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.pdf

OSID开放科学标识码Jun.20232023年6 月Journal of Jinan University(Natural Science&Medicine Edition)No.3Vol.44第44卷第3期暨南大学学报（自然科学与医学版）miR-9-5p靶向调控ZAK对结直肠癌细胞增殖、迁移和侵袭的影响宋北平，蔡梦婷，李光义，易丹*（湖南省人民医院湖南师范大学附属第一医院1.核医学科；2.胃肠外科，湖南长沙410 0 0 5）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、W e s t e r mb l o t 检测结直肠癌细胞系（HCT116、SW 2 6 0、H T 2 9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-9-5pmimic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（P0.05）；在结直肠癌临床组织样本中,ZAK蛋白水平亦显著上调（P0.05）;mi R-9-5p 的表达水平在癌细胞系及组织样本中下调（P0.05）。双荧光素酶报道基因实验显示miR-9-5p能显著影响ZAK3UTR野生型表达载体的荧光素酶活性（P0.05），过表达miR-9-5pmimic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P0.05），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P0.05）。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。关键词miR-9-5p；ZA K；结直肠癌；增殖；迁移；侵袭【中图分类号】R735.3+5;R735.3+7文献标志码 A文章编号1000-9965(2023)03 0248-11D0I:10.11778/j.jdxb.20220242The impact of miR-9-5p targeted ZAK to regulate the proliferation,migration and invasion of colorectal cancer cellsSONG Beiping,(CAI Mingting,ILI Guangyi,)YI Dan1*(1.Department of Nuclear Medicine,2.Department of Gastrointestinal Surgery,Hunan Provincial Peoples Hospital/the First Affilated Hospital of Hunan Normal University,Changsha 410005,Hunan,China)AbstractObjective:To elucidate the molecular mechanism by which miR-9-5p regulates the收稿日期2022-08-03【基金项目湖南省卫生健康委科研计划项目（2 0 2 10 2 0 6 1339）作者简介宋北平（ORCID：0 0 0 0-0 0 0 1-950 9-6 6 98），男，研究方向：核医学、结直肠肿瘤，E-mail:SBP通信作者：易丹（0 RCID：0 0 0 0-0 0 0 1-550 5-8 40 0），男，主任医师，研究方向：核素显像诊断及甲亢、肿瘤转移、疤痕、血管瘤的核素治疗,E-mail:249宋北平，等靶向调控ZAK对结直肠癌细胞增殖、迁移和侵袭的影响m1R-第3期proliferation,migration,and invasion abilities of colorectal cancer cells by targeting the expression ofZAK.Methods:The level of miR-9-5p and mRNA or protein expression levels of ZAK in colorectalcancer cell lines(HCT116,SW260,HT29 and LoVo)and clinical tissue samples were detected byqPCR 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 regulatoryrelationship between miR-9-5p and ZAK.Lipofection was used to co-transfect colorectal cancer cell linesHCT116 and SW620 with miR-9-5p mimics and/or ZAK plasmids.The effects of the miR-9-5p/ZAKsignaling axis on cell proliferation were observed using CCK-8 and colony formation assays,while theeffects on cell migration and invasion abilities were examined using Transwell assays.Results:Comparedwith human normal epithelial cell lines,the mRNA and protein levels of ZAK were significantly up-regulated in colorectal cancer cell lines(P 0.05),and ZAK protein level was up-regulated incolorectal cancer tissue samples(P 0.05).The expression level of miR-9-5p was down-regulated incancer cell lines and tissue samples(P0.05).Dual-luciferase reporter gene experiment showed thatmiR-9-5p significantly affected the luciferase activity of the wild-type ZAK 3UTR expression vector(P 0.05).Overexpression of the miR-9-5p mimic fragment inhibited ZAK protein expression,while theinhibitor fragment promoted ZAK protein expression.CCCK-8,colony formation and Transwellexperiments demonstrated that overexpression of ZAK significantly promoted the growth,clony formation,migration and invasion ability of colorectal cancer cell lines(all P0.05),while co-transfection ofmiR-9-5p counteracted the promoting effect of ZAK on cell function(all P0.05).Conclusion:ZAK ishighly expressed in colorectal cancer,while miR-9-5p is down-regulated;ZAK is a target gene regulatedby miR-9-5p;The miR-9-5p/ZAK signaling axis collectively regulates the growth,migration andinvasion of rectal cancer cells,involved in the occurrence and development of colorectal cancer.Key wordsmiR-9-5p;ZAK;colorectal cancer;proliferation;migration;invasion结直肠癌（colorectalcancer，CRC）是一种常见的消化道恶性肿瘤，其发病率位于恶性肿瘤的第3位，流行病学统计显示，全球范围内结直肠癌的患者数量呈逐年增加趋势，每年发病率接近140万人,约有6 9390 0 例死亡 1-2 。确诊时患者一般属于中、晚期，其5年生存率一般在50%左右，预后较差。目前为止，结直肠癌发生发展及转移的分子机制尚不明确，呕待阐明。ZAK(leucine-zipper and sterile-alpha motif kinase）由Liu等 3 在2 0 0 0 年首次克隆并表达出来，归属混合谱系激酶（mixed lineagekinase，M LK）家族，具有有丝裂原活化蛋白激酶（mitogen-activatedprotein kinase kinase kinase，M A P3K）的功能。ZAK激酶与肿瘤的关系密切,是多种肿瘤的癌基因 4-5。然而在结直肠中,ZAK表达水平的调控机制不清，有待探讨。微小RNA（mi RNA s）是一类进化上高度保守、长约2 2 个核苷酸的非编码单链小分子RNA，通过与靶mRNAs的特异性结合而致其降解或抑制其翻译，对基因进行转录后的调控 6 。miR-9与结直肠癌的发生发展密切相关,是一种抑癌基因 7 ,但其调控的靶基因仍未完全阐明。生物信息学分析预测发现，miR-9-5p在ZAK基因的3 UTR区存在潜在的靶向序列,提示miR-9-5p可能调控ZAK，尚需实验进行验证。本研究旨在探讨miR-9-5p调控ZAK在结直肠癌中的作用及其分子机制，分析该信号轴对结直肠癌细胞增殖、迁移和侵袭能力的影响，为临床治疗结直肠癌提供潜在靶点和理论依据。1材料与方法1.1试剂与仪器人正常肠上皮细胞（NCM460）及结直肠癌细250第44卷暨南大学学报（目然科学与医学版）胞系（HCT116、SW 2 6 0、H T 2 9及LoVo）购自中国科学院上海细胞库。选取湖南省人民医院2 0 18年1月至2 0 19年12 月标本31例，其中结直肠癌组织及癌旁组织14对，对照组织包括癌旁组织及3例肠息肉组织。DMEM细胞培养液、胎牛血清等购自美国Gibco公司；转染用Lipofectamin2000购自Invitrogen公司；qPCRmiRNA及基因试剂盒均购自天根生化科技（北京）公司；miR-9-5pmimic模拟