Table 4_Analysis of the correlation between RFC4 expression and tumor immune microenvironment and prognosis in patients with cervical cancer.xlsx

BackgroundReplication factor C subunit 4 (RFC4) plays a critical role in the initiation and progression of some cancers; however, its relationship with tumor-infiltrating immune cells in cervical cancer (CC) has not been comprehensively analyzed. This study aimed to determine whether RFC4 overexpression affects overall survival in CC and to explore its impact and potential mechanisms on the tumor immune microenvironment.
MethodsData from Genotype-Tissue Expression database (GTEx) and Cancer Genome Atlas (TCGA) database were used as the exploration set. Datasets from the Gene Expression Omnibus (GEO) were used as the validation set. We also validated the expression of the RFC4 protein in the Human Protein Atlas (HPA) database and a real cohort. Clinical data on CC were evaluated for their association with RFC4 using TCGA and GEO databases. Possible relationships amongst RFC4, immune cells, and related genes were investigated using Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) and Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression (ESTIMATE). GO and KEGG pathway enrichment analyses were used to explore potential mechanisms. Tumor immune dysfunction and exclusion (TIDE) scores were used to predict the immunotherapeutic response to RFC4.
ResultsIn the exploration, validation, and real cohort datasets, RFC4 expression was significantly elevated in CC tissues compared to that in normal tissues. Survival analysis based on TCGA and GEO datasets showed that CC patients with high RFC4 expression had a better prognosis than those with low expression. RFC4 expression was strongly correlated with some immunostimulators and immunoinhibitors. RFC4 expression was significantly negatively correlated with activated mast cell immune infiltration, activated CD4 memory T cells, M0 macrophages, and resting natural killer (NK) cells and significantly positively associated with activated dendritic cells, resting dendritic cells, and plasma cells.
ConclusionRFC4 is highly expressed in CC tissues. However, patients with high RFC4 expression in CC have a better prognosis, possibly because RFC4 exerts antitumor effects by affecting the immunostimulatory tumor microenvironment, such as immunostimulatory and dendritic cell infiltration.

背景 复制因子C亚基4（Replication factor C subunit 4, RFC4）在部分癌症的发生与进展中发挥关键作用，但目前尚未对其与宫颈癌（cervical cancer, CC）肿瘤浸润免疫细胞的关联开展全面分析。本研究旨在明确RFC4过表达是否会影响宫颈癌患者的总生存期，并探讨其对肿瘤免疫微环境的影响及潜在机制。
方法 研究数据来自基因型-组织表达数据库（Genotype-Tissue Expression database, GTEx）与癌症基因组图谱（Cancer Genome Atlas, TCGA），作为探索数据集；基因表达综合数据库（Gene Expression Omnibus, GEO）数据集作为验证数据集。本研究还在人类蛋白质图谱（Human Protein Atlas, HPA）数据库及一项真实临床队列中验证了RFC4蛋白的表达水平。借助TCGA与GEO数据库，评估宫颈癌临床数据与RFC4表达的相关性。通过基于估算RNA转录本相对子集的细胞类型识别算法（Cell-type Identification by Estimating Relative Subsets of RNA Transcripts, CIBERSORT）以及恶性肿瘤组织基质与免疫细胞表达定量分析工具（Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression, ESTIMATE），探究RFC4、免疫细胞及相关基因间的潜在关联。采用基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路富集分析，探索潜在作用机制。使用肿瘤免疫功能异常与排斥评分（Tumor Immune Dysfunction and Exclusion, TIDE）预测与RFC4表达相关的免疫治疗响应情况。
结果 在探索集、验证集及真实队列数据集内，宫颈癌组织中的RFC4表达水平显著高于正常对照组织。基于TCGA与GEO数据集的生存分析显示，RFC4高表达的宫颈癌患者预后优于低表达患者。RFC4表达与部分免疫刺激因子及免疫抑制因子显著相关。RFC4表达与活化肥大细胞免疫浸润、活化CD4记忆性T细胞、M0巨噬细胞及静息自然杀伤（natural killer, NK）细胞浸润呈显著负相关，而与活化树突状细胞、静息树突状细胞及浆细胞浸润呈显著正相关。
结论 RFC4在宫颈癌组织中呈高表达状态。但宫颈癌患者中RFC4高表达者预后更佳，这可能是因为RFC4通过调控免疫刺激型肿瘤微环境（如促进免疫刺激因子表达及树突状细胞浸润）发挥抗肿瘤作用。