Table_1_Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer.docx

Early diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC) have been challenging. Signaling cascades involving the Wingless-type (WNT) gene family play important biological roles and show prognostic value in various cancers, including NSCLC. On this basis, this study aimed to investigate the significance of WNTs in the prognosis and tumor immunity in NSCLC by comprehensive analysis. Expression and methylation levels of WNTs were obtained from the ONCOMINE, TIMER, and UALCAN. The dataset obtained from The Cancer Genome Atlas (TCGA) was utilized for prognostic analysis. cBioPortal was used to perform genetic alterations and correlation analysis of WNTs. R software was employed for functional enrichment and pathway analysis, partial statistics, and graph drawing. TRRUST was used to find key transcription factors. GEPIA was utilized for the analysis of expression, pathological staging, etc. Correlative analysis of immune infiltrates from TIMER. TISIDB was used for further immune infiltration validation analysis. Compared with that of normal tissues, WNT2/2B/3A/4/7A/9A/9B/11 expressions decreased, while WNT3/5B/6/7B/8B/10A/10B/16 expressions increased in lung adenocarcinoma (LUAD); WNT2/3A/7A/11 expressions were lessened, while WNT2B/3/5A/5B/6/7B/10A/10B/16 expressions were enhanced in squamous cell lung cancer (LUSC). Survival analysis revealed that highly expressed WNT2B and lowly expressed WNT7A predicted better prognostic outcomes in LUAD and LUSC. In the study of immune infiltration levels, WNT2, WNT9B, and WNT10A were positively correlated with six immune cells in LUAD; WNT1, WNT2, and WNT9B were positively correlated with six immune cells in LUSC, while WNT7B was negatively correlated. Our study indicated that WNT2B and WNT7A might have prognostic value in LUAD, and both of them might be important prognostic factors in LUSC and correlated to immune cell infiltration in LUAD and LUSC to a certain extent. Considering the prognostic value of WNT2B and WNT7A in NSCLC, we validated their mRNA and protein expression levels in NSCLC by performing qRT-PCR, western blot, and immunohistochemical staining on NSCLC pathological tissues and cell lines. This study may provide some direction for the subsequent exploration of the prognostic value of the WNTs and their role as biomarkers in NSCLC.

非小细胞肺癌（non-small cell lung cancer, NSCLC）的早期诊断与预后预测始终颇具挑战。包含无翅型（Wingless-type, WNT）基因家族的信号通路在多种癌症（包括NSCLC）中发挥重要生物学功能，并展现出预后价值。基于此，本研究旨在通过综合分析，探究WNTs在NSCLC预后与肿瘤免疫中的意义。本研究从ONCOMINE、TIMER及UALCAN数据库获取WNTs的表达与甲基化水平数据；采用来自癌症基因组图谱（The Cancer Genome Atlas, TCGA）的数据集开展预后分析；借助cBioPortal工具完成WNTs的遗传变异与相关性分析；使用R软件进行功能富集分析、通路分析、统计学检验及图表绘制；通过TRRUST数据库筛选关键转录因子；利用GEPIA数据库开展表达水平、病理分期等相关分析；针对TIMER数据库中的免疫浸润数据开展相关性研究，并采用TISIDB数据库进行进一步的免疫浸润验证分析。与正常组织相比，肺腺癌（lung adenocarcinoma, LUAD）中WNT2、2B、3A、4、7A、9A、9B、11的表达水平下调，而WNT3、5B、6、7B、8B、10A、10B、16的表达水平上调；肺鳞状细胞癌（squamous cell lung cancer, LUSC）中WNT2、3A、7A、11的表达水平降低，而WNT2B、3、5A、5B、6、7B、10A、10B、16的表达水平升高。生存分析结果显示，在LUAD与LUSC中，高表达的WNT2B与低表达的WNT7A均提示更良好的预后结局。在免疫浸润水平分析中，LUAD内的WNT2、WNT9B与WNT10A的表达水平与6种免疫细胞呈正相关；LUSC内的WNT1、WNT2与WNT9B的表达水平与6种免疫细胞呈正相关，而WNT7B则呈负相关。本研究结果表明，WNT2B与WNT7A在LUAD中可能具备预后价值，二者在LUSC中也可能作为重要的预后因子，并在一定程度上与LUAD及LUSC的免疫细胞浸润相关。鉴于WNT2B与WNT7A在NSCLC中的预后价值，本研究通过对NSCLC病理组织及细胞系开展实时定量聚合酶链反应（qRT-PCR）、蛋白质印迹法（western blot）及免疫组化染色，验证了二者在NSCLC中的mRNA与蛋白表达水平。本研究可为后续探究WNTs在NSCLC中的预后价值及其作为生物标志物的作用提供一定的研究方向。