Long Non-coding RNA GAS5 Regulates Proliferation And Activation Of Cardiac Fibroblasts Via MiR-21 In Rats

Objective Atrial fibrillation is a kind of common arrhythmia clinically,which is associated with the occurrence of a variety of heart diseases such as heart valve diseases.Cardiac fibrosis is a pathological physiological basis of AF and an important feature of myocardial remodeling.Long non-coding RNA is a hot research topic in recent years,which plays an important role in the process of regulating of cell growth and apoptosis and works is associated with micro RNAs.This topic is focus on Lnc RNA GAS5,by influencing the expression of GAS5 to detect the downstream target gene and protein expression and observes the activation and proliferation of rat cardiac fibroblasts;meanwhile,exploring the possible mechanisms of GAS5.Methods The SD rats randomly divided in two groups,30 rat each,named cardiac fibrosis Group A and the normal control Group N.Cardiac fibrosis in rats model was constructed by the method of abdominal subcutaneous injection of isopropyl adrenaline for 1 weeks,and Group N was injected with same amount of normal saline.HE and Masson staining to observe the pathological and collagen changes in rat heart tissue,and calculating Collagen volume fraction(CVF).q RT-PCR was uesd to detect the expression of GAS5 and mi R-21.Ectracting cardiac fibroblasts from neonate SD rats,CFs were transfected with p EGFP-C1-GAS5,GAS5 si RNA and their negative control being used Lipofectamin 2000,cells were harvested 48 h later.q RT-PCR was uesd to detect the expression of GAS5 and mi R-21,and the m RNA expressions of Col1A1 and α-SMA.The expressions of Col1A1,α-SMA and MMP-2 in protein level were determined by western blot.The activities of cells proliferation were confirmed by MTT assay.Results Compared with control Group N,HE and Masson staining showed the accumulation of the extracellular matrix and the formation of cardiac fibrosis,meanwhile,the collagen volume fraction increased.It showed that the expression of GAS5 was lower than Group N,while mi R-21 was higher.After transfect cardiac fibroblasts with p EGFP-C1-GAS5 and its negative control p EGFP-C1,q RT-PCR showed that the expression of Lnc RNA GAS5 than the control CFs increased significantly,mi R-21,Col1A1 and α-SMA expressed lower than control CFs,while the MMP-2 higher.Results from western blot demonstrated significant decrease of α-SMA and Col1A1 in the group that cells transfected with p EGFP-C1-GAS5,while MMP-2 was high.MTT assay results suggested that in the experimental group after transfected 24 h and 48 h,the CFs proliferation activity decreased significantly than the negative and no-treatment control group.After transfecting cardiac fibroblasts with GAS5 si RNA and its negative control,q RT-PCR showed that the expression of GAS5 than the control CFs decreased significantly,mi R-21,Col1A1 and α-SMA expressed higher than control CFs,while the MMP-2 lower.Results from western blot demonstrated significant increase of α-SMA and Col1A1 in the group that cells transfected with GAS5 si RNA,while MMP-2 was low.MTT assay results suggested that in the experimental group after transfected 24 h and 48 h,the CFs proliferation activity increased significantly than the negative and no-treatment control group.Conclusion In the SD rats cardiac fibrosis model induced by ISO,the expression of GAS5 decreased while mi R-21 increased.In CFs,overexpression of GAS5 can significantly inhibit the expression of mi R-21,decrease the accumulation of the extracellular matrix,while the expression of MMP-2 increased.In the situation of lowexpression of GAS5,the higher mi R-21 can inhibit the expression of MMP-2,α-SMA and Col1A1 were higher.These indicate that GAS5 may inhibit the formation of cardiac fibrosis by functioning mi R-21.These provide powerful basis for further explore the role of Lnc RNAs in cardiac fibrosis.