目的:探讨癌相关成纤维细胞(CAFs)在胃癌(GC)肿瘤微环境中的作用及其分子机制。
方法:首先评估GC患者CAFs的表达丰度,并进行生存分析。随后,癌症基因组图谱(TCGA)数据用于差异分析,生存分析,和EPIC分析,同时下载单细胞数据(GSE183904)用于CAF的差异分析。临床数据汇集,单变量和多变量Cox分析,对临床GC组织样品进行免疫荧光,以探索患者CAFs中RCN3的表达。Westernblot和定量聚合酶链反应(qPCR)检测RCN3的表达。通过染色质免疫沉淀(CHIP)实验探索RCN3,PCSK6和STAT1之间的关系,通过检测生物标志物M1/M2检测基因对巨噬细胞极化的影响。
结果:发现与正常组相比,GC中的CAFs明显更高。揭示TCGA差异分析的结果,观察到GC表现出RCN3表达水平的显著上调。临床统计表明RCN3表达水平升高与肿瘤大小的T期分类之间呈正相关。此外,发现RCN3对胃癌患者的总生存期有显著影响,作为独立的预后指标。单细胞数据分析显示PCSK6在巨噬细胞中高表达,和来自GC患者的样品的免疫荧光染色显示GC组织中巨噬细胞的细胞膜上PCSK6的表达增加。随后的细胞实验证实RCN3蛋白可以调节PCSK6的表达,PCSK6通过STAT1调节巨噬细胞极化。
结论:在GC中,CAFs通过RCN3/PCSK6/STAT1通路调节巨噬细胞极化。
OBJECTIVE: To explore the role and molecular mechanism of cancer-associated fibroblasts (CAFs) in the tumor microenvironment of gastric cancer (GC).
METHODS: The expression of CAFs in GC patients was first assessed for abundance, and survival analysis was performed. Subsequently, The Cancer Genome Atlas (TCGA) data were used for differential analysis, survival analysis, and EPIC analysis, while single-cell data (GSE183904) were downloaded for differential analysis of CAFs. Clinical data pooling, univariate and multivariate Cox analysis, and immunofluorescence were carried out on clinical GC tissue samples to explore RCN3 expression within patient CAFs. Western blot and quantitative polymerase chain reaction (qPCR) were used to detect the expression of RCN3. The relationship between RCN3, PCSK6, and STAT1 was explored by chromatin immunoprecipitation (CHIP) experiments, and the effects of the genes on macrophage polarization were detected by detecting biomarkers of biological M1/M2.
RESULTS: CAFs in GC were found to be significantly higher compared to the normal group. Revealing the results of TCGA differential analysis, it was observed that GC exhibited a substantial upregulation in the expression levels of RCN3. The clinical statistics indicate a positive correlation between an elevated level of RCN3 expression and the T-stage classification of tumor size. In addition, RCN3 was found to have a significant impact on the overall survival of patients with gastric cancer, acting as an independent prognostic indicator. Analysis of single-cell data showed high expression of PCSK6 in macrophages, and immunofluorescence staining of samples from GC patients showed increased expression of PCSK6 on the cell membranes of macrophages in GC tissues. The subsequent cellular experiments confirmed RCN3 protein can regulate the expression of PCSK6, and PCSK6 regulates macrophage polarization through STAT1.
CONCLUSIONS: CAFs regulate macrophage polarization through the RCN3/PCSK6/STAT1 pathway in GC.