Single-cell multi-omics and spatial transcriptomics reveal the transcriptional regulatory landscape of clear cell renal cell carcinoma

Background: Clear cell renal cell carcinoma (ccRCC) represents the most aggressive form of renal cell carcinoma (RCC), distinguished by pronounced intratumoral heterogeneity, extensive metabolic reprogramming, and marked resistance to conventional therapeutic approaches. This study aimed to comprehensively characterize the cellular heterogeneity, epigenetic regulation, and transcription factor (TF) networks in ccRCC by integrating multi-omics data, and to identify functional key genes with prognostic and therapeutic significance. Methods: Single-cell RNA sequencing (scRNA-seq), single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq), and spatial transcriptomics (ST) were integrated to comprehensively explore cellular heterogeneity, epigenetic regulation, and TF networks in ccRCC. To uncover dynamic alterations in gene expression during cellular differentiation, single-cell pseudotime analysis and gene set enrichment analysis (GSEA) were performed. Furthermore, the functional significance of Y-box binding protein 3 (YBX3) in ccRCC cells was experimentally validated. Results: Single-cell transcriptomic profiling revealed 16 distinct cell populations within the ccRCC tumor microenvironment (TME), including ccRCC tumor cells, exhausted CD8+ T cells (Exhau CD8+ T cells), and macrophages. The scATAC-seq analysis demonstrated cell type-specific chromatin accessibility in immune cells, whereas ccRCC tumor cells exhibited reduced accessibility at immune-related genes, such as cluster of differentiation 2 (CD2). Epigenetic profiling further indicated that differentially accessible chromatin peaks in ccRCC cells were primarily enriched within intronic and exonic regions, implicating key TFs, including hepatocyte nuclear factor 1-beta (HNF1B) and the FOS-JUNB complex. An integrated analysis of scRNA-seq and scATAC-seq datasets identified five critical genes, YBX3, cubilin (CUBN), small nucleolar RNA host gene 8 (SNHG8), acetyl-CoA acyltransferase 2 (ACAA2), and protein kinase AMP-activated catalytic subunit α2 (PRKAA2), that were significantly associated with ccRCC prognosis. Pathway enrichment analysis revealed their involvement in metabolic reprogramming and tumor progression. Functional assays further confirmed that YBX3 knockdown inhibited ccRCC cell proliferation and migration. Conclusions: This study elucidates cellular heterogeneity, the epigenetic regulatory landscape, and the key genes driving ccRCC progression. The integration of multi-omics data offers novel insights into precise diagnostic strategies and therapeutic interventions, highlighting the pivotal role of genes such as YBX3.