Integrated Weighted Gene Co-Expression Network and Single-Cell RNA Sequencing Analyses Reveal the Prognostic Significance of Hypoxia in Gastric Cancer

<b>Background:</b> Hypoxia is a key driver of cancer progression. However, its specific prognostic significance in gastric cancer (GC) remains insufficiently characterized. <b>Methods:</b> Single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), univariate Cox regression, and least absolute shrinkage and selection operator (LASSO) regression were employed to identify a hypoxia-related prognostic signature. Subsequently, immune microenvironment profiling and single-cell RNA sequencing analyses were employed to further characterize the biological characteristics of the signature. In addition, quantitative real-time polymerase chain reaction (qPCR) was used to validate the expression levels of key hypoxia-associated genes in human GC tissues. <b>Results:</b> Elevated hypoxia levels were linked to worse survival outcomes in GC patients. Through integrated WGCNA, Cox, and LASSO analyses, a hypoxia-related prognostic signature (HYS) consisting of four genes-<i>SPARC</i>, <i>AXL</i>, <i>NRP1</i>, and <i>VCAN</i>-was established. Patients in the HYS-high group exhibited markedly poorer overall survival than their HYS-low counterparts [<i>p</i> = 0.000126, hazard ratio (HR) = 1.936]. Moreover, the HYS-high group exhibited increased infiltration of resting CD4⁺ memory T cells, monocytes, M2 macrophages, and resting mast cells, as well as elevated expression of immunosuppressive molecules, including <i>PDCD1LG2</i> and <i>HAVCR2</i>. Single-cell RNA sequencing analysis revealed that the signature genes were predominantly expressed in cancer-associated fibroblasts. Consistently, qPCR analysis in five paired GC and para-carcinoma tissues confirmed higher expression of these genes in tumor samples (<i>p</i> < 0.01). <b>Conclusions:</b> Our findings indicate that hypoxia is a critical determinant of prognosis in GC and is closely associated with an immunosuppressive tumor microenvironment, highlighting its potential value as a prognostic biomarker and therapeutic target.