猪E2F3基因3′UTR双荧光素酶报告基因质粒构建及其与miR-10a-5p的靶向验证

Construction of porcine E2F3 gene 3'UTR dual luciferase reporter plasmid and its targeting verification with miR-10a-5p

为了鉴定miR-10a-5p与猪E2F3基因的靶向关系,试验从生物信息miRNA Base数据库查询miR-10a-5p的相关信息,并运用MEGA 6.0软件分析其保守性,通过Target Scan生物信息学网站预测其潜在的靶基因及结合位点,然后PCR扩增E2F3基因3′非编码区(3′UTR)片段,构建E2F3的野生型(E2F3-3′UTR-wt)和突变型(E2F3-3′UTR-mut)双荧光素酶报告基因质粒,并分别与miR-10a-5p mimics、mimics NC共转染至293T细胞中,检测双荧光素酶活性变化。结果表明:miR-10a-5p在7个物种间具有高度保守性,成熟序列均为UACCCUGUAGAUCCGAAUUUGU;Target Scan生物信息学网站预测到miR-10a-5p具有包括E2F3基因在内的共241个潜在的靶基因;E2F3基因片段经PCR扩增获得大小约1 288 bp的清晰条带,测序结果与预期一致;转染miR-10a-5p mimics能显著降低猪E2F3基因野生型质粒双荧光素酶活性(P<0.05)。说明猪E2F3基因3′UTR双荧光素酶报告基因质粒构建成功,且其与miR-10a-5p之间存在靶向关系。

In order to identify the targeting relationship between porcine miR-10 a-5 p and E2F3 genes, the information of miR-10 a-5 p was extracted from miRNA Base database and its conservation was analyzed by MEGA 6.0. The potential target genes and binding sites of miR-10 a-5 p were predicted by the Target Scan bioinformatics website. Then the E2F3-3′UTR was amplified by PCR, recombinant plasmid of dual-luciferase reporter gene of wild type(E2F3-3′UTR-wt) and mutant type(E2F3-3′UTR-mut) of E2F3 were constructed and co-transfected with mimics and mimics NC of miR-10 a-5 p into 293 T cells, respectively, and dual-luciferase activity was measured. The result of biological analysis showed that miR-10 a-5 p was highly conserved among seven species, and the mature sequences were UACCCUGUAGAUCCGAAUUUGU.MiR-10 a-5 p has 241 potential target genes, including E2F3, as predicted by Target Scan bioinformatics website. A 1288 bp of E2F3 gene was amplified by PCR, the result of sequencing was consistent with expectation. MiR-10 a-5 p mimics could significantly decrease the activity of wild-type plasmid dual-luciferase of E2F3 in pigs(P<0.05). These results indicated that the pig E2F3 gene 3′-UTR dual-luciferase vector was successfully constructed, and there was a targeting relationship between it and miR-10a-5p.