膜穿孔蛋白E在上皮性卵巢癌组织中的表达水平及其临床意义

Expression level of gasdermin E in epithelial ovarian cancer tissue and its clinical significance

目的:探讨膜穿孔蛋白E (GSDME)在上皮性卵巢癌(EOC)组织中的表达水平,阐明GSDME对EOC发生发展的影响。方法:基于肿瘤基因组图谱(TCGA)和基因型-组织表达(GTEx)数据库,利用R语言分析正常组织和EOC组织中GSDME mRNA表达水平及其表达水平与EOC发生的关系。利用差异性分析、基因本体论(GO)分析、京都基因与基因组百科全书(KEGG)通路富集分析、基因集富集分析(GSEA)、蛋白-蛋白互作(PPI)网络、HUB基因互作网络和免疫浸润分析探讨与GSDME相关联的基因、信号通路及其对EOC发生发展的影响。收集40例EOC患者组织病理切片,按病理分级分为低级别(中分化及高分化,24例)组和高级别(低分化,16例)组,按照对顺铂(DDP)的敏感程度分为DDP耐药组和DDP敏感组。另收集正常卵巢、良性卵巢瘤和交界性卵巢癌组织切片各5例作为对照,利用免疫组织化学(IHC)法检测所有组织切片中GSDME的表达强度。取10对新鲜的卵巢癌组织和癌旁组织,采用Western blotting法检测2组样本中GSDME蛋白表达水平。体外培养Skov3和Skov3/DDP细胞,采用Western blotting法检测细胞中GSDME蛋白表达水平,分析其与卵巢癌耐药的关系。结果:TCGA和GTEx数据库中非配对样本分析,与正常卵巢组织比较,EOC组织中GSDME mRNA表达水平明显降低(P<0.05),且高级别组EOC组织中GSDME mRNA表达水平低于低级别组(P<0.05)。GSDME单基因差异分析,上调基因有460个,下调基因有329个[Log_(2)差异倍数(FC)的绝对值大于1,即|Log_(2)FC|>1,P<0.05]。功能富集及免疫浸润分析,GSDME高表达可以上调循环免疫球蛋白介导的体液免疫反应通路、白细胞迁移通路和免疫球蛋白复合体通路的活性,促进肿瘤微环境中免疫细胞的浸润。IHC染色,GSDME与EOC的病理组织分级及耐药有关,其中DDP敏感组EOC组织中GSDME表达强度明显高于DDP耐药组(P<0.05)。Western blotting法检测,与癌旁组织比较,EOC组织中GSDME蛋白表达水平明显降低(P<0.05);与Skov3细胞比较,Skov3/DDP细胞中GSDME蛋白表达水平明显降低(P<0.05)。结论:GSDME与EOC的恶性程度和耐药表现有密切关联。GSDME低表达是导致肿瘤免疫浸润下降、EOC恶化及耐药的一个关键因素。GSDME可能成为治疗EOC的潜在分子靶点。

Objective:To discuss the expression level of gasdermin E(GSDME)in the epithelial ovarian cancer(EOC)tissue,and to clarify the effect of GSDME on the occurrence and development of EOC.Methods:The expressions of GSDME mRNA in normal tissue and EOC tissue was detected by R language based on the Cancer Genome Atlas(TCGA)and Genotype-Tissue Expression(GTEx)Databases;the correlation between the expression level of GSDME and the occurrence of EOC was analyzed.The difference analysis,Gene Ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway richment analysis,Gene Set Enrichment analysis(GSEA),protein-protein interaction(PPI)network,HUB gene interaction network,and immune infiltration analysis were used to discuss the genes and signaling pathways associated with GSDME and their effects on the occurrence and development of EOC.The pathological tissue sections of 40 EOC patients were collected and divided into low-grade group(well-differentiated and moderately-differentiated,n=24)and high-grade group(poorly differentiated,n=16)based on the pathological grades;according to the sensitivity to cisplatin(DDP),the pathological tissue sections were divided into DDP resistant group and DDP sensitive group.In addition,5 tissue sections of normal ovary,benign ovarian tumor,and 5 tissue sections of borderline ovarian carcinoma were collected as controls.The expression intensities of GSDME in all the tissue sections were detected by immunohistochemistry(IHC)method.Besides,10 pairs of fresh ovarian cancer and paracancerous tissues were selected,and the expression levels of GSDME protein in the samples in two groups were detected by Western blotting method.The ovarian cancer Skov3 cells and Skov3/DDP cells were cultured in vitro,and the expression levels of GSDME protein in the cells were detected by Western blotting method and the relationship between the expression level and DDP-resistance of ovarian cancer was analyzed.Results:The analysis results of unpaired samples in the TCGA and GTEx Databases showed that compared with normal ovarian tissue,the expression level of GSDME mRNA in EOC tissue was significantly decreased(P<0.05),and the expression of GSDME mRNA in EOC tissue in high-grade group was higher than that in low-grade group(P<0.05).The GSDME single gene difference analysis results showed that there were 460 up-regulated and 329 down-regulated genes|(Log_(2) fold charge(FC)|>1,P<0.05).The functional enrichment and immune infiltration analysis results showed that high expression of GSDME could upregulate the activities of humoral immunie response pathways,leukocyte migration pathway and immunoglobulin complex pathway mediated by circulating immunoglobulin and promote the infiltration of immune cells in tumor microenvironment.The IHC staining results showed that GSDME was related to the pathological grade and drug resistance of EOC;the expression intensity of GSDME in EOC tisssue in DDP sensitive group was higher than that in DDP resistant group(P<0.05).The Western blotting results showed that compared with paracancerous tissue,the expression level of GSDME protein in EOC tissue was significantly decreased(P<0.05);compared with Skov3 cells,the expression level of GSDME protein in the Skov3/DDP cells was significantly decreased(P<0.05).Conclusion:GSDME is closely related to the malignancy and cisplatin resistance of EOC.The low expression of GSDME is a key factor leading to the decreasing of tumor immune infiltration,EOC deterioration and resistance.GSDME may be a potential molecular target for the treatment of EOC.