MicroRNA-363-5p Regulates Proliferation of Cardiac Fibroblasts and Expression of Fibrosis-Related Proteins through Targeting THBS3
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 angiotensin Ⅱ(Ang Ⅱ)treatment.Methods HCF were cultured and divided into the following groups:control group(normal HCF),AngⅡ group(treated with 1 x 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),and α-SMA(P＜0.001),Collagen1(P＜0.001),and Collagen3(P＜0.001)in the Ang Ⅱ group 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.
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