猪miR-23a靶向调控Smad3基因表达的研究

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。

Study on Regulation of Gene Expression of Smad3 Gene by miR-23a in Pig

To determine whether the miR-23a regulate Smad3 gene,wild-type and mutant-type dual-luciferase reporter vectors containing Smad3-3'-UTR (psiCHECK^TM-2-W-Smad3-3'-UTR and psiCHECK^TM-2-M-Smad3-3'-UTR) were constructed using Not Ⅰ and Xho Ⅰ,miR-23a mimics,miR-23a inhibitor 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-23a mimic was significantly lower than negative control (P < 0.05).In PK-15 cells transfected with miR-23a mimic,the expression levels of Smad3 mRNA and Smad3 protein were significantly down-regulated (P < 0.05).While there was no significant difference in the expression level of Smad3 protein between PK-15 cells tansfected with miR-23a inhibitor and its negative control (P > 0.05).The results indicated that miR-23a could regulate directely the expression of its target gene Smad3.