非酒精性脂肪肝病靶分子Mst1相关调节miRNA的筛选及鉴定  

Screening of Mst1-related miRNAs Targeting Nonalcoholic Fatty Liver Disease

作　　者：李雨涵 栾延松 祁慧 李建宁 宋辉[1,3] 杨怡 LI Yuhan;LUAN Yansong;QI Hui;LI Jianning;SONG Hui;YANG Yi(Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Ningxia Medical University, Yinchuan 750004,China;Institute of Endocrinology,Ningxia Medical University, Yinchuan 750004,China;Department of Cell Biology and Genetics,School of Basic Medical Science,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院生物化学与分子生物学系,银川750004 [2]宁夏医科大学内分泌学研究所,银川750004 [3]宁夏医科大学基础医学院细胞生物学与遗传学系,银川750004

出　　处：《宁夏医科大学学报》2019年第3期224-229,235,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81670798);2017"西部一流"建设项目开放课题

摘　　要：目的探讨非酒精性脂肪肝病(nonalcoholic fatty liver disease,NAFDL)中Mst1的调控机制。方法利用TargetScan、miRDB等数据库从脂肪源性miRNAs中筛选出可能靶向鼠源Mst1 3’UTR区的miRNAs;通过Western blot初步验证miRNA对Mst1的负性调控效应;采用双荧光素酶报告基因技术确认miRNAs与Mst1的靶向关系;棕榈酸(PA)诱导AML-12小鼠肝脏细胞株成功构建NAFLD细胞模型,证实NAFLD模型中miRNAs通过调控Mst1影响NAFLD脂代谢进程。结果 Western blot实验显示miR-199a-5p、miR-342-3p和miR-691干预后Mst1的蛋白表达水平下调,Luciferases实验证明miR-199a-5p、miR-342-3p和miR-691存在直接靶向Mst1的效应,在NAFLD细胞模型中进一步证实miR-199a-5p、miR-342-3p和miR-691影响肝脏脂代谢进程。由此获得有潜在价值的负性调控miRNAs:miR-199a-5p、miR-342-3p和miR-691。结论 miR-199a-5p、miR-342-3p和miR-691能够靶向并负性调控小鼠肝脏Mst1并参与NAFLD脂代谢进程。Objective Using Mst1,a key molecule of nonalcoholic fatty liver disease(NAFDL),as a study object,the bioinformatics methods were used to predict the specific regulation relationship between the mice liver Mst1 and the fatty circulating miRNAs(mmu-miRNAs).It will provide a basis for discussing the regulation mechanism of Mst1 in NAFLD.Methods Target Scan and microRNADB databases were used to screen microRNAs that might target the Mst1 3'UTR region of mouse origin from adipose-derived microRNAs.The negative regulatory effect of microRNAs on Mst1 was preliminarily verified by Western blot.The targeting relationship between microRNAs and Mst1 was confirmed by double luciferase reporter gene technology,and palmitic acid(PA)induced the successful construction of NAFLD cell model in AML-12 mouse liver cell lines,confirming that miRNAs in the NAFLD model affect the lipid metabolism process of NAFLD by regulating Mst1.Results Western blot analysis showed that the expression of Mst1 was down-regulated by miR-199a-5p,miR-342-3p and miR-691,and Luciferase test proved that miR-199a-5p,miR-342-3p and miR-691 had the effect of directly targeting Mst1,which was further confirmed in the NAFLD cell model that miR-199a-5p,miR-342-3p and miR-691 could affect liver lipid metabolism.Finally,we obtained potentially valuable negative regulatory miRNAs:miR-199a-5p,miR-342-3p and miR-691.Conclusion miR-199a-5p,miR-342-3p and miR-691 can target and negatively regulate Mst1 in mouse liver and participate in the process of lipid metabolism in NAFLD.

关 键 词：Mst1 非酒精性脂肪肝病 生物信息学 MIRNAS 

分 类 号：R575[医药卫生—消化系统]