LncRNA NORAD Regulates the Proliferation,Migration,Invasion,and Apoptosis of Intracranial Aneurysm Vascular Smooth Muscle Cells through the miR-513b-5p/GREM1 Axis
Objective:To study the mechanism of long non-coding RNA DNA damage induced non-coding RNA(LncRNA NORAD)regulating the proliferation,migration,invasion,and apoptosis of vascular smooth muscle cells(VSMC)in intracranial aneurysm(IA)through the miR-513b-5p/GREM1 axis.Methods:Real-time fluorescence quantitative PCR was used to detect the expression of LncRNA NORAD,miR-513b-5p,and GREM1 in human intracranial aneurysm tissue and normal tissue.Human vascular smooth muscle cells(VSMCs)were isolated,cultured in vitro,and randomly separated into control group,LncRNA NORAD siRNA group,miR-513b-5p mimics group,co-transfection(LncRNA NORAD siRNA+miR-513b-5p inhibitor)group,and co-transfection negative control(LncRNA NORAD siRNA negative control+miR-513b-5p inhibitor negative control)group.After grouping and transfection,real-time fluorescence quantitative PCR was used to detect the expression of LncRNA NORAD,miR-513b-5p,and GREM1 mRNA of cells in each group.The proliferation of cells in each group was detected by CCK-8 method and immunofluorescence staining.Hoechst 33342 staining and immunofluorescence staining were used to detect cell apoptosis in each group.Cell migration and invasion in each group were detected by cell scratch assay and Transwell assay.The expression of EMT marker proteins N-cadherin,E-cadherin,and Vimentin of cells in each group was detected by western Blotting.Dual-luciferase reporter assay was used to analyze the targeting regulation of miR-513b-5p by LncRNA NORAD and the targeted regulation of GREM1 by miR-513b-5p in VSMC.Results:Compared with normal tissue,the expression of LncRNA NORAD and GREM1 mRNA in intracranial aneurysm tissue obviously increased(P＜0.05),and the expression of miR-513b-5p obviously decreased(P＜0.05).Compared with control group,the expression of GREM1 mRNA,proliferation rate,Ki67 positive rate,migration rate,invasion number,and the expression of N-cadherin and Vimentin proteins in cells decreased in LncRNA NORAD siRNA group and miR-513b-5p mimics group(P＜0.05),the expression of miR-513b-5p,apoptosis rate,Bax/Bcl 2,and the expression of E-cadherin protein increased(P＜0.05);there was no obvious difference in each index in the co-transfection negative control group(P＞0.05).Compared with LncRNA NORAD siRNA group,the expression of GREM1 mRNA,proliferation rate,Ki67 positive rate,migration rate,invasion number,and the expression of N-cadherin and Vimentin proteins in cells increased in the co-transfection group(P＜0.05),the expression of miR-513b-5p,apoptosis rate,Bax/Bcl-2,and the expression of E-cadherin protein decreased(P＜0.05).Conclusion:Knockdown of LncRNA NORAD can reduce the expression of GREM1 by up-regulating the expression of miR-513b-5p,thereby inhibiting the proliferation,invasion,and migration of VSMCs,and promoting their apoptosis.
intracranial aneurysmlong non-coding RNA DNA damage induced non-coding RNA,LncRNA NORADmiR-513b-5pGREM1vascular smooth muscle cellexperimental study
万方数据
维普
