1、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
2、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（
3、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
4、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
5、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 th
6、e 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
7、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 a
8、nd 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 an
9、d 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
10、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 sample
11、s(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 exp
12、ression.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 ce
13、ll 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 devel
14、opment 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-zipp
15、er 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
16、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
