CDCA7通过与MCM3相互作用介导胰腺癌细胞增殖、侵袭及迁移  

CDCA7 mediates proliferation, invasion and migration of pancreatic cancer cells by interacting with MCM3

作　　者：邓路 何志伟 朱昌毫 陈世裕 刘燕青 李琳 孙诚谊 喻超 DENG Lu;HE Zhiwei;ZHU Changhao;CHEN Shiyu;LIU Yanqing;LI Lin;SUN Chengyi;YU Chao(School of Clinical Medicine,Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Department of Hepatobiliary Surgery,Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Key Laboratory of Liver,Gallbladder,Pancreas and Spleen,Guizhou Medical University,Guiyang 550004,Guizhou Province,China;Guizhou Provincial Institute of Liver,Gallbladder,Pancreas and Spleen Diseases,Guiyang 550004,Guizhou Province,China)

机构地区：[1]贵州医科大学临床医学院,贵州贵阳550004 [2]贵州医科大学附属医院肝胆外科,贵州贵阳550004 [3]贵州医科大学肝胆胰脾重点实验室,贵州贵阳550004 [4]贵州省肝胆胰脾疾病研究所,贵州贵阳550004

出　　处：《肿瘤》2021年第1期24-35,共12页Tumor

基　　金：国家自然科学基金资助项目(编号:81860505;81860506);贵州省科技计划资助项目[编号:黔科合LH字(2016)7229;黔科合平台人才(2017)5404];贵州省研究生导师工作室建设资助项目[编号:黔教研合GZS(2016)09]。

摘　　要：目的:探讨细胞分裂周期相关蛋白7(cell division cycle-associated protein 7,CDCA7)对人胰腺癌细胞增殖、侵袭及迁移能力的影响,及其可能的作用机制。方法:利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)和基因表达谱数据动态分析(Gene Expression Profiling Interactive Analysis,GEPIA)数据库分析胰腺癌组织及癌旁组织中CDCA7表达水平的差异,并采用实时荧光定量PCR和蛋白质印迹法分别检测CDCA7 mRNA和蛋白在人正常胰腺导管上皮细胞HPDE和人胰腺癌Bxpc-3、CFPAC-1、PANC-1、MIA PaCa-2、Capan2及SW1990细胞中的表达水平。构建干扰CDCA7基因表达的重组慢病毒,稳定转入PANC-1细胞,并分别采用实时荧光定量PCR和蛋白质印迹法检测干扰效率。干扰CDCA7基因表达后,分别采用CCK-8法和平板克隆实验检测对PANC-1细胞增殖能力的影响,细胞划痕愈合实验及Transwell小室法检测对PANC-1细胞迁移及侵袭能力的影响;进一步采用蛋白质印迹法检测干扰CDCA7表达后对上皮-间质转化相关蛋白波形蛋白(Vimentin)和E-钙黏蛋白(E-cadherin),以及细胞周期蛋白D1(cyclin D1)和cyclin E1表达水平的影响。通过生物信息学网站(STING和GEPIA)预测CDCA7与微型染色体维持蛋白3(minichromosome maintenance protein 3,MCM3)的关系,并进一步采用免疫共沉淀法检测CDCA7与MCM3的相互作用,采用蛋白质印迹法检测干扰CDCA7表达后MCM3蛋白表达水平的变化。结果:CDCA7在胰腺癌组织中的表达水平明显高于癌旁组织(P<0.05);CDCA7 mRNA及蛋白在胰腺癌PANC-1、MIA PaCa-2和SW1990细胞中的表达水平均明显高于人正常胰腺导管上皮细胞HPDE(P值均<0.05)。干扰CDCA7表达后,PANC-1细胞中CDCA7 mRNA和蛋白的表达水平明显下降(P值均<0.05),PANC-1细胞的增殖、侵袭及迁移能力明显降低(P值均<0.05);Vimentin、cyclin D1和cyclin E1蛋白的表达水平均明显下调,而E-cadherin蛋白的表达水平明显上调(P值均<0.05)。CDCA7与MCM3�Objective:To investigate the effects of cell division cycle-associated protein 7(CDCA7)on the proliferation,invasion and migration of human pancreatic cancer cells and its possible mechanism.Methods:The expression level of CDCA7 in pancreatic cancer tissues and adjacent tissues was analyzed by Cancer Genome Atlas(TCGA)and Gene Expression Profiling Interactive Analysis(GEPIA)databases.The expression level of CDCA7 mRNA and protein in human normal pancreatic ductal epithelial HPDE cells and human pancreatic cancer cell lines Bxpc-3,CFPAC-1,PANC-1,MIA PaCa-2,Capan2 and SW1990 were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.A recombinant lentivirus interfering with CDCA7 gene expression was constructed and stably transfected into PANC-1 cells,and the interference efficiency was detected by real-time fluorescence quantitative PCR and Western blotting,respectively.After interfering with CDCA7 gene expression,CCK-8 and colony formation assay were used to detect the proliferation of PANC-1 cells,the invasion and migration capacities of PANC-1 cells were detected by wound healing and Transwell assays.The expression levels of epithelial-mesenchymal transition-related proteins Vimentin,E-cadherin,cell cycle protein cyclin D1 and cyclin E1 were detected by Western blotting.The correlation between CDCA7 and minichromosome maintenance protein 3(MCM3)was analyzed by STING and GEPIA databases.Co-immunoprecipitation assay was used to detected the interaction between CDCA7 and MCM3.The expression level of MCM3 protein after CDCA7 gene interfering was detected by Western blotting.Results:The expression level of CDCA7 in pancreatic cancer tissues was significantly higher than that in the adjacent tissues(P<0.05),and the expression level of CDCA7 mRNA and protein in PANC-1,MIA PaCa-2 and SW1990 cells were significantly higher than that in HPDE cells(all P<0.05).After CDCA7 silencing,the proliferation,invasion and migration capacities of PANC-1 cells were significantly decreased(all P<0.05);Af

关 键 词：胰腺肿瘤 细胞分裂周期相关蛋白7 细胞增殖 细胞运动 

分 类 号：R735.9[医药卫生—肿瘤]