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Abstract：
BACKGROUND: N6-methyladenosine (m6A) methylation modification exists in Epstein-Barr virus (EBV) primary infection, latency, and lytic reactivation. It also modifies EBV latent genes and lytic genes. EBV-associated gastric cancer (EBVaGC) is a distinctive molecular subtype of GC. We hypothesized EBV and m6A methylation regulators interact with each other in EBVaGC to differentiate it from other types of GC.
OBJECTIVE: To investigate the mechanisms of m6A methylation regulators in EBVaGC to determine the differentiating factors from other types of GC.
METHODS: First, The Cancer Gene Atlas and Gene Expression Omnibus databases were used to analyze the expression pattern of m6A methylation regulators between EBVaGC and EBV-negative GC (EBVnGC). Second, we identified Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment of m6A-related differentially expressed genes. We quantified the relative abundance of immune cells and inflammatory factors in the tumor microenvironment (TME). Finally, cell counting kit-8 cell proliferation test, transwell test, and flow cytometry were used to verify the effect of insulin-like growth factor binding protein 1 (IGFBP1) in EBVaGC cell lines.
RESULTS: m6A methylation regulators were involved in the occurrence and development of EBVaGC. Compared with EBVnGC, the expression levels of m6A methylation regulators Wilms tumor 1-associated protein, RNA binding motif protein 15B, CBL proto-oncogene like 1, leucine rich pentatricopeptide repeat containing, heterogeneous nuclear ribonucleoprotein A2B1, IGFBP1, and insulin-like growth factor 2 binding protein 1 were significantly downregulated in EBVaGC (P < 0.05). The overall survival rate of EBVaGC patients with a lower expression level of IGFBP1 was significantly higher (P = 0.046). GO and KEGG functional enrichment analyses showed that the immunity pathways were significantly activated and rich in immune cell infiltration in EBVaGC. Compared with EBVnGC, the infiltration of activated CD4+ T cells, activated CD8+ T cells, monocytes, activated dendritic cells, and plasmacytoid dendritic cells were significantly upregulated in EBVaGC (P < 0.001). In EBVaGC, the expression level of proinflammatory factors interleukin (IL)-17, IL-21, and interferon-γ and immunosuppressive factor IL-10 were significantly increased (P < 0.05). In vitro experiments demonstrated that the expression level of IGFBP1 was significantly lower in an EBVaGC cell line (SNU719) than in an EBVnGC cell line (AGS) (P < 0.05). IGFBP1 overexpression significantly attenuated proliferation and migration and promoted the apoptosis levels in SNU719. Interfering IGFBP1 significantly promoted proliferation and migration and attenuated the apoptosis levels in AGS.
CONCLUSIONS: m6A regulators could remodel the TME of EBVaGC, which is classified as an immune-inflamed phenotype and referred to as a \"hot\" tumor. Among these regulators, we demonstrated that IGFBP1 affected proliferation, migration, and apoptosis.
OBJECTIVE: To investigate the mechanisms of m6A methylation regulators in EBVaGC to determine the differentiating factors from other types of GC.
METHODS: First, The Cancer Gene Atlas and Gene Expression Omnibus databases were used to analyze the expression pattern of m6A methylation regulators between EBVaGC and EBV-negative GC (EBVnGC). Second, we identified Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment of m6A-related differentially expressed genes. We quantified the relative abundance of immune cells and inflammatory factors in the tumor microenvironment (TME). Finally, cell counting kit-8 cell proliferation test, transwell test, and flow cytometry were used to verify the effect of insulin-like growth factor binding protein 1 (IGFBP1) in EBVaGC cell lines.
RESULTS: m6A methylation regulators were involved in the occurrence and development of EBVaGC. Compared with EBVnGC, the expression levels of m6A methylation regulators Wilms tumor 1-associated protein, RNA binding motif protein 15B, CBL proto-oncogene like 1, leucine rich pentatricopeptide repeat containing, heterogeneous nuclear ribonucleoprotein A2B1, IGFBP1, and insulin-like growth factor 2 binding protein 1 were significantly downregulated in EBVaGC (P < 0.05). The overall survival rate of EBVaGC patients with a lower expression level of IGFBP1 was significantly higher (P = 0.046). GO and KEGG functional enrichment analyses showed that the immunity pathways were significantly activated and rich in immune cell infiltration in EBVaGC. Compared with EBVnGC, the infiltration of activated CD4+ T cells, activated CD8+ T cells, monocytes, activated dendritic cells, and plasmacytoid dendritic cells were significantly upregulated in EBVaGC (P < 0.001). In EBVaGC, the expression level of proinflammatory factors interleukin (IL)-17, IL-21, and interferon-γ and immunosuppressive factor IL-10 were significantly increased (P < 0.05). In vitro experiments demonstrated that the expression level of IGFBP1 was significantly lower in an EBVaGC cell line (SNU719) than in an EBVnGC cell line (AGS) (P < 0.05). IGFBP1 overexpression significantly attenuated proliferation and migration and promoted the apoptosis levels in SNU719. Interfering IGFBP1 significantly promoted proliferation and migration and attenuated the apoptosis levels in AGS.
CONCLUSIONS: m6A regulators could remodel the TME of EBVaGC, which is classified as an immune-inflamed phenotype and referred to as a \"hot\" tumor. Among these regulators, we demonstrated that IGFBP1 affected proliferation, migration, and apoptosis.
摘要：
背景：EB病毒（EBV）原发感染中存在N6-甲基腺苷（m6A）甲基化修饰，延迟，和裂解剂的再激活。它还修饰EBV潜伏基因和裂解基因。EBV相关胃癌（EBVaGC）是一种独特的GC分子亚型。我们假设EBV和m6A甲基化调节因子在EBVaGC中相互作用，以将其与其他类型的GC区分开。
目的：研究m6A甲基化调节因子在EBVaGC中的作用机制，以确定与其他类型GC的区别因素。
方法：首先，癌症基因图谱和基因表达综合数据库用于分析EBVaGC和EBV阴性GC(EBVnGC)之间m6A甲基化调节因子的表达模式。第二,我们确定了基因本体论（GO）和京都基因和基因组百科全书（KEGG）m6A相关差异表达基因的功能富集。我们定量了肿瘤微环境（TME）中免疫细胞和炎症因子的相对丰度。最后,细胞计数试剂盒-8细胞增殖试验,Transwell测试,并采用流式细胞术验证胰岛素样生长因子结合蛋白1(IGFBP1)在EBVaGC细胞系中的作用。
结果：m6A甲基化调节因子参与了EBVaGC的发生和发展。与EBVnGC相比，m6A甲基化调节因子Wilms肿瘤1相关蛋白的表达水平，RNA结合基序蛋白15B,CBL原癌基因样1，富含亮氨酸的五肽重复序列，异质核核糖核蛋白A2B1、IGFBP1和胰岛素样生长因子2结合蛋白1在EBVaGC中显著下调(P<0.05)。IGFBP1表达水平较低的EBVaGC患者的总生存率明显较高(P=0.046)。GO和KEGG功能富集分析表明，EBVaGC中免疫途径被显著激活并富含免疫细胞浸润。与EBVnGC相比，活化的CD4+T细胞的浸润，激活的CD8+T细胞，单核细胞,激活的树突状细胞，和浆细胞样树突状细胞在EBVaGC中显著上调(P<0.001)。在EBVaGC,促炎因子白细胞介素(IL)-17、IL-21、干扰素-γ和免疫抑制因子IL-10的表达水平显著升高(P<0.05)。体外实验表明，IGFBP1在EBVaGC细胞系（SNU719）中的表达水平显着低于EBVnGC细胞系（AGS）（P<0.05）。IGFBP1过表达显著减弱SNU719细胞增殖和迁移，促进细胞凋亡。干扰IGFBP1显著促进AGS细胞的增殖和迁移，降低细胞凋亡水平。
结论：m6A调节剂可以重塑EBVaGC的TME，它被归类为一种免疫发炎的表型，被称为“热”肿瘤。在这些监管机构中，我们证明IGFBP1影响增殖，迁移,和凋亡。
目的：研究m6A甲基化调节因子在EBVaGC中的作用机制，以确定与其他类型GC的区别因素。
方法：首先，癌症基因图谱和基因表达综合数据库用于分析EBVaGC和EBV阴性GC(EBVnGC)之间m6A甲基化调节因子的表达模式。第二,我们确定了基因本体论（GO）和京都基因和基因组百科全书（KEGG）m6A相关差异表达基因的功能富集。我们定量了肿瘤微环境（TME）中免疫细胞和炎症因子的相对丰度。最后,细胞计数试剂盒-8细胞增殖试验,Transwell测试,并采用流式细胞术验证胰岛素样生长因子结合蛋白1(IGFBP1)在EBVaGC细胞系中的作用。
结果：m6A甲基化调节因子参与了EBVaGC的发生和发展。与EBVnGC相比，m6A甲基化调节因子Wilms肿瘤1相关蛋白的表达水平，RNA结合基序蛋白15B,CBL原癌基因样1，富含亮氨酸的五肽重复序列，异质核核糖核蛋白A2B1、IGFBP1和胰岛素样生长因子2结合蛋白1在EBVaGC中显著下调(P<0.05)。IGFBP1表达水平较低的EBVaGC患者的总生存率明显较高(P=0.046)。GO和KEGG功能富集分析表明，EBVaGC中免疫途径被显著激活并富含免疫细胞浸润。与EBVnGC相比，活化的CD4+T细胞的浸润，激活的CD8+T细胞，单核细胞,激活的树突状细胞，和浆细胞样树突状细胞在EBVaGC中显著上调(P<0.001)。在EBVaGC,促炎因子白细胞介素(IL)-17、IL-21、干扰素-γ和免疫抑制因子IL-10的表达水平显著升高(P<0.05)。体外实验表明，IGFBP1在EBVaGC细胞系（SNU719）中的表达水平显着低于EBVnGC细胞系（AGS）（P<0.05）。IGFBP1过表达显著减弱SNU719细胞增殖和迁移，促进细胞凋亡。干扰IGFBP1显著促进AGS细胞的增殖和迁移，降低细胞凋亡水平。
结论：m6A调节剂可以重塑EBVaGC的TME，它被归类为一种免疫发炎的表型，被称为“热”肿瘤。在这些监管机构中，我们证明IGFBP1影响增殖，迁移,和凋亡。
