铁死亡抑制基因在食管癌中的高表达分析OA北大核心CSTPCDMEDLINE
Ferroptosis suppressor genes are highly expressed in esophageal cancer to inhibit tumor cell ferroptosis
目的 通过生物信息学分析和实验验证探讨铁死亡基因在食管鳞癌中的作用.方法 从基因表达数据库(GEO)下载食管鳞癌数据集GSE161533和GSE20347,采用R软件分析获得差异基因(DEGs),将DEGs与铁死亡数据库(FerrDb)中的基因取交集获得食管癌铁死亡相关基因,将获得食管癌铁死亡相关基因进行基因本体论(GO)和京都基因与基因组百科全书(KEGG)分析、String构建蛋白互作网络(PPI)、Cytoscape筛选铁死亡核心基因及抑制基因,筛选获得食管癌铁死亡抑制基因在癌症基因图谱(TCGA)中进行验证.应用qPCR检测食管癌铁死亡抑制基因在正常食管细胞和食管癌细胞中的表达水平,在TE1食管癌细胞系中下调6个食管癌铁死亡抑制基因,分为对照组(Control组)、siRNA阴性对照组(Negative组)和每个siRNA干扰组(包括RRM2-siRNA组、GCLC-siRNA组、TFRC-siRNA组、TXN-siRNA组、SLC7A11-siRNA组和EZH2-siRNA组),对照组不加任何转染试剂,其余各组使用Lipofectamine 2000(终浓度为3 μL/mL)和siRNA(终浓度为 100 nmol/L)的转染体系转染细胞,转染后分别进行流式细胞学实验、CCK8实验以验证食管癌铁死亡抑制基因对食管癌细胞功能的影响,Western blotting实验验证食管癌铁死亡抑制基因对铁死亡进程的影响.结果 共获得58个食管癌铁死亡相关基因,GO分析显示其主要参与谷胱甘肽跨膜转运、铁离子输运和细胞凋亡等生物学过程,KEGG富集分析显示其主要参与铁死亡、谷胱甘肽代谢、抗叶酸抵抗等信号通路.PPI网络包含54个节点和74条边,局部聚类系数为0.522,PPI富集值为P<0.001.RRM2、TFRC、TXN、EZH2、SLC7A11、GCLC 6个食管癌铁死亡抑制基因,在TCGA数据集的食管癌组织中表达高于正常食管组织(P<0.01),与正常食管细胞系比较,这些基因在食管癌细胞系中表达升高(P<0.05).在食管癌TE1细胞中,应用siRNA分别下调RRM2、TFRC、TXN、EZH2、SLC7A11、GCLC可抑制细胞增殖(P<0.05)、促进细胞凋亡(P<0.05),铁死亡进程标志蛋白GPX4、FIH1表达下调(P<0.05)、ACSL4表达上调(P<0.05).结论 铁死亡抑制基因在食管癌中的高表达可能阻碍了食管癌细胞的铁死亡进程,导致肿瘤发生,抑制这些基因可恢复铁死亡进程,促进细胞凋亡,铁死亡抑制基因有望成为食管癌治疗的新靶点.
Objective To explore the role of ferroptosis-related genes in regulating ferroptosis of esophageal squamous cell carcinoma(ESCC).Methods ESCC datasets GSE161533 and GSE20347 were downloaded from the Gene Expression Omnibus(GEO)to identify the differentially expressed genes(DEGs)using R software.ESCC ferroptosis-related genes obtained by intersecting the DEGs with ferroptosis-related genes from FerrDb were analyzed using GO and KEGG analyses,protein-protein interaction(PPI)network analysis,and core gene identification through Cytoscape.The identified ferroptosis suppressor genes were validated using TCGA database,and their expression levels were detected using RT-qPCR in cultured normal esophageal cells and ESCC cells.Six ferroptosis suppressor genes(RRM2,GCLC,TFRC,TXN,SLC7A11,and EZH2)were downregulated with siRNA in ESCC cells,and the changes in cell proliferation and apoptosis were assessed with CCK8 assay and flow cytometry;Western blotting was performed to examine the changes in ferroptosis progression of the cells.Results We identified a total of 58 ESCC ferroptosis-related genes,which involved such biological processes as glutathione transmembrane transport,iron ion transport,and apoptosis and the ferroptosis,glutathione metabolism,and antifolate resistance pathways.The PPI network included 54 nodes and 74 edges with a clustering coefficient of 0.522 and PPI enrichment P<0.001.Cytoscape identified 6 core ferroptosis suppressor genes(RRM2,TFRC,TXN,EZH2,SLC7A11,and GCLC),which were highly expressed in ESCC tissues in the TCGA dataset and in ESCC cell lines.Downregulating these genes in ESCC TE1 cells significantly inhibited cell proliferation,promoted cell apoptosis,reduced the expression levels of ferroptosis markers GPX4 and FIH1,and increased the expression of ACSL4.Conclusion High expression of ferroptosis suppressor genes in ESCC may cause arrest of ferroptosis progression to facilitate tumor development,and inhibiting these genes can restore ferroptosis and promote cell apoptosis,suggesting their value as potential therapeutic targets for ESCC.
王元国;张鹏
天津医科大学总医院心胸外科,天津 300052
食管鳞癌铁死亡细胞凋亡
esophageal squamous cell carcinomaferroptosisapoptosis
《南方医科大学学报》 2024 (007)
1389-1396 / 8
天津市自然科学基金京津冀基础研究合作专项(20JCZXJC00190)
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