微小RNA-1266对钠通道蛋白表达和分布的影响

Abstract

目的探讨心房颤动(AF)相关微小RNA-1266-5p(miR-1266-5p)与AF相关钠离子通道蛋白α亚基编码基因SCNA5的靶向调控关系。方法构建含有预测结合位点3'非翻译区(3'UTR)序列的靶基因,将其与阴性对照质粒以及miRNA共转染入人胚肾HEK293细胞,实验分为空白对照组、阴性对照组、实验一组(pc DNA3. 1-SCN5A 3'UTR改建质粒+miR-1266)和实验二组(pc DNA3. 1-SCN5A WT质粒,不含3'UTR端+miR-1266),荧光定量PCR法和Western blot法检测各组钠通道蛋白SCN5A mRNA和Nav1. 5蛋白表达变化,免疫荧光实验观察SCN5A Nav1. 5蛋白表达和分布变化。结果与空白对照组和阴性对照组比较,实验一组SCN5A mRNA表达分别下调49. 4%和46. 0%,差异有统计学意义(0. 557±0. 016 vs 1. 101±0. 132和1. 031±0. 020,P <0. 05),而实验二组SCN5A mRNA表达无显著变化(P> 0. 05);实验一组SCN5A Nav1. 5蛋白表达下降,而实验二组SCN5A Nav1. 5蛋白表达无显著变化;实验一组和实验二组的SCN5A Nav1. 5蛋白分布都无显著变化。结论 SCN5A可能是miR-1266的直接靶基因,miR-1266可能通过负性调控SCN5A表达参与AF电重构。

Objective To study the relationship between AF-related miR-1266-5 p and sodium channel protein α subcoding gene SCNA5. Methods The target gene containing the predictive binding site 3'UTR sequence was constructed and transfected into HEK293 cells with the negative control plasmid and miRNA. The HEK293 cells were divided into blank control group,negative control group,and experimental group A( pcDNA3. 1-SCN5 A 3' UTR plasmid + miR-1266) and experimental group B( pcDNA3. 1-SCN5 A WT plasmid containing no 3' UTR + miR-1266). The expressions of SCN5 A mRNA and Nav1. 5 protein were detected by RT-qPCR and Western blot respectively. The distribution of Nav1. 5 protein expressions was detected with immunofluorescence staining. Results The expression level of SCN5 A mRNA was significantly lower in experimental group A than in blank control group and negative control group( P < 0. 05). No significant difference in expression level of SCN5 A mRNA was found in experimental group B( P > 0. 05). The expression level of SCN5 A Nav1. 5 protein was significantly lower in experimental group A while no significant difference in expression of of SCN5 A Nav1. 5 protein was detected in experimental group B. No significant difference in distribution of SCN5 A Nav1. 5 protein was detected between experimental groups A and B. Conclusion SCN5 A is a direct target gene of miR-1266 which participates in electrical AF remodeling by downregulating the expression of SCN5 A.