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小鼠骨髓间充质干细胞向胰岛素分泌细胞诱导分化过程中调控PDX-1基因表达miRNAs的鉴定
Identification of miRNAs regulating pancreatic duodenal homeobox gene 1 expression during the induction of bone marrow mesenchymal stem cells differentiation into insulin-producing cells
目的 实现小鼠骨髓间充质干细胞（BMSCs）向胰岛素分泌细胞（IPCs）的诱导分化并对分化过程中可能调控胰十二指肠同源异型盒基因-1（PDX-1）基因表达miRNAs进行鉴定。 方法 首先分离培养BMSCs，应用conophylline和尼克酰胺将其诱导分化为IPCs，采用双硫腙（DTZ）染色和免疫荧光检测胰岛素的表达。然后采用靶基因预测软件miRanda和Target Scan对调控PDX-1基因表达miRNAs进行预测并通过双荧光素酶报告基因系统鉴定。实时定量聚合酶链反应（Real-time PCR）检测诱导分化过程中miRNAs及PDX-1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得到4个可能调控PDX-1表达的miRNAs，通过双荧光素酶报告基因系统检测发现其中的miR-149和miR-346能结合到PDX-1 mRNA 的3’UTR并有效抑制其表达。Real-time PCR检测结果表明，miR-149和miR-346的表达水平与PDX-1表达呈负相关。 结论 miR-149和miR-346能负性调控IPCs诱导分化过程中PDX-1的表达。
Objective To identify the miRNAs which may regulate the expression of pancreatic duodenal homeobox gene 1(PDX-1) during the induction of bone marrow mesenchymal stem cells(BMSCs) differentiation into insulinproducing cells. Methods BMSCs were isolated from bone marrow and induced to differentiate into insulin-producing cells using conophylline and nicotinamide. The miRNAs which may regulate the expression of PDX-1 were predicted using miRanda and TargetScan and identified by dual luciferase report system. The expressions of miRNAs and PDX-1 were determined using Real-time PCR during the induction of BMSCs differentiation into insulin-producing cells. Results The induced cells were stained scarlet by DTZ and the expression of insulin was positive by immunofluorescence cytochemistry. Four miRNAs which may regulate the expression of PDX-1 were firstly obtained by bioinformatics.Using dual luciferase reporter system in vitro, two of the four miRNAs, miR-149and miR-346, were proved to effectively inhibit PDX-1 expression, respectively, by binding to the 3’UTR of PDX-1 mRNAs. The Real-time PCR results showed that the expression of miR-149 or miR-346 was negatively correlated with that of PDX-1 mRNA. Conclusion The results indicated that miR-149 and mi-346 may negatively regulate the expression of PDX-1 during the induction of insulin-producing cells.
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