Circular RNA CircRNA₀01131 Inhibits Cardiac Fibrosis Via Sponging MiR-25-3p

Cardiac fibrosis is characterized by net accumulation of extracellular matrix proteins in the cardiac interstitium,and contributes to both systolic and diastolic dysfunction in many cardiac pathophysiologic conditions,eventually to heart failure.Circ RNA is a covalent circularized non-coding RNA,which is involved in the pathogenesis and development of diseases through regulating genes expression.This paper studies the effect and mechanism of circ RNA?001131 on cardiac fibrosis.Objective: To investigate the effect of circ RNA?001131 on the fibrotic phenotype in rat cardiac fibroblasts(CFs)and mechanism involved.Methods: Masson trichrome staining was performed on the myocardium of a rat model of abdominal aorta coarctation(AAC)induced myocardial remodeling.The expression of circ RNA?001131 and its host gene of phosphatase and tension homolog(Pten)in the myocardium of AAC-induced rats and Ang-II-treated cardiac fibroblasts was confirmed by RTq PCR assay.Actinomycin D treatment and RNase R exonuclease digestion were performed to test the stability of circ RNA?001131 in rat CFs.Over-expression of circ RNA?001131 was achieved in cardiac fibroblasts with infection of the recombinant circ RNA?001131 adenovirus,r Ad-circ RNA?001131.The expression of collagen type I alpha 1(Col1a1),collagen type III alpha 1(Col3a1)and smooth muscle ?-2actin(Acta2)was detected in rat CFs by RT-q PCR and Western blot,respectively.The interaction between circ RNA?001131 and mi R-25-3p was identified by dual luciferase reporter assay,RNA pull down assay and RNA antisense purification(RAP)assay.MRNA expression profile chip was to investigate the target gene of mi R-25-3p.The expression of Btg2 in cardiac fibroblasts overexpressing circ RNA?001131 or mi R-25-3p was determined by q PCR assay.The interaction between mi R-25-3p and Btg2 was identified by dual luciferase reporter assay.The expression of cardiac fibrosis related genes and were detected by Western blot in cardiac fibroblasts over-expressing Btg2 or silence Btg2.The expression of p Smad3,p-Akt and p-NF-?Bp65 were detected in cardiac fibroblasts overexpreesing Btg2 or circ RNA-001131.Verified whether si-Btg2 and mi R-25-3p could reverse the inhibition effect of circ RNA?001131 in cardiac fibrosis.Results: Masson staining results showed that collagen in AAC rat myocardium increased significantly.Circ RNA?001131 and Pten were up-regulated in the myocardium of AACinduced rats and in Ang-?-treated rat CFs.Circ RNA?001131 was more stable than its host gene of Pten when it was subjected to actinomycin D and RNase R exonuclease treatment,respectively.The expression of fibrosis associated genes was down-regulated in cardiac fibroblasts with over-expression of circ RNA?001131.Dual luciferase reporter assay,RNA pull down assay and RNA antisense purification(RAP)assay revealed the interaction between mi R-25-3p and circ RNA?001131.MRNA expression profile chip found Btg2 might be the target gene of mi R-25-3p,and q PCR assay revealed Btg2 was upregulated in rat cardiac fibroblasts overexpressed circ RNA?001131 meanwhile downregulated in rat cardiac fibroblasts overexpressed mi R-25-3p.Both Btg2 and circ RNA?001131 inhibited cardiac fibrosis related genes and p-Smad3 but not p-Akt or p-NF-?Bp65,while mi R-25-3p could reverse the inhibitory effect of Btg2.And mi R-25-3p or silence of Btg2(si-Btg2)could reverse the inhibitory effect of circ RNA?001131 on the expression of fibrosis associated genes in rat CFs.Conclusions: Circ RNA?001131 is up-regulated in cardiac fibrosis,and it inhibits the expression of fibrosis-related genes in CFs through sponging mi R-25-3p and regulating Btg2 expression.