miR-375对人类诱导多能干细胞向胰岛素分泌细胞分化的影响  

Effect of miR-375 on differentiation of human induced pluripotent stem cells into insulin-producing cells

作　　者：邓春艳[1] 王晗月 李宁 蒋豆蔻 俞丽娜[1] 欧阳志斌[1] 李富荣[2] DENG Chun-yan;Wang Han-yue;LI Ning;JIANG Dou-kou;YU Li-na;OUYANG Zhi-bin;LI Fu-rong(Biotherapy Room,The Second Clinical Medical College of Jinan University(Shenzhen People’s Hospi⁃tal),Shenzhen 518020,China;Translational Medicine Collaborative Innovation Center,Shenzhen Key Laboratory of Stem Cell Re⁃search and Clinical Transformation,The Second Clinical Medical College of Jinan University(Shenzhen People’s Hospi⁃tal),Shenzhen 518020,China;Department of Pathophysiology,School of Medicine,Jinan University,Guangzhou 510632,China)

机构地区：[1]暨南大学第二临床医学院(深圳市人民医院)生物治疗室,广东深圳518020 [2]暨南大学第二临床医学院(深圳市人民医院)转化医学协同创新中心,深圳市干细胞研究与临床转化重点实验室,广东深圳518020 [3]暨南大学基础医学院病理生理学系,广东广州510632

出　　处：《中国病理生理杂志》2020年第5期877-885,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81670702);广东省自然科学基金资助项目(No.2015A030313762)。

摘　　要：目的:探讨微小RNA-375(miR-375)在调控人类诱导多能干细胞(hiPSCs)分化为胰岛素分泌细胞(IPCs)过程中的作用及可能机制。方法:构建miR-375过表达的慢病毒载体,转染hiPSCs获得miR-375稳定表达的miR-375-hiPSCs,体外诱导其定向分化为IPCs,采用real-time PCR、流式细胞术及ELISA等方法对其标志性基因、分化效率、胰岛素和C肽释放量等进行检测;生物信息学结合萤光素酶报告基因实验预测并验证miR-375的靶基因,real-time PCR及Western blot检测靶基因的表达。结果:过表达miR-375可上调胰岛β细胞标志性转录因子HNF4α、MafA、Pdx1、Pax6、Nkx6.1、glucokinase和insulin的表达,分化效率由对照组的22.3%提高至38.6%;高糖刺激后胰岛素释放量由成年胰岛细胞分泌量的1/8提高到1/5;过表达miR-375可影响靶基因REST和WNT5A的蛋白翻译水平。结论:miR-375可通过干扰靶基因REST和WNT5A的翻译水平,开启胰岛素分泌相关特异性基因的表达,从而促进hiPSCs向IPCs定向分化。AIM:To explore the role of microRNA-375(miR-375)in the process of human induced pluripotent stem cells(hiPSCs)differentiating into insulin-producing cells(IPCs)and its possible mechanism.METHODS:The lentiviral vector with miR-375 over-expression was constructed and miR-375-hiPSCs with stable expression of miR-375 were induced to differentiate into IPCs in vitro.The marker genes,differentiation efficiency,and insulin and C-peptide release were detected,the target genes of miR-375 were predicted and verified by bioinformatics combined with luciferase reporter assay,and the expression of target genes was detected by real-time PCR and Western blot.RESULTS:Over-expression of miR-375 up-regulated the expression of islet β cell markers HNF4α,MafA,Pdx1,Pax6,Nkx6.1,glucokinase and insulin in the hiPSCs,and the efficiency of differentiation into IPCs was increased from 22.3% of control group to 38.6%.After high glucose stimulation,the insulin release was increased from 1/8 to 1/5 of adult islet secretion.REST and WNT5 A were the target genes involved in miR-375 regulating the differentiation of hiPSCs into IPCs.Over-expression of miR-375 affected the expression of REST and WNT5 A at protein translational level rather than mRNA transcriptional level.CONCLUSION:miR-375 interferes with the protein expression of target genes REST and WNT5 A,and then turns on the expression of specific genes related to insulin secretion,thus promoting the directional differentiation of hiPSCs cells into IPCs.

关 键 词：人类诱导多能干细胞 微小RNA-375 胰岛素分泌细胞 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R363.2[医药卫生—基础医学]