[Screening and identification of vascular calcification-associated genes: implication of thymidine kinase 1]

Objective: Investigate key genes influencing vascular calcification through bioinformatics analysis and experimental validation. Methods: Three vascular calcification datasets (GSE159832, GSE229679 and GSE37558) were obtained from the Gene Expression Omnibus database. Subsequently, gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and conventional gene set enrichment analysis (GSEA) were performed on the common differential expressed genes(DEGs). For in vitro validation, a vascular smooth muscle cell calcification model was established by stimulating mouse primary vascular smooth muscle cells with high phosphate and calcium chloride (Pi+CaCl₂). Cells were divided into a control group and a Pi+CaCl₂ group. To investigate the role of TK1, cells were transfected with TK1-targeting siRNA (siTK1) or control siRNA (siControl) prior to Pi+CaCl₂ stimulation, creating siControl+Pi+CaCl₂ and siTK1+Pi+CaCl₂ groups. The association between key DEGs and vascular calcification was assessed at the protein and mRNA levels using Western blot and quantitative real-time PCR, respectively. Changes in the phosphorylation of the downstream effector, AKT (p-AKT/AKT), were also measured. Results: A total of 2275, 449, and 381 DEGs were identified from the three vascular calcification datasets (GSE159832, GSE229679, and GSE37558), respectively. Two common DEGs-phosphoserine aminotransferase 1 and thymidine kinase 1 (TK1)-were identified across all datasets. GO enrichment analysis revealed that TK1 was significantly enriched in pathways related to ribosome biogenesis, assembly, and rRNA processing and maturation. GSEA-KEGG analysis indicated significant enrichment in the PI3K-AKT signaling pathway, pathways in cancer, neurodegenerative diseases, cytoskeleton, and smooth muscle contraction. Conventional GSEA of TK1 further confirmed significant enrichment in pathways including dynein, epithelial tight junctions, axon guidance, and vascular smooth muscle contraction pathways. At the experimental level, both protein and mRNA expression of TK1, along with the p-AKT/AKT ratio, were significantly lower in the Pi+CaCl₂ group compared to the control group (all P<0.05). Furthermore, compared to the siControl+Pi+CaCl₂ group, the siTK1+Pi+CaCl₂ group exhibited decreased expression of differentiation markers, increased expression of calcification markers, and a further reduced p-AKT/AKT ratio (all P<0.05). Conclusion: Integrated bioinformatics and cellular validation demonstrate a correlation between TK1 expression and vascular calcification, suggesting a potential protective role for TK1 in this pathological process.