DataSheet_3_Identification of specific prognostic markers for lung squamous cell carcinoma based on tumor progression, immune infiltration, and stem index.pdf

IntroductionLung squamous cell carcinoma (LUSC) is a unique subform of nonsmall cell lung cancer (NSCLC). The lack of specific driver genes as therapeutic targets leads to worse prognoses in patients with LUSC, even with chemotherapy, radiotherapy, or immune checkpoint inhibitors. Furthermore, research on the LUSC-specific prognosis genes is lacking. This study aimed to develop a comprehensive LUSC-specific differentially expressed genes (DEGs) signature for prognosis correlated with tumor progression, immune infiltration,and stem index.
MethodsRNA sequencing data for LUSC and lung adenocarcinoma (LUAD) were extracted from The Cancer Genome Atlas (TCGA) data portal, and DEGs analyses were conducted in TCGA-LUSC and TCGA-LUAD cohorts to identify specific DEGs associated with LUSC. Functional analysis and protein–protein interaction network were performed to annotate the roles of LUSC-specific DEGs and select the top 100 LUSC-specific DEGs. Univariate Cox regression and least absolute shrinkage and selection operator regression analyses were performed to select prognosis-related DEGs.
ResultsOverall, 1,604 LUSC-specific DEGs were obtained, and a validated seven-gene signature was constructed comprising FGG, C3, FGA, JUN, CST3, CPSF4, and HIST1H2BH. FGG, C3, FGA, JUN, and CST3 were correlated with poor LUSC prognosis, whereas CPSF4 and HIST1H2BH were potential positive prognosis markers in patients with LUSC. Receiver operating characteristic analysis further confirmed that the genetic profile could accurately estimate the overall survival of LUSC patients. Analysis of immune infiltration demonstrated that the high risk (HR) LUSC patients exhibited accelerated tumor infiltration, relative to low risk (LR) LUSC patients. Molecular expressions of immune checkpoint genes differed significantly between the HR and LR cohorts. A ceRNA network containing 19 lncRNAs, 50 miRNAs, and 7 prognostic DEGs was constructed to demonstrate the prognostic value of novel biomarkers of LUSC-specific DEGs based on tumor progression, stemindex, and immune infiltration. In vitro experimental models confirmed that LUSC-specific DEG FGG expression was significantly higher in tumor cells and correlated with immune tumor progression, immune infiltration, and stem index. In vitro experimental models confirmed that LUSC-specific DEG FGG expression was significantly higher in tumor cells and correlated with immune tumor progression, immune infiltration, and stem index.
ConclusionOur study demonstrated the potential clinical implication of the 7- DEGs signature for prognosis prediction of LUSC patients based on tumor progression, immune infiltration, and stem index. And the FGG could be an independent prognostic biomarker of LUSC promoting cell proliferation, migration, invasion, THP-1 cell infiltration, and stem cell maintenance.

引言
肺鳞状细胞癌（Lung Squamous Cell Carcinoma, LUSC）是非小细胞肺癌（Non-Small Cell Lung Cancer, NSCLC）的一种独特亚型。由于缺乏可作为治疗靶点的特异性驱动基因，LUSC患者的预后较差，即便接受化疗、放疗或免疫检查点抑制剂治疗亦是如此。此外，目前针对LUSC特异性预后基因的研究仍较为匮乏。本研究旨在构建一套全面的LUSC特异性差异表达基因（Differentially Expressed Genes, DEGs）特征模型，以关联肿瘤进展、免疫浸润及干细胞指数相关的预后情况。
方法
从癌症基因组图谱（The Cancer Genome Atlas, TCGA）数据门户中提取LUSC与肺腺癌（Lung Adenocarcinoma, LUAD）的RNA测序数据，并在TCGA-LUSC与TCGA-LUAD队列中开展差异表达基因分析，以筛选与LUSC相关的特异性差异表达基因。通过功能分析与蛋白质相互作用网络分析，对LUSC特异性差异表达基因的功能进行注释，并筛选出排名前100的LUSC特异性差异表达基因。采用单因素Cox回归与最小绝对收缩和选择算子（Least Absolute Shrinkage and Selection Operator, LASSO）回归分析，筛选与预后相关的差异表达基因。
结果
本研究共筛选得到1604个LUSC特异性差异表达基因，并构建了经验证的7基因特征模型，其组成基因为FGG、C3、FGA、JUN、CST3、CPSF4及HIST1H2BH。其中FGG、C3、FGA、JUN及CST3与LUSC不良预后相关，而CPSF4与HIST1H2BH则是LUSC患者潜在的良好预后标志物。受试者工作特征（Receiver Operating Characteristic, ROC）分析进一步证实，该基因特征模型可准确预测LUSC患者的总生存期。免疫浸润分析显示，与低风险（Low Risk, LR）LUSC患者相比，高风险（High Risk, HR）LUSC患者的肿瘤浸润程度更高。高、低风险队列间的免疫检查点基因分子表达水平存在显著差异。基于肿瘤进展、干细胞指数及免疫浸润特征，本研究构建了包含19个长链非编码RNA（Long Non-Coding RNA, lncRNA）、50个微小RNA（MicroRNA, miRNA）及7个预后相关差异表达基因的内源竞争RNA（Competing Endogenous RNA, ceRNA）网络，以阐明LUSC特异性差异表达基因作为新型生物标志物的预后价值。体外实验模型证实，LUSC特异性差异表达基因FGG在肿瘤细胞中的表达水平显著升高，且与肿瘤免疫进展、免疫浸润及干细胞指数相关。体外实验模型证实，LUSC特异性差异表达基因FGG在肿瘤细胞中的表达水平显著升高，且与肿瘤免疫进展、免疫浸润及干细胞指数相关。
结论
本研究证实，基于肿瘤进展、免疫浸润及干细胞指数构建的7基因特征模型，对LUSC患者的预后预测具有潜在临床应用价值。此外，FGG可作为LUSC的独立预后生物标志物，其可促进细胞增殖、迁移与侵袭，增强THP-1细胞浸润，并维持干细胞特性。