沉默FOXO1基因对人主动脉血管平滑肌细胞自噬和凋亡的影响
Effect of silencing FOXO1 gene on autophagy and apoptosis of human aortic vascular smooth muscle cells
王琳茹 1张晶 1赵冬婵 1王晋军 1胡文贤1
作者信息
- 1. 青岛大学附属青岛市海慈医院 青岛市中医医院血管外科,山东 青岛 266000
- 折叠
摘要
目的:探讨叉头框转录因子O1(FOXO1)基因对腹主动脉瘤(AAA)血管平滑肌细胞自噬和凋亡的影响,阐明其可能的作用机制.方法:收集19例AAA患者动脉瘤组织(AAA组)及邻近正常主动脉组织(对照组),采用实时荧光定量PCR(RT-qPCR)法检测2组研究对象动脉瘤组织中FOXO1 mRNA表达水平,透射电镜观察 2 组研究对象动脉瘤组织中自噬溶酶体形成情况;Western blotting法检测2组研究对象动脉瘤组织中FOXO1及自噬相关蛋白卷曲螺旋肌球蛋白样B细胞淋巴瘤2(Bcl-2)结合蛋白(Beclin1)、微管相关蛋白1轻链3 α(LC3)和P62蛋白表达水平.体外培养人主动脉血管平滑肌细胞(hVSMCs),并采用FOXO1 siRNA(si-FOXO1)及其阴性对照(si-NC)慢病毒感染hVSMCs,10 μmol·L-1血管紧张素Ⅱ(Ang Ⅱ)联合自噬激活剂雷帕霉素(Rap)进行干预,将细胞分为空白对照组、Ang Ⅱ组、Ang Ⅱ+si-NC组、Ang Ⅱ+si-FOXO1组、Ang Ⅱ+si-NC+ Rap组和Ang Ⅱ+si-FOXO1+Rap组.CCK-8法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡水平,ELISA法检测各组细胞上清中基质金属蛋白酶2(MMP-2)和基质金属蛋白酶9(MMP-9)水平,RT-qPCR法检测各组细胞中FOXO1 mRNA表达水平,Western blotting法检测各组细胞中FOXO1、Bcl-2、Bcl-2 相关X蛋白(Bax)、剪切型含半胱氨酸的天冬氨酸蛋白水解酶 3(Cleaved caspase-3)、Beclin1、LC3 和P62 蛋白表达水平.结果:与对照组比较,AAA组动脉瘤组织中FOXO1 mRNA表达水平升高(P<0.05),自噬溶酶体数量增多(P<0.05),Beclin1蛋白表达水平和LC3 Ⅱ/LC3 Ⅰ比值升高(P<0.05),P62蛋白表达水平降低(P<0.05).与空白对照组比较,Ang Ⅱ组hVSMCs增殖活性降低(P<0.05),细胞凋亡率升高(P<0.05),细胞上清中MMP-2和MMP-9 水平升高(P<0.05),细胞中Bax、Cleaved caspase-3 和Beclin1 蛋白表达水平及LC3 Ⅱ/LC3 Ⅰ比值升高(P<0.05),Bcl-2和P62蛋白表达水平降低(P<0.05);与Ang Ⅱ+si-NC组比较,Ang Ⅱ+si-FOXO1组hVSMCs增殖活性升高(P<0.05),细胞凋亡率降低(P<0.05),细胞上清中MMP-2和MMP-9水平降低(P<0.05),细胞中Bax、Cleaved-caspase-3和Beclin1蛋白表达水平及LC3 Ⅱ/LC3 Ⅰ比值降低(P<0.05),Bcl-2 和P62 蛋白表达水平升高(P<0.05).与Ang Ⅱ+ si-FOXO1组比较,Ang Ⅱ+si-FOXO1+Rap组细胞凋亡率升高(P<0.05),细胞上清中MMP-2和MMP-9水平升高(P<0.05),细胞中Beclin1蛋白表达水平和LC3 Ⅱ/LC3 Ⅰ比值降低(P<0.05),P62蛋白表达水平升高(P<0.05).结论:FOXO1基因沉默可能通过降低自噬水平来提高Ang Ⅱ暴露下hVSMCs增殖活性,并抑制其凋亡,从而参与AAA的发病.
Abstract
Objective:To discuss the effect of forkhead box O1(FOXO1)gene on the autophagy and apoptosis of the vascular smooth muscle cells in abdominal aortic aneurysm(AAA),and to clarify its possible mechanism.Methods:The aneurysm tissue of nineteen AAA patients(AAA group)and adjacent normal aortic tissue of nineteen AAA patients(control group)were collected.Real-time fluorenscence quantitative PCR(RT-qPCR)method was used to detect the expression level of FOXO1 mRNA in aneurysm tissue of the subjects in two groups;transmission electron microscope was used to observe the autophagolysosome formation in aneurysm tissue of the subjects in two groups;Western blotting method was used to detect the expression levels of FOXO1 and autophagy-related proteins B cell lymphoma-2(Bcl-2)-binding protein(Beclin1),microtubule-associated protein 1 light chain 3α(LC3),and P62 proteins in aneurysm tissue of the subjects in two groups.The human aortic vascular smooth muscle cells(hVSMCs)were cultured in vitro and infected with FOXO1 siRNA(si-FOXO1)and its negative control(si-NC)lentivirus,then treated with 10 μmol·L-1 angiotensin Ⅱ(Ang Ⅱ)combined with autophagy activator rapamycin(Rap).The cells were divided into blank control group,Ang Ⅱ group,Ang Ⅱ+ si-NC group,Ang Ⅱ + si-FOXO1 group,Ang Ⅱ+ si-NC + Rap group,and Ang Ⅱ+ si-FOXO1 + Rap group.CCK-8 assay was used to detect the proliferation activities of the cells in various groups;flow cytometry was used to detect the apoptotic rates of the cells in various groups;ELISA method was used to detect the levels of matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9)in the cell supernatant in various groups;RT-qPCR method was used to detect the expression level of FOXO1 mRNA in the cells in various groups;Western blotting method was used to detect the expression levels of FOXO1,Bcl-2,Bcl-2 associated X prorein(Bax),Cleaved-cysteinyl aspartate specific proteinase-3(Cleaved caspase-3),Beclin1,LC3,and P62 proteins in the cells in various groups.Results:Compared with control group,the expression level of FOXO1 mRNA in aneurysm tissue of the subjects in AAA group was increased(P<0.05),the number of autophagolysosomes was increased(P<0.05),the levels of Beclin1 protein and the ratio of LC3 Ⅱ/LC3 Ⅰ were increased(P<0.05),and the expression level of P62 protein was decreased(P<0.05).Compared with blank control group,the proliferation activity of the hVSMCs in Ang Ⅱ group was decreased(P<0.05),the apoptotic rate of the cells was increased(P<0.05),the levels of MMP-2 and MMP-9 in the cell supernatant were increased(P<0.05),the expression levels of Bax,Cleaved caspase-3,and Beclin1 proteins in the cells and the ratio of LC3 Ⅱ/LC3 Ⅰ were increased(P<0.05),and the expression levels of Bcl-2 and P62 proteins were decreased(P<0.05).Compared with Ang Ⅱ+ si-NC group,the proliferation activity of the hVSMCs in Ang Ⅱ+ si-FOXO1 group was increased(P<0.05),the apoptotic rate of the cells was decreased(P<0.05),the levels of MMP-2 and MMP-9 in the cell supernatant were decreased(P<0.05),the expression levels of Bax,cleaved caspase-3,and Beclin1 proteins in the cells and the ratio of LC3 Ⅱ/LC3 Ⅰ were decreased(P<0.05),and the expression levels of Bcl-2 and P62 proteins were increased(P<0.05).Compared with Ang Ⅱ+ si-FOXO1 group,the apoptotic rate of the hVSMCs in Ang Ⅱ+ si-FOXO1 + Rap group was increased(P<0.05),the levels of MMP-2 and MMP-9 in the cell supernatant were increased(P<0.05),the expression level of Beclin1 protein in the cells and the ratio of LC3 Ⅱ/LC3 Ⅰ were decreased(P<0.05),and the expression level of P62 protein was increased(P<0.05).Conclusion:Silencing the FOXO1 gene may increase the proliferation activity of the hVSMCs exposed to Ang Ⅱ by reducing the autophagy level and inhibiting the apoptosis,thus participating in the pathogenesis of AAA.
关键词
腹主动脉瘤/人血管平滑肌细胞/叉头框转录因子O1/自噬/细胞凋亡Key words
Abdominal aortic aneurysm/Human vascular smooth muscle cell/Forkhead box transcription factor O1/Autophagy/Apoptosis引用本文复制引用
基金项目
国家自然科学基金项目(82172095)
山东省科技厅自然科学基金项目(ZR2022ME083)
山东省卫健委中医药科技发展计划项目(2019-WJZD045)
山东省卫健委医药卫生科技发展项目(202104130109)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据