摘要
目的:观察Polo样激酶1(PLK1)在卵巢癌(OC)细胞和组织中的表达情况,以及抑制PLK1表达对耐5-氟尿嘧啶(5-FU)的OC细胞活力和凋亡的影响。方法:在2011年4月~2018年12月的126名OC患者纳入本研究。采用免疫组化法检测PLK1在OC组织及邻近正常卵巢组织中的表达水平。体外构建耐5-FU的OC细胞系(OVCAR3-5FU_(res)和SKOV3-FU_(res))。将PLK1重组质粒和PLK1-sh RNA慢病毒载体(sh-PLK1)分别转染到OVCAR3、SKOV3、OVCAR3-5FU_(res)和SKOV3-5FU_(res)细胞中。采用CCK-8法检测细胞活力;通过串联亲和纯化和质谱分析确定PLK1在OC细胞中的结合蛋白;流式细胞术检测细胞凋亡。结果:在OC细胞系中,PLK1水平显著上调(P<0.05);并且在大多数OC组织中,PLK1表达较癌旁组织显著上调(P<0.05)。Kaplan-Meier分析显示,PLK1高表达OC患者的平均存活时间显著短于PLK1低表达OC患者(P<0.001)。OC中PLK1水平的升高与FIGO分期、肿瘤分级和错配修复缺陷等临床病理特征显著相关(P<0.05)。与阴性对照细胞相比,转染sh-PLK1的细胞活力显著降低(P<0.05);而转染PLK1重组质粒的OVCAR3和SKOV3细胞的活力显著高于对照细胞。通过串联亲和纯化和质谱分析确定人Mut S蛋白同系物2(hMSH2)是PLK1在OC细胞中的结合蛋白,并且h MSH2基因敲除使sh-PLK1转染的OVCAR3-5FU_(res)细胞的凋亡率降低(P<0.01)。结论:抑制PLK1表达通过靶向h MSH2提高了OC细胞对5-FU的敏感性。
AIM:To observe the expression of Polo-like kinase 1(PLK1)in ovarian cancer(OC)cells and tissues,and the effect of PLK1 inhibition on the viabilily and apoptosis of 5-fluorouracil(5-FU)-resistant OC cells.METHODS:Between April 2011 and December 2018,126 OC patients were enrolled in the study.The expression of PLK1 in OC and adjacent normal ovarian tissues was detected by immunohistochemistry.Two 5-FU-resistant OC cell lines OVCAR3-5FUresand SKOV3-FUreswere constructed in vitro.The PLK1 recombinant plasmid and PLK1-sh RNA lentivirus(sh-PLK1)were transfected into OVCAR3,SKOV3,OVCAR3-5FUresand SKOV3-FUrescells.Cell viability was measured by CCK-8 assay.The binding protein of PLK1 in OC cells was determined by tandem affinity purification and mass spec-trometry.Apoptosis was detected by flow cytometry.RESULTS:The PLK1 level was significantly up-regulated in OC cell lines(P<0.05),and in most OC tissues compared with the matched adjacent tissues(P<0.05).Kaplan-Meier analy-sis showed that the mean survival time of OC patients with high PLK1 expression was significantly shorter than that of the patients with low PLK1 expression(P<0.01).The increase in PLK1 level in OC tissues was significantly correlated with FIGO stage,tumor grade and mismatch repair deficiency.Compared with negative control cells,the viability of OVCAR3and SKOV3 cells transfected with sh-PLK1 was significantly decreased,while the viability of the cells transfected with PLK1 recombinant plasmid was significantly higher than that of control cells(P<0.05).Human mut S homolog 2(h MSH2)was identified as the binding protein of PLK1 in OC cells by tandem affinity purification and mass spectrometry.Knockdown of h MSH2 by si-h MSH2 transfection reduced the apoptotic rate of sh-PLK1-transfected OVCAR3-5FUrescells treated with 5-FU(P<0.01).CONCLUSION:Inhibition of PLK1 expression increases the sensitivity of OC cells to 5-FU by targeting h MSH2.
作者
李娟
何苗
袁春兰
李红梅
邓小梅
肖雪
LI Juan;HE Miao;YUAN Chun-lan;LI Hong-mei;DENG Xiao-mei;XIAO Xue(Sichuan Academy of Medical Sciences,Robot Minimally Invasive Center of Sichuan People's Hospital,Chengdu 610072,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2021年第5期871-878,共8页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81903655)。
