一链双靶siRNA对皮肤鳞状细胞癌NET-1和Survivin基因表达及增殖和凋亡的影响研究  

Experimental study on inhibition of NET-1 and Survivin gene expression and proliferation and apoptosis by double target of siRNA in A431 cell line

作　　者：季周婧 张丽丽[1] 张捷[1] JI Zhoujing;ZHANG Lili;ZHANG Jie(Department of Dermatology,Affiliated Hospital of Nantong University,Jiangsu 226001)

机构地区：[1]南通大学附属医院皮肤科,江苏226001

出　　处：《交通医学》2023年第1期14-19,23,共7页Medical Journal of Communications

基　　金：南通市基础研究计划项目(JC12022018)。

摘　　要：目的:探讨一链双靶siRNA对人皮肤鳞癌细胞株(A431)NET-1和Survivin基因的抑制作用以及对细胞增殖、凋亡的影响。方法:构建siRNA NET-1、siRNA Survivin以及同时靶向NET-1和Survivin基因的一链双靶siRNA。采用免疫共沉淀法检测NET-1和Survivin蛋白在A431人皮肤鳞癌细胞中的相互作用。将A431细胞分为siRNA-NET-1组、siRNA-Survivin组、siRNA-NET-1&Survivin组、siRNA-NC组和control组,分别进行细胞转染。qRT-PCR和Western Blot法分别检测细胞内NET-1、Survivin mRNA和蛋白表达,细胞免疫荧光法观察NET-1、Survivin蛋白在细胞内的表达及定位。CCK-8法和流式细胞仪分别检测细胞增殖与凋亡情况。结果:免疫共沉淀法证实NET-1和Survivin蛋白有相互作用。qRT-PCR和Western Blot结果显示,siRNA-NET-1&Survivin组、siRNA-NET-1组和siRNA-Survivin组细胞中NET-1和Survivin mRNA和蛋白表达水平均低于siRNA-NC组及control组,siRNA-NET-1&Survivin组低于siRNA-NET-1组和siRNA-Survivin组,差异均有统计学意义(P<0.05)。CCK-8法检测显示,siRNA-NET-1&Survivin组、siRNA-NET-1组和siRNA-Survivin组细胞增殖水平低于siRNA-NC组和control组,siRNA-NET-1&Survivin组低于siRNA-NET-1组和siRNA-Survivin组,差异均有统计学意义(P<0.05)。流式细胞仪检测显示,siRNA-NET-1&Survivin组、siRNA-NET-1组和siRNA-Survivin组细胞凋亡率高于siRNA-NC组和control组,siRNA-NET-1&Survivin组高于siRNA-NET-1组和siRNA-Survivin组,差异均有统计学意义(P<0.05)。细胞免疫荧光显示,siRNA-NET-1&Survivin组、siRNA-NET-1组和siRNA-Survivin组NET-1和Survivin蛋白表达阳性细胞百分率低于control组,siRNA-NET-1&Survivin组低于siRNA-NET-1和siRNA-Survivin组,差异均有统计学意义(P<0.05)。结论:NET-1和Survivin蛋白存在相互作用,靶向NET-1和Survivin的一链双靶siRNA能同时下调A431细胞NET-1和Survivin基因表达,并能抑制A431细胞增殖,促进细胞凋亡,一链双靶siRNA作用效果优于单靶siRNA。Objective:To investigate the effect of double target of siRNA on NET-1 and Survivin expression and on the proliferation and apoptosis in skin squamous cell carcinoma cell line(A431).Methods:Synthesize the siRNA sequence targeting the single gene(siRNA-NET-1 and siRNA-Survivin)and both NET-1 and Survivin gene(one-chain-double-target siRNA).Detection of the interaction between NET-1 and Survivin in skin squamous cell carcinoma cell line(A431)by co-immunoprecipitation.A431 cells were divided into siRNA-NET-1 group,siRNA-Survivin group,siRNA-NET-1&Survivin group,siRNA-NC group,and control group for cell transfection.The levels of NET-1 and Survivin mRNA and protein expression in A431 cells were detected by qRT-PCR and Western Blot,and their protein localization and expression were documented by Immunofluorescence.The proliferation and apoptosis of A431 cells were detected by CCK-8 assay and flow cytometry(FCM).Results:Co-immunoprecipitation confirmed the interaction between NET-1 and survivin.The qRT-PCR and Western Blot showed that the expression levels of NET-1 and survivin mRNA and protein in cells of siRNA-NET-1&Survivin group,siRNA-NET-1 group,and siRNA-Survivin group were lower than those of siRNA-NC group and control group,the siRNA-NET-1&Survivin group was significantly lower than those of the siRNA-NET-1 group and the siRNA-Survivin group(P<0.05).CCK-8 showed that the level of cell proliferation in siRNA-NET-1&Survivin group,siRNA-NET-1 group,and siRNA-Survivin group was lower than that in siRNA-NC group and control group,the siRNA-NET-1&Survivin group was significantly lower than that in the siRNA-NET-1 group and the siRNA-Survivin group(P<0.05).Flow cytometry showed that the cell apoptosis rate in siRNA-NET-1&Survivin group,siRNA-NET-1 group,and siRNA-Survivin group was higher than that in siRNA-NC group and the control group,the siRNA-NET-1&Survivin group was significantly higher than the siRNA-NET-1 group and the siRNA-Survivin group(P<0.05).Immunofluorescence showed that the percentage of NET-1 and Surv

关 键 词：NET-1 SURVIVIN 一链双靶siRNA A431 RNA干扰 皮肤鳞状细胞癌 

分 类 号：R739.5[医药卫生—肿瘤]