MiR-20b直接靶向3’-UTR调控HIF-1α表达

MiR-20b directly targets 3’-UTR to regulate the expression of HIF-1α

摘要:

目的：探讨微小RNA-20b（microRNA-20b/miR-20b）是否直接靶向低氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）信使RNA的3’-非编码区（3’-untranslated region，3’-UTR）调控其基因表达。方法：采用TargetScan和miRanda算法预测HIF-1α 3’-UTR区是否存在miR-20b的配对序列；构建并鉴定表达HIF-1α 3’-UTR的双荧光素酶基因报告系统（pmiR-RB-ReportTM-HIF-1α 3’-UTR），将重组质粒（recombinant plasmid，RP）与mimics-miR-20b（M）或NC-miR-20b（N）共转染HeLa细胞，本研究共分为RP、RP+M、RP+N、P、P+M和P+N六组，24 h后检测各组双荧光素酶活性的比值。结果：HIF-1α 3’-UTR区存在miR-20b配对区域；成功构建了含HIF-1α 3’-UTR序列的双荧光素酶基因报告载体；重组质粒与mimics-miR-20b共转染的HeLa细胞荧光素酶活性明显低于其他对照组（P=0.022）。结论：miR-20b可以直接靶向结合HIF-1α 3’-UTR区负向调控其表达。

Abstract:

Objective：To investigate whether miRNA-20b（miRNA-20b/miR-20b） may bind to the 3’-untranslated region（3’-UTR） of hypoxia inducible factor-1α（HIF-1α） mRNA to regulate the expression of HIF-1α. Methods：Targetscan and miRanda softwares were used to predict the presence or absence of miR-20b binding site in the HIF-1α 3’-UTR region. The dual luciferase gene re－porter assay system（pmiR-RB-ReportTM-HIF-1α） was constructed and identified. The ratios of dual luciferase activity were detected in HeLa cells 24 hours after mimics-miR-20b（M） or NC-miR-20b（N） and recombinant plasmid（RP） co-transfection. This study was divided into six groups：RP，RP+M，RP+N，P，P+M and P+N groups. Results：There was a miR-20b binding site in the 3’-UTR of HIF-1α mRNA. The double luciferase gene reporter vector containing HIF-1α 3’-UTR was successfully constructed. The dual lu－ciferase activity was significantly lower in recombinant plasmid and mimics-miR-20b co-transfection group than in empty group（P=0.022）. Conclusion：miR-20b can directly bind to HIF-1α 3’-UTR region to regulate the expression of HIF-1α.