人肝脏MST1相关miRNA的筛选、质粒构建与验证  

Screening,Plasmid Construction and Validation of Human Liver MST1-related miRNA

作　　者：栾延松 李雨涵 祁慧 李建宁 宋辉[1,3] 杨怡 LUAN Yansong;LI Yuhan;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5期433-439,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：目的构建与人肝脏丝氨酸/苏氨酸蛋白激酶4(MST1)表达相关的人源性miRNAs(hsa-miRNAs)的重组质粒并进行初步验证,为深入探讨其在肝脏脂代谢途径中的可能作用奠定基础。方法通过TargetScan、miRDB等数据库并参照文献提供的数据库信息筛选出可能靶向人肝脏MST1的脂肪组织来源miRNAs,并通过Westernblot和双荧光素酶报告基因实验进行筛选;进而改建miRNAs过表达载体骨架,并将筛选得到的miRNAs构建过表达载体,获得miRNA的过表达重组质粒;采用棕榈酸(PA)孵育人肝癌细胞株HepG2构建非酒精性脂肪肝(NAFLD)细胞模型,并验证miRNAs对NAFLD细胞模型内MST1表达的调控效应。结果成功筛选出了靶向人肝脏MST1的miRNA:hsa-miR-448和hsa-miR-518a-3p,并构建了miRNAs过表达载体,在NAFLD细胞模型中验证了hsa-miR-448和hsa-miR-518a-3p过表达对脂代谢的影响。结论初步筛选并验证了调节人肝脏MST1表达的人源性miRNAs。证实hsa-miR-448和hsa-miR-518a-3p过表达通过下调MST1及相关信号通路的关键蛋白进而影响NAFLD的进程。Objective To construct and validate the recombinant plasmid of human hepatic serine/threonine protein kinase 4(MST1)related human microRNAs(hsa-microRNAs),and to lay a foundation for further exploring its possible role in liver lipid metabolism.Methods The adipose tissue-derived miRNAs that might be targeted to human liver MST1 were screened by database data such as TargetScan and miRDB and referenced by the literature,and screened by Western blot and dual luciferase reporter gene assay;then the miRNAs overexpression vector backbone was modified.The selected miRNAs were constructed into an overexpression vector to obtain an miRNA overexpressing recombinant plasmid;the human hepatoma cell line HepG2 was incubated with palmitic acid(PA)to construct a nonalcoholic fatty liver(NAFLD)cell model,and the miRNAs were verified to be NAFLD cells.The regulatory effect of MST1 expression in the model was verified.Results Comprehensive analysis screened and validated human miRNAs that regulate human liver MST1 expression:hsa-miR-448 and hsa-miR-518a-3p.It was confirmed that hsa-miR-448 and hsa-miR-518a-3p affect the progression of NAFLD by down-regulating key proteins of MST1 and related signaling pathways.Conclusion Human miRNAs capable of regulating MST1 expression in human liver and affecting lipid metabolism of NAFLD were initially screened and validated.

关 键 词：丝氨酸/苏氨酸蛋白激酶4 非酒精性脂肪肝病 MIRNA 

分 类 号：R575.5[医药卫生—消化系统]