猪VEGFA 3′UTR生物信息学分析及其与miR-361-5p的靶向验证

Bioinformatic analysis of porcine VEGFA 3′ UTR and its targeting validation of miR-361-5p

为了扩增并分析猪卵泡颗粒细胞中血管内皮生长因子A(VEGFA)基因3编码区(3′UTR)序列、构建VEGFA 3′UTR荧光素酶报告质粒以及利用双荧光素酶报告基因验证miR-361-5p与VEGFA 3′UTR的靶向关系,试验通过RT-PCR扩增VEGFA 3′UTR,并用生物信息学方法分析预测其保守性及潜在的互作miRNA,最后将扩增的VEGFA 3′UTR序列克隆至荧光素酶报告载体中,与miR-361-5p mimics(试验组)或NC mimics(对照组)共同转染293细胞,通过双荧光素酶报告系统检测两组细胞的荧光素酶活性,从而鉴定miR-361-5p与VEGFA 3′UTR的靶向关系。结果表明:在猪卵泡颗粒细胞中成功扩增了VEGFA基因3′UTR序列,其在哺乳动物中保守性较高,有多个潜在miRNA结合位点;成功构建了VEGFA基因3′UTR荧光素酶报告质粒,证明了miR-361-5p可以直接作用于VEGFA基因3′UTR,抑制其荧光素酶活性。

To amplify and analyze the 3′untranslated region(3′UTR)of VEGFA gene and construct a luciferase reporter vector containing the VEGFA 3′UTR to determine its interaction with miR-361-5 p via dual luciferase reporter gene system.This tudy amplified 3′UTR of VEGFA with RT-PCR and identified its conservation and potential interacting microRNAs using bioinformatic analysis.Then,the VEGFA 3′UTR fragment amplified by PCR was cloned into luciferase reporter vector.The luciferase reporters containing VEGFA 3′UTR and miR-361-5 p mimics(experimental group)or NC mimics(control group)were co-transfected into 293 cells and then dual-luciferase reporter assay was performed to detect the luciferase activity of the two groups of cells.In conclusion:We successfully amplified the VEGFA 3′UTR from pig granulosa cells.Bioinformatic analysis suggested VEGFA 3′UTR is highly conserved among mammals and contains multiple potential miRNA interaction sites.The luciferase reporter vector containing the VEGFA 3′UTR was constructed successfully,which confirmed that miR-361-5 p can directly affect VEGFA 3′UTR and repress its luciferase activity.