miR-106b靶向调控胰岛素分泌细胞诱导分化过程中Ngn3基因的表达

Expression of Ngn3 gene in differentiation of miR-106b-regulated insulin-producing cells

目的实现骨髓间充质干细胞(bone marrow mesenchymal stem cells,bMSCs)向胰岛素分泌细胞(insulin-producing cells,IPCs)的诱导分化并验证分化过程中miR-106b靶向调控Ngn3基因表达。方法首先,分离培养bMSCs,应用活化素A(activin A)和B细胞素(beta cellulin)将其诱导分化为IPCs。然后,采用靶基因预测软件miRanda和TargetScan对miR-106b和Ngn3基因的靶向匹配关系进行预测并通过双荧光素酶报告基因系统鉴定。qRT-PCR检测诱导分化过程中miR-106b和Ngn3的表达。结果诱导分化后的细胞双硫腙染色呈猩红色,免疫荧光化学显示有胰岛素表达。生物信息学方法预测发现miR-106b和Ngn3基因二者匹配良好,通过双荧光素酶报告基因系统检测发现miR-106b能结合到Ngn3 mRNA的3'UTR并有效抑制其表达。qRT-PCR检测结果表明,miR-106b的表达水平与Ngn3表达呈负相关。结论 miR-106b能调控IPCs诱导分化过程中Ngn3的表达。

Objective To study the differentiation of bone marrow mesenchymal stem cells （bMSCs） into insulin-producing cells （IPCs） and the expression of Ngn3 in differentiation of miR-106b-regulated IPCs. Methods The isolated bMSCs were induced to differentiate into IPCs with activin A and 13 cellulin. Target match relation between miR-106b and Ngn3 was predicted using miranda and TargetScan respectively, and identified with the dual luciferase report system. Expressions of miR-106b and Ngn3 were detected by RT-PCR during the differentiation of bMSCs into IPCs. Results The IPCs were stained scarlet with DTZ. Immunofluorescence cytochemistry showed expression of insulin. Bioinformatics method demonstrated that miR-106b was well matched with Ngn3. Dual luciferase reporter system revealed that miR-106b bound to the 3＇UTR of Ngn3 mRNA could effectively inhibit the expression of Ngn3. RT-PCR displayed that the expression level of miR-106b was negatively related with that of Ngn3 mRNA. Conclusion miR- 106b can regulate the expression of Ngn3 during the differentiation of bMSCs into IPCs.