猪miR-23a靶向调控Smad3基因表达的研究

摘要

To determine whether the miR-23 aregulate Smad3gene, wild-type and mutant-type dual-luciferase reporter vectors containing Smad3-3′-UTR (psiCHECKTM-2-W-Smad3-3′-UTR and psiCHECKTM-2-M-Smad3-3′-UTR) were constructed using Not Ⅰ and Xho Ⅰ, miR-23 amimics, miR-23 ainhibitor and their negative control were transfected with or without dual luciferase reporter vectors in PK-15 cells, luciferase activity were assayed, the mRNA and protein expression levels of Smad3 gene were determined by Real-time PCR and Western blotting, respectively.The results showed that the luciferase activity in PK-15 cells transfected with wild-type dual-luciferase reporter vectors and miR-23 amimic was significantly lower than negative control (P0.05) .The results indicated that miR-23 acould regulate directely the expression of its target gene Smad3.%为了确定miR-23a是否靶向调控Smad3基因, 试验利用NotⅠ和XhoⅠ酶构建包含Smad3-3′-UTR的野生型 (psiCHECKTM-2-W-Smad3-3′-UTR) 和突变型双荧光酶报告载体 (psiCHECKTM-2-M-Smad3-3′-UTR), 并在PK-15细胞中转染miR-23amimics、miR-23ainhibitor及其阴性对照, 采用双荧光酶检测试剂盒检测荧光素酶活性, 用实时荧光定量PCR和Western blotting法分别检测Smad3基因的mRNA和蛋白表达水平.结果表明, 将含Smad3-3′-UTR的野生型和突变型双荧光酶报告载体与miR-23amimic共转染PK-15细胞, 野生型报告质粒表达的荧光素酶活性显著低于其阴性对照组 (P＜0.05);转染miR-23amimics能显著下调Smad3基因mRNA及其蛋白表达水平 (P＜0.05);而转染miR-23ainhibitor组与miR-23ainhibitor阴性对照组相比, Smad3基因蛋白表达差异不显著 (P＞0.05) .综合上述结果可知, 猪miR-23a可靶向作用于Smad3基因.