过表达KLF7对非小细胞肺癌细胞增殖凋亡的影响  

Effect of overexpression of KLF7 on proliferation and apoptosis of human non⁃small cell lung cancer cells

作　　者：吕巧 闵迁 姜露 陈黔 彭锦 代黔 Lv Qiao;Min Qian;Jiang Lu;Chen Qian;Peng Jin;Dai Qian(Clinical Research Center,The Second Affiliated Hospital,Army Medical University,Chongqing 400037,China)

机构地区：[1]陆军军医大学第二附属医院临床医学研究中心,重庆400037

出　　处：《中华肺部疾病杂志(电子版)》2025年第1期1-7,共7页Chinese Journal of Lung Diseases(Electronic Edition)

基　　金：重庆市自然科学基金面上项目(CSTB2023NSCQ⁃MSX0417)。

摘　　要：目的通过构建Krüppel样转录因子7(krüppel⁃like factor 7,KLF7)真核表达载体,分析KLF7对肺癌细胞增殖、凋亡的调控作用,为发现非小细胞肺癌(non⁃small⁃cell lung cancer,NSCLC)治疗靶点提供新的线索。方法通过聚合酶链反应(polymerase chain reaction,PCR)方法克隆KLF7完整序列,酶切、连接、测序等试验方法构建过表达载体并验证表达效率。将A594细胞随机分空载实验为对照组和KLF7过表达为KLF7组,采用流式细胞术检测A594细胞周期、凋亡和活性氧变化,EdU检测A594细胞增殖情况,实时荧光定量PCR及蛋白质印迹法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、G1/S⁃特异性周期蛋白⁃D1(cyclin D1)、B⁃细胞淋巴瘤⁃2(B⁃cell lymphoma⁃2,Bcl⁃2)、Bcl⁃2相关X蛋白(Bcl⁃2⁃associated X protein,Bax)mRNA和蛋白表达水平。结果克隆KLF7 CDS区全长693bp,并连接在pCDNA3.1载体上,构建KLF7过表达载体,提高HEK293T及A594细胞中KLF7的表达水平(P<0.01)。与对照组相比,KLF7组在G1期细胞百分比降低,G2期细胞百分比升高,增殖指数(proliferation index,PI)明显升高,同时EdU阳性细胞比例升高(P<0.01)。与对照组比较,KLF7组活性氧水平受到抑制,细胞凋亡率降低(P<0.01)。与对照组比较,KLF7组PCNA、CyclinD1、Bcl⁃2 mRNA相对表达和蛋白表达水平升高,Bax的mRNA相对表达和蛋白表达水平降低(P<0.01)。结论本文成功构建KLF7过表达载体,肺癌细胞中,KLF7过表达能促进细胞增殖并抑制细胞凋亡,参与肺癌发生,有望成为新的NSCLC生物标志物。Objective To investigate the regulation of KLF7 on the proliferation and apoptosis of lung cancer cells by constructing the KLF7 eukaryotic expression vector,thereby providing novel clues for the discovery of therapeutic targets for non⁃small cell lung cancer(NSCLC).Methods The entire sequence of KLF7 was cloned through PCR,and the overexpression vector was constructed by means of enzyme digestion,ligation,and sequencing,with the expression efficiency verified.A594 cells were randomly divided into the experimental control group(control group)and the KLF7 overexpression group(KLF7 group).Changes in the cell cycle,apoptosis,and reactive oxygen species of A594 cells were detected using flow cytometry,and the proliferation of A594 cells was examined by EdU.The expression levels of Proliferating Cell Nuclear Antigen(PCNA),G1/S⁃specific cyclin⁃D1(CyclinD1),B⁃cell lymphoma⁃2(Bcl⁃2,Bcl⁃2),Bcl⁃2⁃associated X protein(Bax)mRNA and protein were detected by real⁃time quantitative PCR and Western blotting.Results The total length of the CDS region of KLF7 was cloned to 693 bp and linked to the pCDNA3.1 vector to construct the KLF7 overexpression vector,which could significantly enhance the expression level of KLF7 in HEK293T and A594 cells(P<0.01).Compared with the control group,the percentage of cells in the G1 phase decreased,the percentage of cells in the G2 phase increased,the proliferation index(PI)significantly increased,and the percentage of EdU⁃positive cells significantly increased in the KLF7 overexpression group(P<0.01).Compared with the control group,the ROS level was inhibited and the apoptosis rate decreased in the KLF7 overexpression group(P<0.01).Compared with the control group,the relative expression levels of PCNA,CyclinD1,and Bcl⁃2 mRNA and protein increased in the KLF7 overexpression group,while the relative expression levels of Bax mRNA and protein decreased(P<0.01).Conclusion In this study,the KLF7 overexpression vector was successfully constructed.In lung cancer cells,KLF7 overexp

关 键 词：非小细胞肺癌 Krüppel样转录因子7 细胞增殖 细胞凋亡 肺癌细胞 

分 类 号：R734.2[医药卫生—肿瘤]