Table 7_GPR65 is a novel immune biomarker and regulates the immune microenvironment in lung adenocarcinoma.xlsx

BackgroundThe tumor microenvironment (TME) plays a crucial role in the progression of lung adenocarcinoma (LUAD), and it may serve as the best prognostic predictor of LUAD. GPR65 is an extracellular pH-sensing G protein-coupled receptor and a glycosphingolipid receptor, which is engaged in the functions of regulating tumor immunity. However, the prognostic value of GPR65 and its relevance to immune infiltration in LUAD are unknown.
MethodsThe proportion of immune, stromal and tumor cells in LUAD samples was assessed by ESTIMATE algorithm scores with RNA sequence data and clinical information from LUAD patients downloaded from The Cancer Genome Atlas (TCGA) database. We screened differential genes (DEGs) in the immune and stromal components, and then screened modular genes by the WGCNA algorithm, which were intersected with DEGs and incorporated into the LASSO-COX regression model. Additionally, nomogram containing GPR65 and clinical features were constructed for predicting patient prognosis. Then, the correlation between GPR65 and immune cell infiltration was assessed by CIBERSORT, and the impact of hub gene on immunotherapy was determined using correlation analysis between GPR65 and immune checkpoint molecules. Finally, we confirmed the expression of GPR65 in LUAD by Western Blot, Quantitative Real-time PCR and Immunohistochemistry.
ResultsIn our study, we found that low expression of GPR65 was associated with poorer overall survival and primary treatment outcome in patients with LUAD. Moreover, GPR65 expression was found to be closely correlated with multiple tumor infiltrating immune cells (TIICs) and immune checkpoint molecules. Immunohistochemistry and Quantitative Real-time PCR results confirmed that the transcription levels and protein expression levels of GPR65 in LUAD tissues were significantly lower than in normal tissues. Western Blot results showed that the expression of GPR65 in human normal lung epithelial cell lines was significantly higher than the expression level in LUAD cell lines.
ConclusionsGPR65 may be an important immune biomarker in the prognosis and diagnosis of LUAD.

**背景** 肿瘤微环境（tumor microenvironment, TME）在肺腺癌（lung adenocarcinoma, LUAD）的发生发展中发挥关键作用，或可成为肺腺癌最佳的预后预测因子。GPR65是一种细胞外pH感应型G蛋白偶联受体，同时也是糖鞘脂受体，参与调控肿瘤免疫功能。然而，GPR65在肺腺癌中的预后价值及其与免疫浸润的相关性尚不明确。**方法** 本研究从癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据库下载肺腺癌患者的RNA测序数据与临床信息，通过ESTIMATE算法评分评估肺腺癌样本中免疫细胞、基质细胞与肿瘤细胞的占比。首先筛选免疫组分与基质组分中的差异基因（differential genes, DEGs），随后通过加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA）算法筛选模块基因，将模块基因与差异基因取交集后纳入LASSO-COX回归模型。此外，本研究构建包含GPR65与临床特征的列线图，用于预测患者预后。通过CIBERSORT算法评估GPR65与免疫细胞浸润的相关性，并通过GPR65与免疫检查点分子的相关性分析，明确核心基因对免疫治疗的影响。最后，本研究通过蛋白质免疫印迹（Western Blot）、实时定量聚合酶链反应（Quantitative Real-time PCR）与免疫组化（Immunohistochemistry）验证GPR65在肺腺癌中的表达水平。**结果** 本研究发现，肺腺癌患者中GPR65低表达与较差的总生存期及一线治疗结局显著相关。此外，GPR65的表达水平与多种肿瘤浸润免疫细胞（tumor infiltrating immune cells, TIICs）及免疫检查点分子密切相关。免疫组化与实时定量聚合酶链反应结果证实，肺腺癌组织中GPR65的转录水平与蛋白表达水平均显著低于正常组织。蛋白质免疫印迹结果显示，人正常肺上皮细胞系中GPR65的表达水平显著高于肺腺癌细胞系。**结论** GPR65或可成为肺腺癌预后与诊断的重要免疫生物标志物。