MicroRNA-363-5p靶向血小板反应蛋白调控心脏成纤维细胞增殖及纤维化相关蛋白的表达

MicroRNA⁃363⁃5p Regulates Proliferation of Cardiac Fibroblasts and Expression of Fibrosis⁃Related Proteins through Targeting THBS3

目的探究microRNA-363-5p(miR-363-5p)是否通过调控血小板反应蛋白3(THBS3)参与血管紧张素Ⅱ(AngⅡ)处理的人类心脏成纤维细胞(HCF)增殖及纤维化相关蛋白的表达。方法培养HCF,分为对照组(正常HCF),AngⅡ组(1×10-6mol/L AngⅡ处理),mimic-NC组(转染模拟物)、miR-363-5p mimic组(转染miR-363-5p模拟物)、inhibitor-NC组(转染抑制剂阴性对照)、miR-363-5p inhibitor组(转染miR-363-5p抑制剂);pcDNA+mimic-NC组(共转染空载体pcDNA+模拟物)、pcDNA+miR-363-5p mimic组(共转染空载体pcDNA+miR-363-5p模拟物)、pcDNA-THBS3+miR-363-5p mimic组(共转染空载体pcDNA-THBS3+miR-363-5p模拟物)。细胞计数试剂盒(CCK-8)检测细胞增殖活性;蛋白质印迹法检测细胞THBS3蛋白及纤维化相关蛋白;实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测细胞miR-363-5p和THBS3 mRNA;双荧光素酶报告基因实验检测细胞荧光活性。结果与对照组相比,AngⅡ组细胞增殖活性(P<0.05)、α平滑肌肌动蛋白(P<0.001)、Ⅰ型胶原蛋白(P<0.001)、Ⅲ型胶原蛋白(P<0.001)均升高。与mimic-NC组相比,miR-363-5p mimic组细胞增殖活性(P<0.01)、α平滑肌肌动蛋白(P<0.001)、Ⅰ型胶原蛋白(P<0.001)、Ⅲ型胶原蛋白(P<0.001)均降低,且抑制miR-363-5p具有相反作用。双荧光素酶报告实验显示,miR-363-5p靶向负调控THBS3。与pcDNA+mimic-NC组相比,pcDNA+miR-363-5p mimic组细胞增殖活性、α平滑肌肌动蛋白、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白均降低(P均<0.01)。与pcDNA+miR-363-5p mimic组相比,pcDNA-THBS3+miR-363-5p mimic组细胞增殖活性(P<0.05)、α平滑肌肌动蛋白(P<0.01)、Ⅰ型胶原蛋白(P<0.001)、Ⅲ型胶原蛋白(P<0.001)均升高。挽救实验结果显示过表达THBS3减弱miR-363-5p抑制AngⅡ处理导致的细胞增殖及纤维化相关蛋白表达。结论miR-363-5p靶向THBS3抑制HCF增殖及纤维化相关蛋白表达。

Objective To investigate whether microRNA⁃363⁃5p(miR⁃363⁃5p)is involved in the regulation of THBS3 in human cardiac fibroblast(HCF)proliferation and the expression of fibrosis⁃related proteins during angiotensinⅡ(AngⅡ)treatment.Methods HCF were cultured and divided into the following groups:control group(normal HCF),AngII group(treated with 1×10-6 mol/L AngII),mimic⁃NC group(transfected mimic),miR⁃363⁃5p mimic group(transfected miR⁃363⁃5p mimic),inhibitor⁃NC group(transfected inhibitor),miR⁃363⁃5p inhibitor group(transfected miR⁃363⁃5p inhibitor);pcDNA+mimic⁃NC group(co⁃transfected pcDNA+mimic),pcDNA+miR⁃363⁃5p mimic group(co⁃transfected pcDNA+miR⁃363⁃5p mimic),pcDNA⁃THBS3+miR⁃363⁃5p mimic group(co⁃transfected pcDNA⁃THBS3+miR⁃363⁃5p mimic).Cell Counting Kit⁃8(CCK⁃8)was used to detect cell proliferation activity.THBS3 protein and fibrosis⁃related proteins was detected by Western blotting.Real⁃time fluorescence quantitative reverse transcription⁃polymerase chain reaction(qRT⁃PCR)was used to measure miR⁃363⁃5p and THBS3 mRNA in cells.Cell fluorescence activity was detected by double luciferase reporter gene assay.Results Compared to the control group,the cell proliferation activity(P<0.05),andα⁃SMA(P<0.001),Collagen1(P<0.001),and Collagen3(P<0.001)in the AngⅡgroup were elevated.Compared to the mimic⁃NC group,the cell proliferation activity(P<0.01),α⁃SMA(P<0.001),Collagen1(P<0.001),and Collagen3(P<0.001)in the miR⁃363⁃5p mimic group were reduced,and the inhibition of miR⁃363⁃5p had the opposite effect.Dual⁃luciferase reporter assay revealed that miR⁃363⁃5p negatively regulated THBS3.Compared to the pcDNA+mimic⁃NC group,the cell proliferation activity,α⁃SMA,Collagen1,and Collagen3(P<0.01)reduced in the pcDNA+miR⁃363⁃5p mimic group.Compared to the pcDNA+miR⁃363⁃5p mimic group,the cell proliferation activity(P<0.05),α⁃SMA(P<0.01),Collagen1(P<0.001),and Collagen3(P<0.001)were elevated in the pcDNA⁃THBS3+miR⁃363⁃5p mimic group.Overexpression of THBS3 attenuated the inhibition of cell proliferation and fibrosis⁃related protein expression induced by miR⁃363⁃5p in response to AngII treatment.Conclusion MiR⁃363⁃5p inhibits proliferation of HCF and the expression of fibrosis⁃related proteins through targeting THBS3.