MiR-30a-3p靶基因的预测与验证

Prediction and Verification of MiR-30a-3p Target Gene

目的:预测和验证miR-30a-3p的靶基因。方法:利用生物信息学方法预测miR-30a-3p的靶基因;扩增靶基因3'UTR区,与pmirGLO质粒连接构建靶基因融合表达载体后与miR-30a-3p mimics共同转染到人正常滋养细胞系HTR-8/SVneo细胞中,利用双荧光素酶报告基因实验验证miR-30a-3p与靶基因的靶向关系;在HTR-8/SVneo细胞中转染miR-30a-3p mimics,利用Western Blot实验检测靶蛋白表达水平。结果:生物信息学方法预测结果显示胰岛素样生长因子-1(IGF-1)为miR-30a-3p靶基因之一;双荧光素酶报告基因实验显示,IGF-1基因3'UTR区中含有明确的miR-30a-3p结合位点;Western Blot实验结果显示HTR-8/SVneo细胞转染miR-30a-3p mimics后,IGF-1蛋白表达水平明显下调(P<0.05)。结论:miR-30a-3p与IGF-1有确切的靶向关系,miR-30a-3p能够负性调控IGF-1蛋白水平的表达。

Objective：To predict and verify the target gene of miR-30a-3p. Methods：The putative target genes of miR-30a-3p were predicted by bioinformatics approach, The 3＇UTR region of putative target gene was amplified,and cloned into the plasmid containing luciferase report gene pmirGLO, The luciferase reporter vector and miR-30a-3p mimics were co-transfected into HTR-8/SVneo cells, The interaction between miR-30a-3p and target genes was verified by luciferase activity analysis,and the protein expression was tested using Western blot. Results：lGF-1 was one of the putative target genes of miR-30a-3p by bioinformatics analysis. Luciferase activity analysis showed that the 3＇UTR region of IGF-1 gene had miR-30a-3p binding sites, IGF-1 was down-regulated by transfection of miR-30a-3p mimics, The expression of IGF-1 protein in HTR-8/SVneo cells significantly decreased after transfection of miR-30a-3p mimics （ P 〈 0.05 ）. Conclusiona.IGF-1 was a target gene of miR-30a-3p,and miR-30-3p could reversely regulate IGF-1 expression.