The identification of miR?320b target gene
- Author:
- No author available
- Journal Title:
- Chinese Journal of Diabetes
- Issue:
- 1
- DOI:
- 10.3969/j.issn.1006-6187.2023.01.004
- Key Word:
- miR-320b;双荧光素酶报告基因实验;靶基因
Abstract： 目的 寻找miR-320b靶基因,探讨其参与T2DM发生发展的分子机制.方法 4种在线软件Target Scan、MIRDB、DIANA TOOLS和miRmap预测miR-320b靶基因.采用在线软件VENNY2.1选取4种预测结果交集.在线数据库String查询靶基因编码蛋白质间的相关性,筛选其候选靶基因.采取全基因合成方式合成野生型及突变型靶基因双荧光素酶重组Psicheck-2质粒载体,Sanger测序鉴定重组质粒是否构建成功.选取对数生长期293T细胞分为6组:野生型质粒+miRNA阴性对照组(Psicheck2-PTEN-WT+NC)和野生型质粒+miR-320b mimic组(Psicheck2-PTEN-WT+mimic);突变型质粒+miRNA阴性对照组(Psicheck2-PTEN-Mut+NC)和突变型质粒+miR-320b mimic组(Psicheck2-PTEN-Mut+mimic);空质粒载体+miRNA阴性对照组(Psicheck2-NC+NC)和空质粒载体+miR-320b mimic组(Psicheck2-NC+mimic).各组进行相应质粒和miRNAs共转染,检测双荧光素酶活性.结果 4种在线软件Target Scan、MIRDB、DIANA TOOLS和miRmap预测miR-320b靶基因数分别为847、1045、806和6208,VENNY2.1取交集后得到66个共同靶基因.在线数据库String显示,66个靶蛋白之间的相关性主要集中在以人第10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)为核心的蛋白.双荧光素酶报告基因实验显示,Psicheck2-PTEN-WT+miR320b组荧光素酶活性比值低于Psicheck2-PTEN-WT+NC组(P<0.05).结论 miR-320b作用于靶基因PTEN的3'UTR,为探讨miR-320b参与T2DM发生发展提供依据.
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