靶向调控c-SKI的microRNA的筛选及验证

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (1): 60-66.

靶向调控c-SKI的microRNA的筛选及验证

王娟, 李鹏, 吕忠英, 张颖, 曹桂秋^*

新疆医科大学第五附属医院心血管内科, 新疆乌鲁木齐 830001

收稿日期:2018-12-29 修回日期:2019-03-29 出版日期:2020-01-05 发布日期:2019-12-27
通讯作者: ^*caoguiqiu@126.com
基金资助:
新疆医科大学科研创新基金(XYDCX201694); 自治区卫生计生委青年科技人才专项科研项目(2016Y02)

Screening and identification of the microRNA targeting c-SKI

WANG Juan, LI Peng, LYU Zhong-ying, ZHANG Ying, CAO Gui-qiu^*

Department of Cardiology, the fifth Affiliated Hospital of Xinjiang Medical University, Urumchi 830001, China

Received:2018-12-29 Revised:2019-03-29 Online:2020-01-05 Published:2019-12-27
Contact: ^*caoguiqiu@126.com

摘要/Abstract

摘要： 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。

关键词: c-SKI, miR-155a-5p, miR-17a-5p, 双萤光素酶报告系统, 心肌纤维化

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.

Key words: c-SKI, miR-155a-5p, miR-17a-5p, dual-luciferase reporter gene system, myocardial fibrosis

中图分类号:

王娟, 李鹏, 吕忠英, 张颖, 曹桂秋. 靶向调控c-SKI的microRNA的筛选及验证[J]. 基础医学与临床, 2020, 40(1): 60-66.

WANG Juan, LI Peng, LYU Zhong-ying, ZHANG Ying, CAO Gui-qiu. Screening and identification of the microRNA targeting c-SKI[J]. Basic & Clinical Medicine, 2020, 40(1): 60-66.

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