变异促进TM6SF2蛋白降解，减少胆固醇转运而阻碍VLDL合成，影响脂质向肝细胞外运输诱导NAFLD。存在E167K变异→TM6SF2降解→胆固醇转运障碍→VLDL分泌减少→NAFLD的信号途径。Pirola发现TM6SF2与外周血VLDL含量相关，提示我们推测的正确性。为验证该科学假设，本课题①通过NMR、DXMS研究TM6SF2降解机制；②蛋白质谱、GST Pull-down阐明胆固醇转运障碍影响VLDL分泌的信号通路；③探索Crispr纠正E167K改善NAFLD的可行性。可望阐明E167K变异诱导NAFLD的分子机制，为疾病预警及治疗提供潜在靶点。

近年来中国人群非酒精性脂肪肝（NAFLD）的发病率显著上升， 急需加强NAFLD的病因及机制研究。我们前期发现TM6SF2基因E167K变异人群NAFLD发病率升高，因而本课题开展了TM6SF2基因E167K变异与非酒精性脂肪肝相关性及其机制研究。通过实验我们发现：1.TM6SF2基因在体内外NAFLD模型中以及NAFLD患者肝脏中的反馈性的表达上调。2.TM6SF2基因E167K突变相较于野生型TM6SF2降解加速，且可能与溶酶体降解途径相关。3.TM6SF2蛋白与NPC1蛋白两者不存在直接相互作用，但是TM6SF2会影响NPC1表达。4.在NAFLD疾病中TM6SF2基因表达上调可促进细胞内脂肪酸代谢，降低细胞内的脂质积累，改善炎症反应，表明TM6SF2表达上调在NAFLD疾病中对肝脏起保护作用。5.TM6SF2基因E167K突变引起的TM6SF2蛋白降解加速，且通过溶酶体依赖途径降解。6.TM6SF2基因E167K突变引起的TM6SF2蛋白缺乏会导致其对肝脏的保护作用消失，引起C57BL/6J小鼠肝脏脂质沉积增加并且加重肝脏炎症反应。7.TM6SF2蛋白缺乏会导致细胞内存在异常脂肪酸代谢现象，其特点是脂肪酸摄取增多以及从头和成途径增强，并伴有脂肪酸β-氧化水平下降。8.根据其引起的异常脂肪酸代谢现象，使用ACC抑制剂（MK-4074）治疗可减轻TM6SF2蛋白缺乏所引起的NAFLD表型。研究表明E167K突变导致TM6SF2下调或缺失，通过多种途径促进NAFLD的发生。该研究对于进一步理解NAFLD的发生机制，为NAFLD易感人群筛查、NAFLD的诊断及治疗提供了新思路和实验依据。此外，课题组还研究了SAMM50基因多态性与NAFLD的相关性及其潜在机制。