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。

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

王辉, 李章富, 温行, 刘剑利, 李福才

中国医科大学生命科学学院医学遗传学教研室, 沈阳 110122

收稿日期:2020-09-06出版日期:2021-08-30发布日期:2021-07-29
通讯作者:李福才E-mail:fc@cmu.edu.cn
作者简介:王辉(1989-),男,硕士研究生.
基金资助:国家自然科学基金（81272969）


关键词: 肽基脯氨酰顺反异构酶1, 喉癌细胞系, 生物学行为
Abstract: 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-1β group (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. immunofluorescence 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 overexpression 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 centrosome 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.
Key words: peptidylprolyl cis/trans isomerase NIMA-interacting 1, laryngeal cancer cell line, biological behavior
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