铁死亡抑制基因在食管癌中的高表达分析

doi:10.12122/j.issn.1673-4254.2024.07.19

摘要/Abstract

摘要：

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。

关键词: 食管鳞癌, 铁死亡, 细胞凋亡

Abstract:

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.

Key words: esophageal squamous cell carcinoma, ferroptosis, apoptosis

王元国, 张鹏. 铁死亡抑制基因在食管癌中的高表达分析[J]. 南方医科大学学报, 2024, 44(7): 1389-1396.

Yuanguo WANG, Peng ZHANG. Ferroptosis suppressor genes are highly expressed in esophageal cancer to inhibit tumor cell ferroptosis[J]. Journal of Southern Medical University, 2024, 44(7): 1389-1396.

图/表 7

表1 食管癌铁死亡抑制基因及GAPDH基因引物序列

Tab.1 Sequences of the primers for the ferroptosis suppressor genes and GAPDH

Primer	Primer sequence (5'-3')	Product length (bp)
RRM2	Forward Primer:GAACTTATTAGCAGAGATGAGG Reverse Primer:TTCTATCCGAACAGCATTGA	123
GCLC	Forward Primer:TCTCCAGGTGACATTCCAA Reverse Primer:TGACACATAGCCTCGGTAA	127
TFRC	Forward Primer:CTTCCTTCAATCACACTCAG Reverse Primer:ACAGTCTCCTTCCATATTCC	119
TXN	Forward Primer:AGCAGATCGAGAGCAAGA Reverse Primer:GAAGCAACATCCTGACAGT	193
SLC7A11	Forward Primer:CCTGTTGTGTCCACCATC Reverse Primer:AAGATTCCTGCTCCAATGAT	176
EZH2	Forward Primer:AAGGATACAGACAGTGATAGG Reverse Primer:ACATTCTCAGGAGGTTCAAT	164
GAPDH	Forward Primer:GAAGGTGAAGGTCGGAGTC Reverse Primer:GGGTGGAATCATATTGGAAC	152

图1 GSE161533和GSE20347差异基因分析火山图

Fig.1 Volcano map of the DEGs. Blue color represents the downregulated genes (log2FC<-1.0, P<0.05) and red the upregulated genes (log2FC>1.0, P<0.05) in GSE20347 and GSE161533 datasets.

图2 GSE161533和GSE20347差异基因与铁死亡相关基因的韦恩图

Fig.2 Venn diagram of GSE161533 and GSE20347 DEGs and ferroptosis genes.

图3 GO、KEGG功能富集图和PPI网络图

Fig.3 GO and KEGG functional enrichment map and PPI network map. A: Bubble diagram of GO analysis of BP, MF and CC functions. B: Bubble diagram of KEGG analysis of the main signaling pathways. C: PPI network (red indicates up-regulated and green down-regulated genes). D: Core gene network diagram screened by the "Degree" module.

图4 6个食管癌铁死亡抑制基因在TCGA数据集中的表达水平

Fig.4 Expression levels of 6 ferroptosis suppressor genes in TCGA esophageal cancer dataset. **P<0.01, ***P<0.001.

图5 6个食管癌铁死亡抑制基因在食管癌细胞系中的表达水平

Fig.5 Expression levels of 6 ferroptosis suppressor genes in different esophageal cancer cell line. *P<0.05, **P<0.01, ***P<0.001.

图6 敲低铁死亡抑制基因对食管癌细胞增殖和凋亡的影响

Fig.6 Effect of ferroptosis suppressor gene knock-down on proliferation and apoptosis of esophageal carcinoma cells. A, B: Effect of knocking down ferroptosis suppressor genes on TE1 cell apoptosis and proliferation. C-F: Effect of knocking down ferroptosis suppressor genes on GPX4, FIH1 and ACSL4 proteins. *P<0.05 vs Control and Negative group.

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