猪miR-23a靶向调控Smad3基因表达的研究

doi:10.16431/j.cnki.1671-7236.2019.01.023

摘要/Abstract

摘要：

为了确定miR-23a是否靶向调控Smad3基因，试验利用Not Ⅰ和Xho Ⅰ酶构建包含Smad3-3'-UTR的野生型（psiCHECK^TM-2-W-Smad3-3'-UTR）和突变型双荧光酶报告载体（psiCHECK^TM-2-M-Smad3-3'-UTR），并在PK-15细胞中转染miR-23a mimics、miR-23a inhibitor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-23a mimic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-23a mimics能显著下调Smad3基因mRNA及其蛋白表达水平（P < 0.05）；而转染miR-23a inhibitor组与miR-23a inhibitor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。

关键词: miR-23a; Smad3基因; 表达调控

Abstract:

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.

Key words: miR-23a; Smad3 gene; expression regulation

中图分类号:

S813.3

邱梅玉, 翟腾蛟, 黄涛, 李涛, 孙敬礼, 孙晓梅. 猪miR-23a靶向调控Smad3基因表达的研究[J]. 《中国畜牧兽医》, 2019, 46(1): 200-205.

QIU Meiyu, ZHAI Tengjiao, HUANG Tao, LI Tao, SUN Jingli, SUN Xiaomei. Study on Regulation of Gene Expression of Smad3 Gene by miR-23a in Pig[J]. , 2019, 46(1): 200-205.

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