第一部分  肾透明细胞癌微环境相关预后基因的挖掘

目的  越来越多的证据表明肿瘤微环境（tumor microenvironment, TME）在肾透明细胞癌（clear cell renal cell carcinoma, ccRCC）的发生、进展、预后及免疫治疗反应中起着关键作用。本研究旨在利用癌症基因组图谱（the cancer genome atlas, TCGA）数据库中的ccRCC数据挖掘影响ccRCC患者预后的TME相关的关键基因，并探讨其临床意义。

方法  从TCGA数据库中获取ccRCC的转录组数据和临床数据，通过 ESTIMATE算法估算每个样本的免疫成分和基质成分含量。利用“Limma” R包筛选不同免疫成分和基质成分之间的差异表达基因（differentially expressed genes, DEGs），并对获得的DEGs进行基因本体（Gene Ontology, GO）和京都基因和基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）的功能和通路富集分析。通过蛋白互作网络及单因素Cox回归分析筛选与ccRCC预后相关的关键基因，并进一步探究关键基因在肿瘤组织和正常组织中的表达差异情况及其表达水平与ccRCC患者生存及临床病理特征的关系。

结果  TME中免疫成分含量与ccRCC患者的总生存期相关（P<0.05）。研究共筛选出5个TME相关的关键基因（IGLL5、MZB1、HSD11B1、TNFSF13B及PPARGC1A）。ccRCC肿瘤组织中TNFSF13B的表达水平显著高于正常组织，而PPARGC1A的表达水平明显低于正常组织（P<0.001）。TNFSF13B的表达水平与ccRCC患者的肿瘤进展和不良生存预后呈正相关，PPARGC1A的表达水平与ccRCC患者的肿瘤进展和不良生存预后呈负相关（P<0.05）。

结论  TNFSF13B和PPARGC1A的表达水平与ccRCC患者的预后密切相关，有可能成为ccRCC潜在的预后标记物和治疗靶点。

第二部分  TNFSF13B和PPARGC1A在肾透明细胞癌中的表达及对微环境的作用

目的  探究肿瘤微环境（tumor microenvironment, TME）相关的关键基因TNFSF13B和PPARGC1A在肾透明细胞癌（clear cell renal cell carcinoma, ccRCC）中的差异表达水平及预后作用，并进一步探讨两个基因的表达对ccRCC微环境的影响及可能的机制。

方法  1. 从基因表达汇编（gene expression omnibus, GEO）数据库中获取ccRCC数据集的转录组数据和临床资料。回顾性收集本院2019年10月至2021年7月期间35例ccRCC患者的病理石蜡组织切片和新鲜组织样本用于免疫组化、实时定量PCR和免疫印迹研究。验证TNFSF13B和PPARGC1A在临床ccRCC样本中的真实表达水平以及其表达对ccRCC患者预后的影响。2. 从癌症基因组图谱（the cancer genome atlas, TCGA）数据库中获取ccRCC的转录组数据，利用基因集富集分析（Gene set enrichment analysis, GSEA）对TNFSF13B和PPARGC1A高低表达组样本进行富集分析。3. 应用CIBERSORT算法计算TCGA数据库ccRCC肿瘤样本中22种肿瘤浸润免疫细胞（tumor-infiltrating immune cells, TICs）的比例，并通过差异性分析和相关性分析探究TNFSF13B和PPARGC1A表达与TICs的关系。4. 进一步探讨TNFSF13B和PPARGC1A表达与ccRCC肿瘤突变负荷、免疫检查点表达水平以及免疫治疗反应的相关性。

结果  1. 进一步明确了ccRCC肿瘤组织中TNFSF13B的表达水平显著高于正常组织，PPARGC1A的表达水平显著低于正常组织，并且TNFSF13B高表达和PPARGC1A低表达的患者表现出更差的预后。2. GSEA显示TNFSF13B和PPARGC1A高表达组的基因均显著富集于免疫相关活动。3. TNFSF13B和PPARGC1A的表达均与TME中多种类型的TICs浸润比例相关，例如CD8 T细胞和调节性T细胞的比例与TNFSF13B的表达呈正相关，与PPARGC1A的表达呈负相关。4. TNFSF13B和PPARGC1A的表达与ccRCC肿瘤突变负荷、多个免疫检查点的表达水平以及药物治疗敏感性均显著相关。

结论  TNFSF13B 和PPARGC1A的表达水平与ccRCC患者的生存预后密切相关。二者的表达可能在ccRCC肿瘤微环境重塑中发挥重要作用，有望成为ccRCC潜在的治疗靶点和免疫治疗反应预测标记物。

Part I Identification of Tumor Microenvironment-Related Prognostic Genes in Clear Cell Renal Cell Carcinoma

Objective Increasing evidence suggests that the tumor microenvironment (TME) plays crucial roles in carcinogenesis, cancer progression, prognosis and immunotherapeutic efficacy in clear cell renal cell carcinoma (ccRCC). In this study, we comprehensively analyzed ccRCC RNA-sequencing data from The Cancer

Genome Atlas (TCGA) database to identify candidate prognostic TME-related genes involved in ccRCC, and to further explore their clinical significance.

Methods The transcriptome and clinical data of ccRCC were extracted from the TCGA database, and the immune and matrix components of each sample were estimated by the ESTIMATE algorithm. The "Limma" R package was used to screen the differentially expressed genes (DEGs) between immune and matrix components, and the obtained DEGs were subjected to functional and pathway enrichment analysis by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The key genes associated with ccRCC prognosis were screened by protein interaction network and univariate Cox regression analysis. We further investigated the differential expression of key genes in tumor and normal tissues and their expression levels in relation to the survival and clinicopathological characteristics of ccRCC patients.

Results The content of immune components in TME was correlated with the overall survival of ccRCC patients (P<0.05). A total of five TME-related pivotal genes (IGLL5, MZB1, HSD11B1, TNFSF13B, and PPARGC1A) were identified. Compared to the normal tissues, the expression level of TNFSF13B in ccRCC tumor tissues was significantly higher, while the expression level of PPARGC1A was significantly lower (both P<0.001). Meanwhile, the expression level of TNFSF13B was positively correlated with tumor progression and poor survival prognosis in ccRCC patients, and the expression level of PPARGC1A was negatively correlated with tumor progression and poor survival prognosis in ccRCC patients (both P<0.05).

Conclusion The expression levels of TNFSF13B and PPARGC1A are closely related to the prognosis of ccRCC patients, and have the potential to be the prognostic markers and therapeutic targets for ccRCC.

Part II The Effect of the Expression of TNFSF13B and PPARGC1A on the Clear Cell Renal Cell Carcinoma Microenvironment

Objective To investigate the differential expression levels and prognostic roles of TNFSF13B and PPARGC1A, key genes related to the tumor microenvironment (TME), in clear cell renal cell carcinoma (ccRCC), and to further explore the effects of the two genes on the ccRCC microenvironment and the underlying mechanisms.

Methods 1. Transcriptomic and clinical data of the ccRCC datasets were obtained from the gene expression omnibus (GEO) database. Retrospectively collected pathological paraffin tissue sections and fresh tissue samples from 35 ccRCC patients from our hospital between October 2019 and July 2021 for immunohistochemistry, real-time quantitative PCR, and western blotting studies. To verify the true expression levels of TNFSF13B and PPARGC1A in clinical ccRCC tissues and the impact of their expression on the prognosis of ccRCC patients. 2. The transcriptomic data of ccRCC was obtained from the cancer genome atlas (TCGA) database. Gene set enrichment analysis (GSEA) was conducted among the samples of TNFSF13B and PPARGC1A high and low expression groups to investigate potential pathways and functions. 3. The CIBERSORT algorithm was applied to estimate the abundance of 22 tumor-infiltrating immune cells (TICs) in ccRCC tumor samples from the TCGA database. Correlations between TNFSF13B and PPARGC1A expression and TICs were evaluated using differential analysis and correlation analysis. 4. Associations between TNFSF13B and PPARGC1A expression and tumor mutational burden, immune checkpoints expression levels, and response to immunotherapy involved in ccRCC were further explored.  

Results 1. The results further validated that the expression levels of TNFSF13B in ccRCC tumor tissues were significantly higher and that PPARGC1A were significantly lower than in normal tissues, and patients with high TNFSF13B expression and low PPARGC1A expression probably exhibited worse prognosis. 2. GSEA revealed that genes in the groups with high TNFSF13B and PPARGC1A were enriched mainly in immune-related activities. 3. By combining difference and correlation analyses, the expression of both TNFSF13B and PPARGC1A were found to be significantly associated with multiple types of TICs. For example, the proportion of CD8 T cells and regulatory T cells were positively correlated with TNFSF13B expression and negatively correlated with PPARGC1A expression. 4. TNFSF13B and PPARGC1A expression were significantly correlated with the tumor mutational burden, several immune checkpoints expression, and drug sensitivity involved in ccRCC.

Conclusion The expression levels of TNFSF13B and PPARGC1A are closely related to the survival prognosis of ccRCC patients. They may play significant roles in the remodeling of the ccRCC microenvironment, and are expected to be potential therapeutic targets and predictive markers of the immunotherapeutic response in ccRCC.