目的：在小鼠胰岛β细胞系MIN6细胞中分析miRNA⁃24、 miRNA⁃34a和miRNA⁃375对Neurod1／BETA2的表达调控以及作用方式。方法设计并合成针对小鼠Neurod1基因3′非翻译区（3′untranslated re⁃gion，3′UTR）序列的引物，克隆于pMIR⁃REPORTTM荧光素酶报告基因载体中；用miRanda软件预测可能调控Neurod1表达的miRNAs，挑选在β细胞中有相关功能研究的miRNAs并委托公司合成pre⁃miRNAs；脂质体法转染至MIN6细胞中，荧光素酶报告基因技术分析这些miRNA对Neurod1的作用；定量RT⁃PCR分析Neurod1的mRNA水平； Western 印迹检测Neurod1的蛋白表达水平； GSIS检测干扰Neurod1后MIN6细胞的功能。结果成功制备小鼠Neurod1基因的3′UTR荧光素酶报告基因载体；选取了预测在Neurod1的3′UTR有互补结合位点的3个miRNAs （miRNA⁃24、 miR⁃34a和miR⁃375），并挑选一个没有互补结合位点的miRNA （miR⁃29a）作为阴性对照；在MIN6细胞中过表达pre⁃miRNA⁃24、 pre⁃miR⁃34a和pre⁃miR⁃375能够抑制Neurod1基因3′UTR荧光素酶报告基因的活性，而过表达阴性对照 miRNA⁃29a则不能抑制其活性；pre⁃miRNA⁃24、 pre⁃miR⁃34a和pre⁃miR⁃375还可以不同程度抑制Neurod1的蛋白表达；在MIN6细胞中干扰Neurod1蛋白表达能够降低细胞的GSIS功能。结论 miRNA⁃24、 miR⁃34a和miR⁃375能够抑制小鼠胰岛β细胞系MIN6细胞中Neurod1的蛋白表达。%Objective To analyze whether miRNA⁃24 , miRNA⁃34 a and miRNA⁃375 could regulate Neurod1/BETA2 expression in pancreaticβ⁃cell line MIN6 cells�Methods The primers of Neurod1 3′UTR template DNA sequences were designed, synthesized, and cloned into Luciferase reporter vector pMIR⁃REPORTTM�MiRanda software was used to predict potential miRNAs regula⁃ting Neurod1 , some of which were selected to be verified experimentally�Dual luciferase reporter gene system was used to analyze the effect of miRNAs against Neurod1 3′UTR�Quantitative RT⁃PCR was performed to analyze mRNA level of Neurod1 after miRNA transfection�Western blot assay was used to evaluate the protein level of Neurod1�Results The luciferase reporter vector pMIR⁃Neu⁃rod1⁃3′UTR was constructed and sequenced�According to miRanda software, miRNA⁃24, miRNA⁃34a, and miRNA⁃375 were predicted to bind to the 3′UTR of Neurod1 gene�MiRNA⁃29a, which could not bind to the 3′UTR of Neurod1 , was selected as a negative control�Overexpression miRNA⁃24, miRNA⁃34a, and miRNA⁃375 could reduce the luciferase activity of Neurod1 3′UTR, whereas miRNA⁃29a could not regulate its activity�Overexpression of miRNA⁃24, miRNA⁃34a, and miR⁃NA⁃375 repressed Neurod1 protein levels at different degree�Silencing of Neurod1 repressed basal and stimulated levels of insulin in pancreatic β⁃cell line MIN6 cells�Conclusions MiRNA⁃24 , miRNA⁃34a, and miRNA⁃375 can repress Neurod1 protein expression in MIN6 cells.
展开▼
