RE-1序列沉默转录因子/Kv1.3通路调控血管平滑肌细胞表型转化促进腹主动脉瘤形成的机制  

Alternative splicing of RE-1 silencing transcription factor mediates vascular smooth muscle cell phenotypic switch during progression of abdominal aortic aneurysm via releasing Kv1.3 potassium channel

作　　者：王预立 姚永杰 陈亮 刘宇婷 李一男[1] 杨硕菲[1] 薛冠华[1] 张岚[1] Wang Yuli;Yao Yongjie;Chen Liang;Liu Yuting;Li Yinan;Yang Shuofei;Xue Guanhua;Zhang Lan(Department of Vascular Surgery,Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200127,China)

机构地区：[1]上海交通大学医学院附属仁济医院血管外科,200127

出　　处：《中华实验外科杂志》2022年第3期455-458,共4页Chinese Journal of Experimental Surgery

基　　金：国家青年科学基金(81800415);上海市自然科学基金(20ZR1471800);上海交通大学各类基金/转化医学交叉重点A类(YG2019ZDA12)。

摘　　要：目的探讨可变剪接调控RE-1序列沉默转录因子(REST)在血管平滑肌细胞(VSMC)表型转化及腹主动脉瘤(AAA)发生发展中起到的作用。方法检测人AAA组织和小鼠AAA模型及对照正常动脉中膜VSMC形态和弹性纤维改变;实时定量反转录聚合酶链反应(RT-qPCR)、蛋白质免疫印迹法(western blot)检测AAA组织及对照正常动脉组织中REST及其剪接异构体REST4的表达。RT-qPCR和Western blot检测沉默REST对人原代主动脉平滑肌细胞(HASMC)收缩型标志物α-SMA、合成型标志物波形蛋白(VIM)以及钾离子通道Kv1.3蛋白表达水平的影响。细胞增殖检测法(CCK-8)和细胞结晶紫染色实验检测沉默REST后HASMC增殖能力的改变。t检验验证定量数据的统计学差异。结果人和小鼠AAA中膜平滑肌细胞形态由梭型向分支型转化,AAA组REST表达低于对照组、REST4表达高于对照组(REST=0.143±0.086,t=5.672,P<0.05;REST=115.245±8.909,t=21.460,P<0.01)。沉默REST后,α-SMA表达量低于对照组,VIM和Kv1.3表达量高于对照组(α-SMA=0.335±0.030,t=9.711,P<0.01;VIM=10.177±0.728,t=15.170,P<0.01;Kv1.3=13.427±1.103,t=14.690,P<0.01),同时沉默REST后,VSMC增殖能力高于对照组(A_(450):2.41±0.27比3.85±0.14,t=6.744,P<0.01;光密度值:29.00±2.46比348.25±27.36,t=16.440,P<0.01)。上述作用可被Kv1.3特异性抑制剂逆转(α-SMA=2.448±0.498,t=4.314,P<0.05;VIM=0.758±0.030,t=4.018,P<0.05)。结论AAA中膜平滑肌细胞中REST可变剪接上调,抑制REST功能,激活Kv1.3表达,进而诱导VSMC表型转化。Objective To explore the molecular mechanism of vascular smooth muscle cell(VSMC)phenotypic switch induced by RE-1 silencing transcription factor(REST)alternative splicing during progression of abdominal aortic aneurysm(AAA).Methods Histology staining was used to observe VSMCs morphological changes and degradation of elastic fibers of both human and mouse AAA media.Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting were performed to detect the expression level of REST and its AS variant REST4 in the media of AAA tissue.After knockdown of REST,the expression level ofα-SMA,Vimentin(VIM)and Kv1.3 was detected by RT-qPCR and Western blotting.The cell proliferation ability was measured by cell counting kit-8(CCK-8)assay and crystal violet staining.Student’s t test was applied for statistical analysis.Results Downregulation of REST and upregulation of REST4 were found in AAA tissue,demonstrating the increase in REST AS during development of AAA(REST=0.143±0.086,t=5.672,P<0.01;REST4=115.245±8.909,t=21.460,P<0.01).The Knockdown of REST gene increased the expression of Kv1.3 and induced change of VSMC phenotypic biomarker(α-SMA=0.335±0.030,t=9.711,P<0.01;VIM=10.177±0.728,t=15.170,P<0.01;Kv1.3=13.427±1.103,t=14.690,P<0.01).Meantime,the decreased REST improved HASMCs proliferation and migration ability(A450:2.41±0.27 vs.3.85±0.14,t=6.744,P<0.01;IOD:29.00±2.46 vs.348.25±27.36,t=16.440,P<0.01).VSMC phenotypic switch induced by REST AS could be inverted by blocking Kv1.3(α-SMA=2.448±0.498,t=4.314,P<0.05;VIM=0.758±0.030,t=4.018,P<0.05).Conclusion REST AS was up-regulated in the media of AAA.REST AS could induce VSMC phenotypic switch and improve VSMCs proliferation ability by releasing Kv1.3 contributing to the progression of AAA.

关 键 词：腹主动脉瘤 平滑肌细胞 钾离子通道 

分 类 号：R543.16[医药卫生—心血管疾病]