Integrated bioinformatics analysis and experimental validation of SRF as a potential senescence biomarker in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is closely linked to cellular senescence. Identifying senescence-related biomarkers is crucial for discovering potential diagnostic markers and therapeutic strategies for MASLD. Senescence-related genes from GenAge were integrated with GEO datasets (GSE89632, GSE63067). Overlapping genes among differentially expressed genes, WGCNA modules, and senescence sets were screened using five machine learning algorithms (LASSO, Random Forest, Boruta, GBM, SVM-RFE). Immune infiltration and functional enrichment were assessed by single-sample gene set enrichment analysis (ssGSEA) and gene set enrichment analysis (GSEA), respectively. Finally, the expression of these four hub genes was confirmed by RT-qPCR in animal models, cell lines, and clinical samples, while SRF protein levels were further validated by Western blotting. Single-cell RNA sequencing (scRNA-seq) was utilized for cell-type-specific characterization and in silico virtual knockout of SRF. The pathogenic role of SRF was investigated through in vitro knockdown assays. A total of 46 genes associated with both MASLD and cellular senescence were identified. After screening by ML and validation with external datasets, SRF, ATF3, ME1, and GADD45G were determined to be key genes. Experimental validation demonstrated significantly downregulated expression of SRF, ATF3, and GADD45G in MASLD, whereas ME1 was upregulated. Single-cell analysis confirmed predominant expression of SRF in hepatocytes, with marked downregulation during MASLD progression. Furthermore, in silico virtual knockout of SRF in single-cell clusters revealed a dramatic transcriptomic shift toward a pro-steatotic and pro-senescence state, consistent with the observed bulk RNA-seq profiles. Critically, SRF knockdown significantly exacerbated both lipid deposition and cellular senescence in hepatocyte steatosis models, confirming its functional involvement in MASLD pathogenesis. SRF deficiency accelerates hepatocellular senescence and exacerbates MASLD progression. Our results identify SRF as a potential biomarker and therapeutic target for MASLD.