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BACKGROUND

Colorectal cancer is considered as the second most common cancer worldwide. Studies have shown that m6A RNA methylation abnormalities play an important role in the pathogenesis of many human diseases, including cancer. The current study was designed to characterize the mutation of m6A related genes and explore their prognostic role in colorectal cancer.

METHODS

RNA-seq data and somatic mutation data of TCGA-COAD and TCGA-READ were downloaded from UCSC xena for comprehensive analysis. M6A related genes were selected from previous literatures, including "Writer" protein (METTL3, METTL5, METTL14, METTL16, ZC3H13, RBM15, WTAP, KIAA1429), "Reader" protein YTHDF1, YTHDF2, YTHDF3, YTHDC1, YTHDC2, HNRNPC, IGF2BP1, IGF2BP2, IGF2BP3), and "Eraser" protein (FTO, ALKBH5). Kaplan-Meier diagrams were used to explore the correlation between m6A-related genes and colorectal cancer prognosis. The correlations between m6A-related genes and clinical parameters and immune-related indicators were explored by Spearman correlation analysis. And finally, the expression patterns of five key genes (RBMX, FMR1, IGF2BP1, LRPPRC and YTHDC2) were detected by qPCR in CRC specimens.

RESULTS

In CRC, the expressions of m6A-related genes were significantly different between CRC and normal control except METTL14, YTHDF2, YTHDF3. Some of CRC patients (178 in 536) have a m6A-related genes mutation. ZC3H13 has highest mutation frequency of all m6A-related genes. M6A-related genes mainly enrich in regulation of mRNA metabolic process pathway. Patients with high expressions of FMR1, LRPPRC, METTL14, RBMX, YTHDC2, YTHDF2, YTHDF3 have poor prognosis in CRC. There was a significant correlation between the FMR1, LRPPRC, RBMX, YTHDC2, IGF2BP1 expression and the clinical characteristics of CRC. In addition, these genes are significantly associated with immune-related indicators. According to the expression patterns of FMR1, LRPPRC, RBMX, YTHDC2, and IGF2BP1, patients with CRC were clustered into two groups, and their survival was significantly different. By evaluating the tumor microenvironment in two clusters using ssGSEA, expressions of immune checkpoints and GSVA enrichment analysis, we observed that the immune and stem cell index of two cluster were much different. The qPCR results showed that RBMX expression was markedly elevated in cancerous tissues than in the normal colonic tissues.

CONCLUSION

Our study identified novel prognostic markers associated with immune of CRC cancer patients. Moreover, the potential mechanisms of prognostic markers in regulating the etiology of CRC cancer were investigated. These findings enrich our understanding of the relationships between m6a related genes and CRC, and may provide novel ideas in the therapy of CRC patients.

BACKGROUND

METHODS

RESULTS

CONCLUSION

背景

结直肠癌被认为是全球第二常见的癌症。研究表明，m6A RNA 甲基化异常在许多人类疾病的发病机制中发挥着重要作用，包括癌症。本研究旨在对 m6A 相关基因的突变进行特征描述，并探讨其在结直肠癌中的预后作用。

方法

从 UCSC xena 下载 TCGA-COAD 和 TCGA-READ 的 RNA-seq 数据和体细胞突变数据进行综合分析。m6A 相关基因是从先前的文献中选择的，包括“Writer”蛋白（METTL3、METTL5、METTL14、METTL16、ZC3H13、RBM15、WTAP、KIA A1429）、“Reader”蛋白 YTHDF1、YTHDF2、YTHDF3、YTHDC1、YTHDC2、HNRNPC、IGF2BP1、IGF2BP2、IGF2BP3）和“Eraser”蛋白（FTO、ALKBH5）。使用 Kaplan-Meier 图探讨 m6A 相关基因与结直肠癌预后的相关性。通过 Spearman 相关性分析探讨 m6A 相关基因与临床参数和免疫相关指标的相关性。最后，通过 qPCR 检测 CRC 标本中五个关键基因（RBMX、FMR1、IGF2BP1、LRPPRC 和 YTHDC2）的表达模式。

结果

在结直肠癌中，除了 METTL14、YTHDF2 和 YTHDF3 之外，CRC 和正常对照之间 m6A 相关基因的表达差异有统计学意义。部分 CRC 患者（536 例中有 178 例）存在 m6A 相关基因的突变。ZC3H13 是所有 m6A 相关基因中突变频率最高的。m6A 相关基因主要富集在调节 mRNA 代谢过程途径中。FMR1、LRPPRC、METTL14、RBMX、YTHDC2、YTHDF2 和 YTHDF3 高表达的患者在 CRC 中预后较差。FMR1、LRPPRC、RBMX、YTHDC2、IGF2BP1 的表达与 CRC 的临床特征之间存在显著相关性。此外，这些基因与免疫相关指标显著相关。根据 FMR1、LRPPRC、RBMX、YTHDC2 和 IGF2BP1 的表达模式，将 CRC 患者聚类为两组，其生存情况有显著差异。通过使用 ssGSEA 评估两组的肿瘤微环境，进行 GSVA 富集分析，我们观察到两组的免疫和干细胞指数有很大差异。qPCR 结果表明，RBMX 在癌组织中的表达明显高于正常结肠组织。