基于TCGA数据库探讨CDC20在宫颈癌组织中的表达及其对癌细胞自噬的影响  

Exploring the expression of CDC20 in cervical cancer tissues based on TCGA database and its effect on cancer cell autophagy

作　　者：莫艳秀 徐欧欧 李梁政 喻坤林 贺嘉旖 王岐本 MO Yanxiu;XU Ouou;LI Liangzheng;YU Kunlin;HE Jiayi;WANG Qiben(School of Basic Medical Science,Xiangnan University,Hunan Chenzhou 423000,China;College of Clinical Studies,Xiangnan University,Hunan Chenzhou 423000,China)

机构地区：[1]湘南学院基础医学院,湖南郴州423000 [2]湘南学院临床学院,湖南郴州423000

出　　处：《现代肿瘤医学》2025年第3期375-387,共13页Journal of Modern Oncology

基　　金：湖南省教育厅科学研究项目(湘教通[2023]361号,编号:23A0589);湖南省大学生创新训练计划项目(湘教通[2024]191号,编号:4854);湘南学院2020高层次人才科研启动基金(编号:2021A20)。

摘　　要：目的:探讨细胞分裂周期蛋白20(cell division cycle protein 20,CDC20)在宫颈癌(cervical cancer,CC)组织中的表达,并分析CDC20对宫颈癌细胞自噬的影响。方法:基于TCGA数据库分析CC组织中CDC20的表达以及对宫颈癌患者生存率和临床分期的影响,GSEA富集分析CDC20与自噬通路的相关性,通过STRING分析蛋白质-蛋白质相互作用(PPI)网络,ssGSEA算法和Spearman's分析免疫细胞浸润。免疫组化检测宫颈癌和癌旁组织中CDC20的表达水平。CRISPR-cas9技术设计双靶点敲除sgRNA2&7-CDC20基因并构建其细胞稳株。分别设置sgRNA-NC组、sgRNA-CDC20组、sgRNA-NC组+血清饥饿24 h和sgRNA-CDC20+血清饥饿24 h组。透射电镜(TEM)检测细胞中自噬小体的变化,免疫荧光、qRT-PCR和Western Blot法检测自噬基因p62、Beclin 1和LC3的细胞定位、mRNA和蛋白表达水平。结果:与癌旁组织相比,宫颈癌组织中CDC20呈高表达,其高表达与患者的年龄、临床分期、免疫浸润和不良预后(P<0.05)以及较短的总生存期(P<0.001)高度相关。GSEA提示CDC20高表达参与自噬等九个通路。通过STRING数据库筛选到53个CDC20的互作蛋白。免疫组化结果显示CDC20在宫颈癌组织中的表达量显著高于癌旁组织。TEM结果显示敲除CDC20可诱导宫颈癌C33A细胞发生自噬;免疫荧光显示p62、Beclin 1和LC3主要定位在细胞质中,CDC20敲除和血清饥饿24 h均能观察LC3的荧光强度有明显增多且具有叠加效应;qRT-PCR和Western Blot分析结果都显示敲除CDC20后增加细胞内酸性自噬体囊泡的形成和显著性增强自噬相关蛋白LC3B-II/I的表达(P<0.05)。结论:CDC20在宫颈癌组织和细胞中均高表达并影响自噬,可能是CC的独立预后标志物,CDC20有望成为分子靶向治疗的新靶点。Objective:To explore the expression of cell division cycle protein 20(CDC20) in cervical cancer(CC) tissues and analyzing the effect of CDC20 on autophagy in cervical cancer cells.Methods:The TCGA data were used to analyze CDC20 expression in CC.tissues and the effects of C.DC20 expression on survival ratio and clinical stage of cervical cancer patients.The correlation of CDC20 with autophagy pathway was analyzed by GSEA enrichment analysis.The protein-protein interaction(PPI) network of CDC20 was anaylyzed using STRING database,and immune cell infiltration was analyzed by ssGSEA and Spearmans analysis.CDC20 expression in cervical carcinoma and adjacent tissues were detected by immunohistochemistry method.The sgRNA2&7-CDC20 dual-gene knockout stable cell strain was constructed by CRISPR-cas9 method.The experiments were divided into sgRnA-NC,sgRNACDC20,sgRNA-NC+serum-starved for 24 h group and sgRNA-CDC20+serum-starved for 24 h group.Changes in autophagosomes in cells were detected by transmission electron microscopy(TEM).The subcellular localization,mRNA,and protein expression levels of the autophagy genes p62,Beclin 1,and LC3 were determined by immunofluorescence,qRT-PCR,and Western Blot analysis,respectively.Results:CDC.20 was highly expressed in cervical cancer tissues compared to adjacent tissues,which was highly correlated with patient age,clinical stage,immune infiltration,and poor prognosis(P <0.05),and shorter overall survival(P <0.001).GSEA analysis suggested that high expression of CDC20 participates in nine pathways including autophagy pathway,and the STRING database analysis suggested that 53 proteins could interact with CDC20.Immunohistochemistry results showed that CDC20 expression was significantly higher in cervical cancer tissues than in adjacent normal tissues.TEM results showed that CDC20 knockdown induced autophagy in cervical cancer C33A cells.Immunofluorescence results showed that p62,Beclin 1 and LC3 were mainly localized in the cytoplasm,and the knockdown of CDC20 or serum starvation for 2

关 键 词：TCGA 宫颈癌 自噬 预后 CDC20 

分 类 号：R737.33[医药卫生—肿瘤]