Proteomic Analysis Reveals Lipid Metabolism Disruption and Key Targets in ARPE-19 Cells after RNF13 Knockdown

Background Despite RNF13 is dysregulated in retinal degeneration models, its precise role in retinal function is not well understood. Previous studies suggest RNF13 may affect cellular pathways, including lipid metabolism, in the retina. However, the molecular mechanisms underlying its effects remain unclear. This study aims to identify the pathways regulated by RNF13 and key molecular targets involved in retinal degeneration. Methods To investigate the impact of RNF13 on the retina, the ARPE-19 cell line was transduced with lentivirus-RNF13-RNAi to interfere RNF13 expression. Cell viability was assessed via CCK-8 assay. Differentially expressed proteins (DEPs) were identified using tandem mass tag proteomics and further analyzed with KEGG pathway analysis, Gene Ontology (GO) annotation, and protein-protein interaction network analysis. Western blotting, qRT-PCR, and parallel reaction monitoring (PRM) were used to validate and identify RNF13-regulated targets. Oil Red O staining and CCK-8 were employed to assess phenotypic changes induced by siRNA-mediated SCD knockdown. Results A total of 340 DEPs were identified, with 80 downregulated and 260 upregulated after RNF13 knockdown. GO analysis showed that DEPs were enriched in "membrane" components and linked to “cellular processes” and “metabolic processes”, involving “binding” and “catalytic activity.” KEGG pathway analysis revealed significant disruptions in metabolic and PPAR signaling pathways. Western blotting, qRT-PCR, and PRM validation indicated that proteins in PPAR signaling pathway, such as SCD, were potential downstream targets regulated by RNF13. SCD knockdown demonstrated significant reduction in Oil Red O staining area and inhibition of cellular proliferation. Conclusions RNF13 knockdown primarily disrupts lipid metabolism via interference with the PPAR signaling pathway. SCD emerged as a key target among multiple PPAR-related proteins, suggesting its important role in retinal degeneration.