Jichu yixue yu linchuang (Jan 2020)
Screening and identification of the microRNA targeting c-SKI
Abstract
Objective To screen and validate the microRNA targeting c-SKI and associated with fibrosis. Methods Bioinformatics 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-155a-5p/miR-17a-5p) were selected. Building SKI-3 ′-UTR wild type (c-SKI-wt) and mutant (c-SKI-mut) plasmid,miR-155a-5p/miR-17a-5p mimics and mimics NC,miR-155a-5p/miR-17a-5p inhibitor and inhibitor NC were co-transfected into the 293T cells. The c-SKI-wt or c-SKI-mut. Dual-luciferase reporter assay was performed to detect the luciferase activity in different groups.miR-155a-5p,miR-17a-5p 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-155a-5p and miR-17a-5p 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-155a-5p/ miR-17a-5p mimics group (P＜0.05),and the c-SKI reporter gene luciferase activity was obviously upregulated in miR-155a-5p and miR-17a-5p inhibitor group (P＜0.05).3)Compared with the NC group, the expression of c-SKI significantly down-regulated in miR-155a-5p and miR-17a-5p mimics group, but up-regulated in miR-155a-5p and miR-17a-5p inhibitor group as detected by Western blot(P＜0.01). Conclusions miR-155a-5p and miR-17a-5p can target c-SKI, and regulate the expression of c-SKI in myocardial fibroblasts.
