LINC00205靶向miR-514a-3p/NF-κB对血管平滑肌细胞生物学功能的作用

The effect of LINC00205 targeting miR-514a-3p/NF-κB on the biological function of vascular smooth muscle cells

目的LINC00205是否通过调控miR-514a-3p/NF-κB通路而参与动脉粥样硬化发生过程尚未清楚。文章旨在探讨LINC00205对血管紧张素II(AngII)诱导的血管平滑肌细胞(HA-VSMC)增殖、凋亡、迁移的影响及其对miR-514a-3p/NF-κB通路的调控作用。方法采用AngII诱导的HA-VSMC细胞建立动脉粥样硬化模型为AngII组。将正常培养的细胞作为正常组,将si-con、si-LINC00205、miR-con、miR-514a-3p mimics、anti-miR-con、anti-miR-514a-3p及si-LINC00205与anti-miR-con、si-LINC00205、anti-miR-514a-3p转染至AngII诱导的HA-VSMC细胞,分别为si-con组、si-LINC00205组、miR-con组、miR-514a-3p组、anti-miR-con组、anti-miR-514a-3p组、si-LINC00205+anti-miR-con组、si-LINC00205+anti-miR-514a-3p组。采用qRT-PCR法检测LINC00205、miR-514a-3p的表达量;采用MTT实验、Transwell小室实验分别检测细胞增殖、迁移能力;采用流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测LINC00205与miR-514a-3p的靶向关系;Western blot法检测CyclinD1、MMP2、MMP9、Cleaved-caspase-3、p-p65、p-IкBα、p65、IкBα蛋白表达量。将20只大鼠随机分为对照组(普通喂养)及AS组(蛋氨酸喂养,建立AS模型),检测颈动脉LINC00205、miR-514a-3p、p-p65、p-IкBα、p65、IкBα各蛋白表达。结果与正常组凋亡率比较,AngII组明显降低(P<0.05);与si-con组凋亡率比较,si-LINC00205组明显升高(P<0.05)。AngII组HA-VSMC细胞迁移数较正常组明显升高[(153±14)vs(67±6),P<0.05],si-LINC00205组较si-con组明显降低[(72±7)vs(158±15),P<0.05]。与正常组比较,AngII组CyclinD1、MMP2、MMP9蛋白水平明显升高(P<0.05),Cleaved-caspase-3蛋白水平明显降低(P<0.05);与si-con组比较,si-LINC00205组CyclinD1、MMP2、MMP9蛋白水平明显降低(P<0.05),Cleaved-caspase-3蛋白水平明显升高(P<0.05)。miR-514a-3p组A值、迁移数、CyclinD1、MMP2、MMP9蛋白水平较miR-con组明显降低(P<0.05),凋亡率、Cleaved-caspase-3蛋白水平明显升高(P<0.05);anti-miR-514a-3p组CyclinD1、MMP2、MMP9蛋白水平较anti-miR-con组升高(P<0.05),Cleaved-caspase-3蛋白水平降低(P<0.05)。与si-LINC00205+anti-miR-con组比较,si-LINC00205+anti-miR-514a-3p组CyclinD1、MMP2、MMP9蛋白水平升高(P<0.05),Cleaved-caspase-3蛋白水平降低(P<0.05)。与对照组比较,AS组LINC00205、p-p65、p-IкBα蛋白水平明显升高(P<0.05),miR-514a-3p蛋白水平降低(P<0.05)。结论LINC00205表达降低能够有效调控miR-514a-3p/NF-κB通路,从而能够有效抑制因AngII介导的HA-VSMC细胞迁移及繁殖,并可诱导细胞凋亡。

Objective Whether LINC00205 participates in the process of atherosclerosis by regulating the miR-514a-3p/NF-κB pathway remains unclear.This study aims to investigate the effects of LINC00205 on proliferation,apoptosis and migration of vascular smooth muscle cells(HA VSMC)induced by angiotensin II(AngII)and its regulation of miR-514a-3p/NF-κB pathway.Methods The atherosclerosis model of HA-VSMC cells induced by AngII was established as AngII group.Using the normal cultured cells as the normal group,si-con,si-LINC00205,miR-con,miR-514a-3p mimics,anti miR con,anti-miR-514a-3p,si-LINC00205+anti miR-con,si-LINC00205+anti-miR-514a-3p were transfected into ANGII-induced HA VSMC cells as AngII+si-con group,AngII+si-LINC00205 group,AngII+miR-con group,AngII+miR-514a-3p group,AngII+anti-miR-con group,AngII+anti-miR-514a-3p group,AngII+si-LINC00205+anti-miR-con group,AngII+si-LINC00205+anti-miR-514a-3p group,respectively.The expression levels of LINC00205 and miR-514a-3p were detected by qRT PCR.MTT assay and Transwell assay were used to detect cell proliferation and migration.The apoptosis rate was detected by flow cytometry.The targeting relationship between LINC00205 and Mir 514a 3p was detected by dual luciferase report assay.The protein expressions of CyclinD1,MMP2,MMP9,Cleaved caspase 3,p p65,p IкBα,p65 and IкBαwere detected by Western blot.The expression of LINC00205,miR 514a-3p,p p65,p IкBα,p65,IкBαin the carotid artery of 20 rats were randomly divided into control group(normal feeding)and AS group(methionine feeding).Results Compared with normal group,the apoptosis rate of AngII group was significantly decreased(P<0.05).Compared with AngII+Si Con group,the apoptosis rate of AngII+Si LINC00205 group was significantly increased(P<0.05).The migration number of HA VSMC in AngII group was significantly higher than that in normal group[(153±14)vs(67±6),P<0.05].The parameters of AngII+Si LINC00205 were significantly lower than that of AngII+Si CON[(72±7)vs(158±15),P<0.05].Compared with normal group,the protein levels of CyclinD1,MMP2 and MMP9 in AngII group were significantly increased(P<0.05),and Cleaved-caspase-3 protein level was significantly decreased(P<0.05).Compared with AngII+Si Con group,the protein levels of CyclinD1,MMP2 and MMP9 in AngII+Si LINC00205 group were significantly decreased(P<0.05),but the protein levels of Cleaved caspase 3 were significantly increased(P<0.05).Compared with AngII+Si LINC00205+anti miR con group,the protein levels of CyclinD1,MMP2 and MMP9 were increased in AngII+Si LINC00205+Anti miR-514a-3p group(P<0.05).Cleaved-caspase-3 protein level was decreased(P<0.05).Compared with control group,the protein levels of LINC00205,p p65 and p IкBαin AS group were significantly increased(P<0.05),while the protein levels of miR 514a 3p were decreased(P<0.05).Conclusion The decreased expression of LINC00205 can effectively regulate the Mir 514a-3p/NF-κB pathway,which can effectively inhibit the migration and reproduction of HA VSMC mediated by AngII,and induce cell apoptosis.