miR-9-5p靶向调控ZAK对结直肠癌细胞增殖、迁移和侵袭的影响  

The impact of miR-9-5p targeted ZAK to regulate the proliferation,migration and invasion of colorectal cancer cells

作　　者：宋北平 蔡梦婷 李光义[2] 易丹[1] SONG Beiping;CAI Mengting;LI Guangyi;YI Dan(Department of Nuclear Medicine,Hunan Provincial People's Hospital/the First Affilated Hospital of Hunan Normal University,Changsha 410005,Hunan,China;Department of Gastrointestinal Surgery,Hunan Provincial People's Hospital/the First Affilated Hospital of Hunan Normal University,Changsha 410005,Hunan,China)

机构地区：[1]湖南省人民医院/湖南师范大学附属第一医院核医学科,湖南长沙410005 [2]湖南省人民医院/湖南师范大学附属第一医院胃肠外科,湖南长沙410005

出　　处：《暨南大学学报（自然科学与医学版）》2023年第3期248-258,共11页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：湖南省卫生健康委科研计划项目(202102061339)。

摘　　要：目的:阐明miR-9-5p通过靶向调控ZAK表达从而影响结直肠癌细胞增殖、迁移和侵袭能力的分子机制。方法:通过qPCR、Western blot检测结直肠癌细胞系(HCT116、SW260、HT29及LoVo)及结直肠癌患者组织样本中miR-9-5p表达水平及ZAK的mRNA和蛋白表达水平。生物信息学方法分析miR-9-5p与ZAK结合的靶向序列。双荧光素酶基因报告实验检测miR-9-5p对ZAK的靶向调控关系。脂质体法将miR-9-5p mimic或/和ZAK质粒共转染结直肠癌细胞系HCT116及SW620,通过CCK-8及克隆形成实验观察miR-9-5p/ZAK信号轴对细胞增殖的影响,通过Transwell观察对细胞迁移和侵袭能力的影响。结果:与人正常上皮细胞系相比,在结直肠癌细胞系中ZAK的mRNA及蛋白水平显著上调(P<0.05);在结直肠癌临床组织样本中,ZAK蛋白水平亦显著上调(P<0.05);miR-9-5p的表达水平在癌细胞系及组织样本中下调(P<0.05)。双荧光素酶报道基因实验显示miR-9-5p能显著影响ZAK 3’UTR野生型表达载体的荧光素酶活性(P<0.05),过表达miR-9-5p mimic片段可抑制ZAK蛋白表达,而inhibitor片段促进ZAK蛋白表达。CCK-8、克隆形成实验及Transwell实验表明,过表达ZAK可显著促进结直肠癌细胞系细胞的增殖、克隆形成、细胞迁移和侵袭能力(均P<0.05),而同时过表达miR-9-5p则可抵消ZAK对细胞功能的促进作用(均P<0.05)。结论:结直肠癌中ZAK呈高表达,而miR-9-5p呈低表达;ZAK是miR-9-5p的靶向调控基因;miR-9-5p/ZAK信号轴共同调控结直肠癌细胞的增殖、迁移和侵袭,参与结直肠癌的发生发展。Objective:To elucidate the molecular mechanism by which miR-9-5p regulates the proliferation,migration,and invasion abilities of colorectal cancer cells by targeting the expression of ZAK.Methods:The level of miR-9-5p and mRNA or protein expression levels of ZAK in colorectal cancer cell lines(HCT116,SW260,HT29 and LoVo)and clinical tissue samples were detected by qPCR and western blot.Bioinformatics analysis was employed to analyze the target sequences of miR-9-5p binding to ZAK.Dual-luciferase reporter assays were conducted to investigate the regulatory relationship between miR-9-5p and ZAK.Lipofection was used to co-transfect colorectal cancer cell lines HCT116 and SW620 with miR-9-5p mimics and/or ZAK plasmids.The effects of the miR-9-5p/ZAK signaling axis on cell proliferation were observed using CCK-8 and colony formation assays,while the effects on cell migration and invasion abilities were examined using Transwell assays.Results:Compared with human normal epithelial cell lines,the mRNA and protein levels of ZAK were significantly upregulated in colorectal cancer cell lines(P<0.05),and ZAK protein level was up-regulated in colorectal cancer tissue samples(P<0.05).The expression level of miR-9-5p was down-regulated in cancer cell lines and tissue samples(P<0.05).Dual-luciferase reporter gene experiment showed that miR-9-5p significantly affected the luciferase activity of the wild-type ZAK 3'UTR expression vector(P<0.05).Overexpression of the miR-9-5p mimic fragment inhibited ZAK protein expression,while the inhibitor fragment promoted ZAK protein expression.CCCK-8,colony formation and Transwell experiments demonstrated that overexpression of ZAK significantly promoted the growth,clony formation,migration and invasion ability of colorectal cancer cell lines(all P<0.05),while co-transfection of miR-9-5p counteracted the promoting effect of ZAK on cell function(all P<0.05).Conclusion:ZAK is highly expressed in colorectal cancer,while miR-9-5p is down-regulated;ZAK is a target gene regulated by miR-9-5p;The mi

关 键 词：miR-9-5p ZAK 结直肠癌 增殖 迁移 侵袭 

分 类 号：R735.35[医药卫生—肿瘤] R735.37[医药卫生—临床医学]