慢病毒介导的CCDC80基因敲除通过降低Aib1表达抑制卵巢癌细胞增殖  

Lentivirus-mediated CCDC80 knock-out inhibits proliferation of ovarian cancer cells by decreasing Aib1 expression

作　　者：王卓文 陈子彦 杨柳青 胡汉寅 石子晴 林仙德 钱若文轩 郭燕君[1] 潘巍巍[1] WANG Zhuo-wen;CHEN Zi-yan;YANG Liu-qing;HU Han-yin;SHI Zhi-qing;LIN Xian-de;QIAN Ruo-wen-xuan;GUO Yan-jun;PAN Wei-wei(Department of Biology,College of Medicine,Jiaxing University,Jiaxing 314001,China)

机构地区：[1]嘉兴学院医学院生物教研室

出　　处：《中国病理生理杂志》2019年第5期777-783,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.31871402; No.81402162;浙江省自然科学基金资助项目(No.LY17H160060);浙江省大学生科技创新项目(No.2018R417024)

摘　　要：目的:探讨含卷曲螺旋结构域蛋白80(coiled-coil domain-conaining protein 80,CCDC80)基因敲除对人卵巢癌ES-2细胞增殖和凋亡的影响。方法:慢病毒介导的CRISPR/Cas9技术敲除卵巢癌ES-2细胞中的CCDC80基因,基因组测序检测CCDC80的敲除效率;细胞增殖曲线、细胞划痕实验和软琼脂克隆形成实验检测CCDC80基因敲除对细胞增殖、迁移和克隆形成能力影响;Annexin V/PI染色和流式细胞术检测CCDC80基因敲除后细胞周期和凋亡的变化;Western blot法分析增殖相关蛋白表达量;裸鼠荷瘤模型体内实验研究CCDC80基因敲除对卵巢癌细胞增殖的影响。结果:DNA测序结果显示慢病毒介导的CRISPR/Cas9方法可以在卵巢癌ES-2细胞中敲除CCDC80基因。CCDC80基因敲除可以显著抑制细胞增殖、克隆形成和迁移能力(P<0.01)。此外,CCDC80基因敲除可以减少G_1期细胞数目,增加S和G_2期细胞数,促进细胞凋亡(P<0.01)。裸鼠体内研究显示,CCDC80基因敲除可以显著抑制ES-2细胞体内增殖(P<0.01),免疫组化结果显示CCDC80基因敲除细胞形成的肿瘤组织DNA损伤蛋白p-H2AX表达增加,而增殖标志物BrdU表达量减少。Western blot结果显示CCDC80基因敲除细胞p-histone H3表达量减少,p-ERK1/2表达增加(P<0.01)。qPCR结果显示CCDC80基因敲除细胞中Aib1 mRNA表达显著降低(P<0.01)。结论:CCDC80基因敲除抑制了ES-2细胞增殖,促进细胞凋亡,其机制可能与影响Aib1蛋白表达及MAPK信号通路有关。AIM:To explore the role of coiled-coil domain-conaining protein 80(CCDC80)gene deletion in the proliferation and apoptosis of human ovarian cancer ES-2 cells.METHODS:Lentivirus-mediated CCDC80 deletion in ovarian cancer cells was conducted by CRISPR/Cas9 method.Genomic sequencing was used to detect knock-out efficiency.The proliferation and colony formation of CCDC80 deletion cells were determined by cell growth curve and soft agar assay.The migration of CCDC80 deletion cells was measured by cell scratch assay.The apoptosis and cell cycle were analyzed by Annexin V/PI staining and flow cytometry.The protein levels of p-histone H3 and p-ERK1/2 were determined by Western blot.Nude mouse model was established to detect the tumorigenic capacity of CCDC80 deletion cells in vivo.RESULTS:Genomic sequencing results showed that CCDC80 was efficiently knocked out in ES-2 cells.CCDC80 deletion significantly repressed the proliferation,migration and colony formation of ES-2 cells(P<0.01).CCDC80 deletion increased the apoptosis rate and affected G 1 and S progression(P<0.01).CCDC80 deletion repressed the cell proliferation(P<0.01)in vivo.IHC results showed that CCDC80 deletion increased DNA damage and decreased cell proliferation.Western blot results showed that the protein level of p-histone H3 was decreased,while the protein level of p-ERK1/2 was increased in CCDC80 deletion cells(P<0.01).qPCR results showed that CCDC80 deletion significantly decreased Aib1 mRNA expression(P<0.01).CONCLUSION:Genetically CCDC80 deletion represses ES-2 cell proliferation,migration and colony formation,and promotes cell apoptosis by decreasing Aib1 expression.

关 键 词：CCDC80基因 卵巢癌 细胞增殖 细胞凋亡 AIB1蛋白 

分 类 号：R737.31[医药卫生—肿瘤] R363.2[医药卫生—临床医学]