双荧光素酶报告基因技术验证miR-31对LATS2的靶向调控作用

Dual luciferase reporter gene technology for verifying targeted regulation effect of miR-31 on LATS2

目的通过双荧光素酶报告基因技术验证miR-31对大肿瘤抑制因子2(LATS2)的靶向调控作用,观察miR-31在心肌细胞肥大过程中的可能影响。方法运用Targetscan软件预测大鼠miR-31对LATS2的靶向调控作用;以双荧光素酶报告质粒为工具载体,分别插入人工合成的大鼠LATS2基因野生型3′UTR(LATS2-3′UTR-WT)及突变型3′UTR(LATS2-3′UTR-MU)片段,构建LATS2-3′UTR-WT及LATS2-3′UTR-MU双荧光素酶报告质粒;重组双荧光素酶报告质粒分别与miR-31过表达质粒共转染293T细胞,检测荧光素酶活性并分析miR-31对LATS2的靶向调控作用;血管紧张素Ⅱ(AngⅡ)诱导体外心肌细胞肥大模型,RT-qPCR检测miR-31、LATS2及心肌细胞肥大基因心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)的表达,F肌动蛋白荧光探针观察心肌细胞形态变化。结果Targetscan软件预测结果显示,大鼠miR-31与LATS2基因3′UTR存在互补结合位点;经酶切及基因测序鉴定,成功构建LATS2-3′UTR-WT及LATS2-3′UTR-MU双荧光素酶报告质粒;与LATS2-3′UTR-MU+miR-31组相比,LATS2-3′UTR-NC+miRNA-31组荧光素酶活性无明显变化(0.98±0.03vs.1.00±0.03,P>0.05),而LATS2-3′UTR-MT+miR-31组与LATS2-3′UTR-NC+miR-31组相比,荧光素酶活性显著降低(0.74±0.02vs.1.00±0.03,P<0.01);AngⅡ诱导48h后可检测到心肌细胞肥大基因ANP(P<0.01)及β-MHC表达上调(P<0.05),心肌细胞肥大过程中miR-31表达显著上调(P<0.01),LATS2基因明显下调(P<0.05),96h后心肌细胞表面积明显增大。结论miR-31可通过与LATS2基因3′UTR互补结合实现其对LATS2靶向调控作用,miR-31靶向作用LATS2可能参与调控心肌细胞的肥大。

Objective To verify the targeted regulation effect of miR-31 on LATS2 by dual luciferase reporter gene technology and to observe its possible influence during cardiomyocyte hypertrophy process. Methods The Targetscan software was used to predict the targeted regulation effect of rat miR-31 on LATS2.The recombinant double luciferase reporter plasmid served as the tool vector and were inserted by the fragments of wild-type 3′UTR (LATS2-3′UTR-WT) or mutant-type 3′UTR (LATS2-3′UTR-MU) of rat synthetic LATS2 gene for constructing the double luciferase vectors of LATS2-3′UTR-WT and LATS2-3′UTR-MU respectively.The recombinant luciferase reporter plasmids and the plasmids overexpressing miR-31 were co-transfected into 293T cells,and the luciferase activity was detected and the targeted regulatory effect of miR-31 on LATS2 was analyzed.The in vitro cardiomyocyte hypertrophic model was induced by AngⅡ.The expressions of miR-31,LATS2,myocardial hypertrophy genes ANP and β-MHC were detected by RT-qPCR.The change of cardiomyocyte morphology was observed by F-actin fluorescence probe. Results The Targetscan software prediction results showed that the complementary binding site existed between rat miR-31 and 3′UTR of LATS2 gene.In the verification of enzyme digestion and gene sequencing,the dual luciferase reporter vectors carrying LATS2-3′UTR-WT and LATS2-3′UTR-MU were successfully constructed.Compared to the LATS2-3′ UTR-MU+miR-31 group,the luciferase activity of LATS2-3′ UTR-NC+miR-31 group showed no significant change (0.98±0.03 vs. 1.00±0.03, P >0.05).However,compared with the LATS2-3′ UTR-NC+miR-31 group,the luciferase activity of the LATS2-3′ UTR-MU+miR-31 group was significantly decreased (0.74±0.02 vs . 1.00±0.03, P <0.01).The myocardial hypertrophy genes ANP and β-MHC expression up-regulation could be detected after AngⅡ induction for 48 h ( P <0.01, P <0.05),In the cardiomyocyte hypertrophy process,the miR-31 expression was significantly up-regulated( P <0.01),while the LATS2 gene was down-regulated obviously ( P < 0.05 ).The surface area of cardiomyocyte was increased significantly after 96 h. Conclusion miR-31 realizes the targeted regulation effect on LATS2 by the complementation binding with 3 ′UTR of LATS2 gene,the miR-31 targeted effect on LASTS2 may be involved in the regulation of cardiomyocyte hypertrophy.