Promoter Hypermethylation-Driven NPHS2 Silencing Promotes Immune Escape and Sunitinib Resistance in Clear Cell Renal Cell Carcinoma.

Objective: Renal cell carcinoma is a common malignancy of the urinary system. In this study, we analyzed a public clear cell renal cell carcinoma (ccRCC) dataset and identified Nephrosis 2, idiopathic, steroid-resistant (NPHS2) as a candidate gene to investigate whether epigenetic dysregulation of NPHS2 is associated with tumor microenvironment remodeling. Methods: Differential expression analysis was first performed on GSE68417 using GEO2R. In addition, clinical samples and cell-based assays were used to evaluate changes in NPHS2 expression and promoter methylation following 5'-Aza-CdR treatment. Subsequently, 786-O and A498 cells were obtained, and sunitinib-resistant 786-O/R and A498/R sublines were established. Lentiviral vectors with abnormal expression of NPHS2 were transfected into 786-O, A498, 786-O/R, and 498-R cells to detect changes in biological behaviors such as cell activity and epithelial-mesenchymal transition (EMT). Finally, a ccRCC tumor-bearing mouse model was constructed and intervened with lentiviral vectors with abnormal expression of NPHS2. Results: A total of 159 differentially expressed genes (DEGs) were identified in the GSE68417 dataset, and NPHS2 was included in all morphological changes related to cell epigenetic modifications. 5'-Aza-CdR markedly increased NPHS2 protein expression in 786-O cells (p < 0.05). Meanwhile, there was a high methylation phenomenon in the promoter region of the NPHS2 gene in ccRCC tissue (p < 0.05). In vitro, NPHS2 overexpression suppressed malignant cell behavior, attenuated EMT, and weakened immune evasion in ccRCC cells (p < 0.05). Finally, upregulating NPHS2 could inhibit the growth of ccRCC (p < 0.05) without causing significant changes in liver and kidney functions or malignant pathological damage in mice. Conclusion: These findings support an association between promoter hypermethylation and NPHS2 downregulation in ccRCC and suggest that NPHS2 loss may contribute to immune-evasive features and reduced sunitinib sensitivity. Further multicohort and mechanistic studies are warranted.