靶向调控c-SKI的microRNA的筛选及验证

目的筛选和验证靶向调控c-SKI并与纤维化相关的microRNA(miRNA)。方法生物信息学方法预测并结合文献报道,筛选出靶向c-SKI的候选miRNAs,RT-qPCR检测人心肌成纤维细胞(HCFBs)中候选miRNAs和c-SKI的表达,筛选出抑制作用最显著的miRNA;构建c-SKI-3′-UTR野生型(c-SKI-wt)和突变型(c-SKI-mut)载体,分别与miR-155a-5p/miR-17a-5p的模拟物、抑制剂及对照在人胚肾上皮细胞(HEK293T)中共转染,双萤光素酶报告系统检测各组荧光素酶活性;接着,分别将miR-155a/miR-17a-5p mimics和inhibitor转染至人心肌成纤维细胞(HCFBs),Western blot检测各组细胞c-SKI的表达。结果 1)经筛选miR-155a-5p和miR-17a-5p对c-SKI的抑制作用最明显(P<0.01);2)与NC组相比,miR-155a-5p/miR-17a-5p mimics组萤光素酶活性均显著下降(P<0.05),miR-155a-5p/miR-17a-5p inhibitor组萤光素酶活性均明显增强(P<0.05);3)与NC组相比,miR-155a-5p/miR-17a-5p mimics组中c-SKI蛋白表达显著下调,miR-155a-5p/miR-17a-5p inhibitor组中c-SKI的表达显著上调(P<0.01)。结论 miR-155a-5p和miR-17a-5p可分别靶向结合c-SKI的3′-UTR,在HCFBs中负性调控c-SKI的表达。

Screening and identification of the microRNA targeting c-SKI

Objective To screen and validate the microRNA targeting c-SKI and associated with fibrosis. MethodsBioinformatics methods was used to predict the candidate miRNAs targeting c-SKI, RT-qPCR was used to detect the expression of candidate miRNAs and c-SKI gene in myocardial fibroblasts, and the most significant miRNAs(miR-155 a-5 p/miR-17 a-5 p) were selected. Building SKI-3 ′-UTR wild type(c-SKI-wt) and mutant(c-SKI-mut) plasmid,miR-155 a-5 p/miR-17 a-5 p mimics and mimics NC,miR-155 a-5 p/miR-17 a-5 p inhibitor and inhibitor NC were co-transfected into the 293 T cells. The c-SKI-wt or c-SKI-mut. Dual-luciferase reporter assay was performed to detect the luciferase activity in different groups.miR-155 a-5 p,miR-17 a-5 p mimics and inhibitor were transfected to myocardial fibroblasts.Western blot was used to detect the expression of c-SKI in each group.Results 1)after the primary screening miR-155 a-5 p and miR-17 a-5 p showed the most obvious inhibition on c-SKI(P<0.01). 2)Compared with the NC group, the c-SKI reporter gene luciferase activity significantly decreased inmiR-155 a-5 p/miR-17 a-5 p mimics group(P<0.05),and the c-SKI reporter gene luciferase activity was obviously upregulated in miR-155 a-5 p and miR-17 a-5 p inhibitor group(P<0.05).3)Compared with the NC group, the expression of c-SKI significantly down-regulated in miR-155 a-5 p and miR-17 a-5 p mimics group, but up-regulated in miR-155 a-5 p and miR-17 a-5 p inhibitor group as detected by Western blot(P<0.01). Conclusions miR-155 a-5 p and miR-17 a-5 p can target c-SKI, and regulate the expression of c-SKI in myocardial fibroblasts.