DataSheet_2_Identification and validation of signature for prognosis and immune microenvironment in gastric cancer based on m6A demethylase ALKBH5.csv

BackgroundN6-methyladenosine (m6A) RNA regulators play important roles in cancers, but their functions and mechanism have not been demonstrated clearly in gastric cancer (GC).
MethodsIn this study, the GC samples with clinical information and RNA transcriptome were downloaded from The Cancer Genome Atlas database. The different expression genes were compared by the absolute value and median ± standard deviation. Samples with complete information were randomly divided into a training dataset and a test dataset. The differential expression genes (DEGs) between ALKBH5-low and ALKBH5-high subgroups were identified in the training dataset and constructed a risk model by Cox and least absolute shrinkage and selection operator regression. The model was testified in test datasets, overall survival (OS) was compared with the Kaplan–Meier method, and immune cell infiltration was calculated by the CIBERSORT algorithm in the low-risk and high-risk subgroups based on the model. The protein levels of ALKBH5 were detected with immunohistochemistry. The relative expression of messenger-ribonucleic acid (mRNA) was detected with quantitative polymerase chain reaction.
ResultsALKBH5 was the only regulator whose expression was lower in tumor samples than that in normal samples. The low expression of ALKBH5 led to the poor OS of GC patients and seemed to be an independent protective factor. The model based on ALKBH5-regulated genes was validated in both datasets (training/test) and displayed a potential capacity to predict a clinical prognosis. Gene Ontology analysis implied that the DEGs were involved in the immune response; CIBERSORT results indicated that ALKBH5 and its related genes could alter the immune microenvironment of GC. The protein levels of ALKBH5 were verified as lowly expressed in GC tissues. SLC7A2 and CGB3 were downregulated with ALKBH5 knockdown.
ConclusionsIn this study, we found that ALKBH5 might be a suppressor of GC; ALKBH5 and its related genes were latent biomarkers and immunotherapy targets.

背景：N6-甲基腺苷（N6-methyladenosine, m6A）RNA调控因子在癌症中发挥重要作用，但其在胃癌（gastric cancer, GC）中的功能与作用机制尚未得到清晰阐明。
方法：本研究从癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库下载了携带完整临床信息与RNA转录组数据的胃癌样本。以绝对值、中位数±标准差作为判定标准，对差异表达基因进行筛选与比较。将信息完整的样本随机划分为训练数据集与测试数据集。在训练集中鉴定出ALKBH5低表达与高表达亚组间的差异表达基因（differentially expressed genes, DEGs），并通过Cox回归与最小绝对收缩和选择算子（least absolute shrinkage and selection operator, LASSO）回归构建风险预测模型。随后在测试集中对该模型进行验证；采用Kaplan-Meier法比较高低风险亚组的总生存期（overall survival, OS），并基于模型通过CIBERSORT算法计算两组的免疫细胞浸润水平。采用免疫组化法检测ALKBH5的蛋白表达水平，通过定量聚合酶链式反应（quantitative polymerase chain reaction, qPCR）检测信使核糖核酸（messenger-ribonucleic acid, mRNA）的相对表达量。
结果：ALKBH5是唯一在肿瘤组织中表达量低于正常组织的调控因子。ALKBH5低表达与胃癌患者较差的总生存期相关，其可作为一项独立的保护性预后因素。基于ALKBH5调控基因构建的风险模型在训练集与测试集中均得到验证，展现出良好的临床预后预测潜力。基因本体（Gene Ontology, GO）富集分析结果显示，上述差异表达基因参与免疫应答过程；CIBERSORT分析结果表明，ALKBH5及其相关基因可重塑胃癌免疫微环境。免疫组化验证结果证实，胃癌组织中ALKBH5的蛋白表达水平显著降低；在ALKBH5敲低的样本中，SLC7A2与CGB3的表达水平明显下调。
结论：本研究发现ALKBH5可能作为胃癌的抑癌因子；ALKBH5及其相关基因可作为潜在的预后生物标志物与免疫治疗靶点。