肽基脯氨酰顺反异构酶1对喉癌细胞系Hep-2细胞生物学行为的影响  

Effect of peptidylprolyl cis/trans isomerase NIMA-interacting 1 on the biological behavior of Hep-2 cells

作　　者：王辉[1] 李章富[1] 温行 刘剑利[1] 李福才[1] WANG Hui;LI Zhangfu;WEN Xing;LIU Jianli;LI Fucai(Department of Medical Genetics,School of Life Sciences,China Medical University,Shenyang 110122,China)

机构地区：[1]中国医科大学生命科学学院医学遗传学教研室,沈阳110122

出　　处：《中国医科大学学报》2021年第8期681-689,共9页Journal of China Medical University

基　　金：国家自然科学基金(81272969)。

摘　　要：目的探讨肽基脯氨酰顺反异构酶1(Pin1)对喉癌细胞系Hep-2细胞中心体扩增以及生物学行为的影响。方法培养人喉癌细胞系Hep-2细胞,构建、转染Pin1 siRNA并分组。Pin1过表达分为转染pcDNA3.1组(NC组)、转染pcDNA3.1-Pin1组(Pin1组)、转染pcDNA3.1-Pin1后加核转录因子(NF-κB/p65)抑制剂(BAY11-7082)组(Pin1+7082组)。Pin1干扰分为转染阴性对照组(NC组)、转染siRNA组(Si-Pin1组)、转染siRNA后加NF-κB/p65激活剂(IL-1β)组(Si-Pin1+IL-1β组)。Western blotting检测Pin1、CDK2及NF-κB/p65蛋白表达,实时PCR检测Pin1及CDK2基因表达,免疫荧光实验检测细胞中心体扩增及NF-κB/p65核转位,流式细胞仪检测Hep-2细胞周期、凋亡、克隆形成,CCK-8实验检测Hep-2细胞增殖能力,Transwell及划痕实验检测Hep-2细胞的迁移能力。结果Pin1过表达促进中心体复制异常、克隆形成、细胞迁移、细胞周期G1/S转化,NF-κB/p65核转位以及CDK2的表达;NF-κB/p65抑制剂能够减弱这些效应。下调Pin1表达能够抑制中心体异常复制、克隆形成、细胞迁移、细胞周期G1/S的转化,NF-κB/p65入核以及CDK2的表达;NF-κB/p65激活剂能够逆转这些作用。结论Pin1可能经NF-κB通路上调CDK2的表达来影响Hep-2细胞的生物学行为。Objective To determine whether peptidylprolyl cis/trans isomerase NIMA-interacting 1(Pin1)affects the centrosome number and biological behavior of Hep-2 cells.Methods Hep-2 cells(human laryngeal cancer cell line)were cultured,constructed,transfected with Pin1 siRNA,and divided into the following groups:Pin1 overexpression,negative control(NC)group(transfected with the pcDNA3.1 plasmid),Pin1 group(transfected with pcDNA3.1-Pin1 plasmid),Pin1+7082 group(transfected with pcDNA3.1-Pin1 plasmid and treated with BAY11-7082).Pin1 knockdown,NC group(transfected with the negative control),Si-Pin1 group(transfected with Pin1 siRNA),Si-Pin1+IL-1βgroup(transfected with Pin1 siRNA and treated with IL-1β).The protein expression levels of Pin1,CDK2,and NF-κB/p65 were determined by Western blotting.The mRNA expression levels of Pin1 and CDK2 were determined by RT-PCR.Immuno-fluorescence was used to detect centrosome abnormality and nuclear translocation of NF-κB/p65.Flow cytometry was used to evaluate the cycle distribution and apoptosis of Hep-2 cells.Hep-2 cell proliferation was evaluated using the CCK-8 assay and clone formation assay.The migration ability of Hep-2 cells was assessed by scratch-wound and transwell assays.Results Results showed that Pin1 overexpres-sion promoted abnormal centrosome amplification,clone formation,cell migration,cell-cycle G1/S transition,nuclear import of NF-κB/p65,and CDK2 expression that could be eliminated by an NF-κB/p65 inhibitor.Upon downregulating Pin1 expression,abnormal centro-some amplification,clone formation,cell migration,cell cycle G1/S transition,nuclear import of NF-κB/p65,and CDK2 expression were all inhibited but could be reversed by treatment with an NF-κB/p65 activator.Conclusion Pin1 can upregulate CDK2 expression through the NF-κB pathways;this may be a mechanism by which Pin1 affects the biological behavior of Hep-2 cells.

关 键 词：肽基脯氨酰顺反异构酶1 喉癌细胞系 生物学行为 

分 类 号：R394[医药卫生—医学遗传学]