周期蛋白依赖激酶抑制因子3通过调控G2/M期检查点促进肝内胆管癌细胞生长并降低对顺铂的敏感性  

CDKN3 reduces the sensitivity of intrahepatic cholangiocarcinoma cells to cisplatin by regulating G2/M checkpoint

作　　者：朱志文 阳亮芳 郑杨 黄海丽 何惠娟 ZHU Zhiwen;YANG Liangfang;ZHENG Yang;HUANG Hail;HE Huijuan(Department of Hepatobiliary Surgery,the Affiliated Hospital of Guangdong Medical University,Zhanjiang 524001,China)

机构地区：[1]广东医科大学附属医院肝胆外科,广东湛江524001

出　　处：《实用医学杂志》2022年第22期2768-2773,2779,共7页The Journal of Practical Medicine

基　　金：国家自然科学基金(编号:81600484);广东省自然科学基金(编号:2018A030307066)。

摘　　要：目的探究周期蛋白依赖激酶抑制因子3(CDKN3)在肝内胆管癌中的表达和产生的影响,及其发挥作用的分子机制。方法生物信息学和免疫组化分析CDKN3的表达情况;将CDKN3干扰质粒、CDKN3过表达质粒和阴性对照质粒转入肝内胆管癌细胞系(HCCC-9810)并通过Western blot检测转染效果;通过克隆实验和划痕实验检测HCCC-9810细胞的增殖和迁移能力;Western blot检测相关通路蛋白表达情况;细胞周期实验分析处于各个时期的细胞比例。结果与正常组织相比,CDKN3在胆管癌组织中呈高表达(P<0.05);KEGG和GO富集分析显示CDKN3与细胞周期G2/M期检查点密切相关;干扰CDKN3后,诱导HCCC-9810细胞G2/M期阻滞,导致增殖和迁移能力减弱(P<0.05);进一步研究发现,沉默CDKN3可下调CDC25C/CDK1轴活性,导致CDK1低活性的磷酸化水平增高;此外,CDKN3过表达可显著降低HCCC-9810细胞对顺铂的敏感性(P<0.05)。结论CDKN3在胆管癌组织中被显著上调,CDKN3可能通过调控G2/M期检查点相关蛋白的表达,从而参与到肝内胆管癌的发生发展和化疗耐药。Objective To investigate the expression and effect of CDKN3 in intrahepatic cholangiocarcinoma and its molecular mechanism.Methods The expression of CDKN33 was analyzed by bioinformatics and immunohistochemistry.CDKN3 interference plasmid,CDKN3 overexpression plasmid and negative control plasmid were transferred into intrahepatic cholangiocarcinoma cell line(HCCC-9810)and the transfection effect was detected by western blot.Proliferation and migration ability of HCCC-9810 cells were detected by colony formation experiment and healing experiment.The expression of related pathway proteins was detected by western blot.Cell cycle asssay was used to analyze the proportion of cells at various stages.Results CDKN3 was highly expressed in cholangiocarcinoma when compared with that in normal tissues(P<0.05).KEGG and GO enrichment analysis showed that CDKN3 was closely related to G2/M checkpoint of cell cycle.Interfering CDKN3 induced G2/M phase arrest of HCCC-9810 cells,resulting in decreased proliferation and migration(P<0.05).Further studies showed that CDKN3silence down-regulated the activity of CDC25C/CDK1 axis,leading to increased phosphorylation level(P<0.05).In addition,CDKN3 overexpression significantly reduced the sensitivity of HCCC-9810 cells to cisplatin(P<0.05).Conclusion CDKN3 is significantly up-regulated in intrahepatic cholangiocarcinoma.CDKN3 may be involved in the development of intrahepatic cholangiocarcinoma by regulating the expression of proteins related to G2/M checkpoint.

关 键 词：肝内胆管癌 CDKN3 细胞周期 化疗耐药 

分 类 号：R735.8[医药卫生—肿瘤]