Abstract：
OBJECTIVE: To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression.
METHODS: We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis.
RESULTS: We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway.
CONCLUSIONS: Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.
METHODS: We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis.
RESULTS: We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway.
CONCLUSIONS: Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.
摘要：
目的：为了揭示结直肠癌（CRC）发展的潜在机制，我们应用生物信息学分析来鉴定关键基因,并通过实验验证了它们在CRC发生和进展中的可能作用.
方法：我们对差异表达基因（DEGs）进行了基因本体论（GO）和京都基因和基因组百科全书（KEGG）途径分析，构建了一个蛋白质-蛋白质相互作用(PPI)网络来寻找前10个枢纽基因，并分析其在结肠腺癌（COAD）和直肠腺癌（READ）中的表达。我们还研究了这些基因与免疫细胞浸润和预后之间的相关性，并使用qRT-PCR和Western印迹验证了SLC9A2在CRC组织和细胞系中的表达。在体外进行功能实验以研究SLC9A2对肿瘤生长和转移的影响。
结果：我们发现了130个DEG，在CRC中45个上调和85个下调。GO分析表明，这些DEGs主要富含与细胞pH调节相关的功能，酶原颗粒，和跨膜转运蛋白活性。KEGG通路分析显示DEGs在胰腺分泌中起关键作用,类风湿性关节炎,和IL-17信号通路。我们鉴定了10个hub基因：CXCL1、SLC26A3、CXCL2、MMP7、MMP1、SLC9A2、SLC4A4、CLCA1、CLCA4和ZG16。GO富集分析表明，这些hub基因主要参与转录的正调控。基因表达分析显示CXCL1、CXCL2、MMP1和MMP7在CRC中高表达,而CLCA1、CLCA4、SLC4A4、SLC9A2、SLC26A3和ZG16的表达水平较低。生存分析显示5个关键基因与CRC的预后显著相关。在CRC组织和细胞系中，SLC9A2的mRNA和蛋白质表达水平均显着降低。重要的是,SLC9A2在SW480细胞中的过表达导致细胞增殖的显著抑制,迁移,和入侵。蛋白质印迹分析显示磷酸化ERK(p-ERK)和磷酸化JNK(p-JNK)蛋白的表达水平显著升高,而SLC9A2过表达后，ERK和JNK的表达水平没有显着变化。相关分析表明SLC9A2表达与MAPK信号通路之间存在潜在联系。
结论：我们的研究表明，SLC9A2通过MAPK通路作为肿瘤抑制因子，可能成为CRC诊断和治疗的潜在靶点。
方法：我们对差异表达基因（DEGs）进行了基因本体论（GO）和京都基因和基因组百科全书（KEGG）途径分析，构建了一个蛋白质-蛋白质相互作用(PPI)网络来寻找前10个枢纽基因，并分析其在结肠腺癌（COAD）和直肠腺癌（READ）中的表达。我们还研究了这些基因与免疫细胞浸润和预后之间的相关性，并使用qRT-PCR和Western印迹验证了SLC9A2在CRC组织和细胞系中的表达。在体外进行功能实验以研究SLC9A2对肿瘤生长和转移的影响。
结果：我们发现了130个DEG，在CRC中45个上调和85个下调。GO分析表明，这些DEGs主要富含与细胞pH调节相关的功能，酶原颗粒，和跨膜转运蛋白活性。KEGG通路分析显示DEGs在胰腺分泌中起关键作用,类风湿性关节炎,和IL-17信号通路。我们鉴定了10个hub基因：CXCL1、SLC26A3、CXCL2、MMP7、MMP1、SLC9A2、SLC4A4、CLCA1、CLCA4和ZG16。GO富集分析表明，这些hub基因主要参与转录的正调控。基因表达分析显示CXCL1、CXCL2、MMP1和MMP7在CRC中高表达,而CLCA1、CLCA4、SLC4A4、SLC9A2、SLC26A3和ZG16的表达水平较低。生存分析显示5个关键基因与CRC的预后显著相关。在CRC组织和细胞系中，SLC9A2的mRNA和蛋白质表达水平均显着降低。重要的是,SLC9A2在SW480细胞中的过表达导致细胞增殖的显著抑制,迁移,和入侵。蛋白质印迹分析显示磷酸化ERK(p-ERK)和磷酸化JNK(p-JNK)蛋白的表达水平显著升高,而SLC9A2过表达后，ERK和JNK的表达水平没有显着变化。相关分析表明SLC9A2表达与MAPK信号通路之间存在潜在联系。
结论：我们的研究表明，SLC9A2通过MAPK通路作为肿瘤抑制因子，可能成为CRC诊断和治疗的潜在靶点。
