Kindlin-2 RNA干扰经Wnt信号通路抑制血管平滑肌细胞增殖  

Kindlin-2 RNA interference inhibits vascular smooth muscle cell proliferation via the Wnt signaling pathway

作　　者：吴校林[1] 朱锐[1] 周青[1] 胡河 朱通建[1] 刘文卫[1] Wu Xiaolin;Zhu Rui;Zhou Qing;Hu He;Zhu Tongjian;Liu Wenwei(Department of Cardiology,Affiliated Hospital of Hubei University of Arts and Science,Xiangyang Central Hospital)

机构地区：[1]襄阳市中心医院、湖北文理学院附属医院心血管内科,襄阳441000

出　　处：《重庆医科大学学报》2020年第4期504-509,共6页Journal of Chongqing Medical University

基　　金：国家自然科学基金青年科学基金资助项目(编号:81600288);湖北省自然科学基金青年科学基金资助项目(编号:2016CFB344)。

摘　　要：目的:观察Kindlin-2 RNA干扰(RNA interference,RNAi)对大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖的影响并探讨相关的作用机制。方法:构建并制备Kindlin-2 siRNA慢病毒载体并感染大鼠VSMC,CCK-8和BrdU技术检测重组Wnt3a蛋白诱导的VSMC增殖情况,实时定量PCR测定VSMC中Kindlin-2、c-Myc和cyclin D1 mRNA的表达水平,免疫共沉淀了解Kindlin-2和β-catenin的关系,Western blot检测VSMC中Kindlin-2、β-catenin、磷酸化β-catenin(Ser675)、GSK-3β和磷酸化GSK-3β(Ser9)蛋白的表达。结果:Kindlin-2 siRNA慢病毒载体能有效感染大鼠VSMC。CCK-8和BrdU结果表明Kindlin-2 RNAi能够显著抑制Wnt3a诱导的VSMC增殖(1.12±0.14 vs. 2.25±0.15,P=0.000;0.162±0.017 vs. 0.288±0.019,P=0.000)。与阴性对照组相比,Kindlin-2 RNAi组和Kindlin-2 RNAi+Wnt3a组中Kindlin-2、c-Myc和cyclin D1 mRNA表达水平均明显下降(0.964±0.014 vs. 0.530±0.029,P=0.000;0.980±0.025 vs. 0.572±0.022,P=0.000;0.979±0.009 vs. 0.590±0.035,P=0.002和0.964±0.014 vs. 0.569±0.027,P=0.000;0.980±0.025 vs. 0.741±0.026,P=0.001;0.979±0.009 vs. 0.769±0.017,P=0.023)。免疫共沉淀证实VSMC中Kindlin-2可以与β-catenin结合,而且Kindlin-2 RNAi+Wnt3a组中Kindlin-2、磷酸化β-catenin(Ser675)和磷酸化GSK-3β(Ser9)蛋白的表达水平较阴性对照+Wnt3a组明显降低(0.468±0.029 vs. 0.725±0.033,P=0.001;1.058±0.109 vs. 1.478±0.045,P=0.001;0.624±0.048 vs. 0.809±0.067,P=0.020)。但各组中总β-catenin和总GSK-3β蛋白水平没有明显变化(F=0.638,P=0.647;F=0.781,P=0.563)。结论:Kindlin-2 RNAi可以通过Wnt信号通路发挥作用并抑制VSMC增殖。Objective:To investigate the effect of Kindlin-2 RNA interference(RNAi)on rat vascular smooth muscle cell(VSMC)proliferation and related mechanism of action. Methods:Kindlin-2 small interfering RNA(siRNA)lentiviral vectors were constructed and then used to infect rat VSMCs. CCK-8 assay and the BrdU technique were used to assess the proliferation of VSMCs induced by recombinant Wnt3 a protein;quantitative real-time PCR was used to measure the mRNA expression of Kindlin-2,c-myc,and cyclinD1 in VSMCs;co-immunoprecipitation was used to evaluate the association between Kindlin-2 and β-catenin;Western blot was used to measure the protein expression of Kindlin-2,β-catenin,phosphor-β-catenin(Ser675),glycogen synthase kinase-3β(GSK-3β),and phosphor-GSK-3β(Ser9)in VSMCs. Results:Kindlin-2 siRNA lentiviral vectors effectively infected rat VSMCs. The CCK-8 and BrdU results indicated that Kindlin-2 RNAi significantly inhibited the proliferation of VSMCs induced by Wnt3 a(1.12±0.14 vs. 2.25±0.15,P=0.000;0.162±0.017 vs. 0.288±0.019,P=0.000). Compared with the negative control group,the Kindlin-2 RNAi group had significantly lower m RNA expression of Kindlin-2,c-Myc,and cyclin D1(Kindlin-2:0.530 ±0.029 vs. 0.964±0.014,P=0.000;c-Myc:0.572±0.022 vs. 0.980±0.025,P=0.000;cyclin D1:0.590±0.035 vs. 0.979±0.009,P=0.002),and the Kindlin-2 RNAi+Wnt3 a group also had significantly lower expression than the negative control group(Kindlin-2:0.569±0.027 vs. 0.964±0.014,P=0.000;c-Myc:0.741±0.026 vs. 0.980±0.025,P=0.001;cyclin D1:0.769±0.017 vs. 0.979±0.009,P=0.023).Co-immunoprecipitation confirmed that Kindlin-2 could bind to β-catenin in VSMCs,and the Kindlin-2 RNAi +Wnt3 a group had significantly lower protein expression of Kindlin-2,phosphor-β-catenin(Ser675),and phosphor-GSK-3β(Ser9)than the negative control+Wnt3 a group(Kindlin-2:0.468±0.029 vs. 0.725±0.033,P =0.001;phosphor-β-catenin:1.058 ±0.109 vs. 1.478±0.045,P=0.001;phosphor-GSK-3β:0.624±0.048 vs. 0.809±0.067,P=0.020). There were no significant chang

关 键 词：Kindlin-2 RNA干扰 血管平滑肌细胞 WNT信号通路 

分 类 号：R541.4[医药卫生—心血管疾病]