Integrated analysis of scRNA-seq and bulk RNA-seq identifies prognostic cell populations and genes in cholangiocarcinoma

Abstract Background Cholangiocarcinoma is a relatively uncommon cancer, accounting for approximately 3% of all gastrointestinal malignancies. However, its global incidence has been steadily rising over the past few decades, and the precise factors driving this increase remain unclear. This study aimed to identify prognosis-related cell populations and genes in cholangiocarcinoma and to further validate selected findings experimentally. Methods In this study, we performed a comprehensive analysis of single-cell RNA sequencing (scRNA-seq) data on cholangiocarcinoma obtained from publicly available databases. The analytical workflow included clustering and cell annotation, GO, KEGG, GSEA analysis, TCGA data acquisition and preprocessing, Scissor analysis, survival analysis, and deconvolution analysis. Key findings were further validated using cell-based experiments. The expression of MT2A and TKT was further validated in clinical cholangiocarcinoma samples by Western blot and qRT-PCR, and the effects of MT2A on cell proliferation, migration, invasion, and apoptosis were assessed by in vitro experiments. Results Our findings revealed a significant imbalance between malignant cells and T cells within cholangiocarcinoma tissues, and a higher abundance of malignant cells was associated with poorer prognosis. The distribution of Scissor-positive cells was highly consistent with that of malignant cells, while differentially expressed genes in Scissor-positive cells, such as MT2A and TKT, were associated with poor prognosis in patients with cholangiocarcinoma. Furthermore, the expression levels of MT2A and TKT were found to be higher in clinical cholangiocarcinoma samples than in normal tissues. Functional experiments further demonstrated that MT2A promoted the proliferation, migration, and invasion of cholangiocarcinoma cells. Conclusions We identified distinct prognosis-related cell populations and differentially expressed genes in cholangiocarcinoma and further confirmed the functional relevance of MT2A through cell-based experiments. These findings provide new insights into the pathogenesis and progression of cholangiocarcinoma and may facilitate the development of targeted therapeutic strategies.