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Published on: January 24, 2017
Proteomic analysis reveals lipid metabolism disruption and key targets in ARPE-19 cells after RNF13 knockdown
Chen Xie1,2, Xin Yu1,2, Liyin Wang3,4,5
1Clinical Research Center, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 311003, Zhejiang, China.
Abstract:
Although RNF13 is known to be dysregulated in models of retinal degeneration, its specific role in retinal pigment epithelium cell function remains unclear. This study aimed to elucidate the RNF13-regulated signalling pathways and identify key molecular targets in vitro. Using lentivirus-mediated RNF13 knockdown in ARPE-19 cells, we observed significant alterations in protein expression using tandem mass tag proteomics and identified 340 differentially expressed proteins (DEPs), among which 80 were downregulated and 260 were upregulated. GO annotation revealed that these DEPs were primarily associated with "membrane" components and linked to "cellular processes" and "metabolic processes" involving "binding" and "catalytic activity". KEGG pathway analysis revealed significant disruptions in metabolic pathways, particularly the PPAR signalling pathway. Subsequent validation via western blotting, qRT-PCR, and parallel reaction monitoring confirmed that SCD expression is downregulated in RNF13-knockdown cells. Furthermore, siRNA-mediated knockdown of SCD expression resulted in a significant reduction in the Oil Red O-stained area and inhibited cellular proliferation. Impaired lipid metabolism and cell viability induced by RNF13 knockdown were restored by SCD overexpression. These findings suggest that RNF13 may influence lipid metabolism through the PPAR signalling pathway in ARPE-19 cells, with a possible involvement of SCD as a potential mediator in this process.
Insights
RNF13 knockdown in retinal pigment epithelium cells disrupts lipid metabolism via the PPAR pathway, with SCD potentially mediating these effects. Restoring SCD levels reverses RNF13 knockdown-induced impairments.
Area of Science:
- Molecular Biology
- Cell Biology
- Ophthalmology
Background:
- RNF13 dysregulation is observed in retinal degeneration models.
- The precise function of RNF13 in retinal pigment epithelium (RPE) cell biology is not well understood.
- Investigating RNF13's role is crucial for understanding RPE cell function and potential therapeutic targets.
Purpose of the Study:
- To elucidate RNF13-regulated signaling pathways in RPE cells.
- To identify key molecular targets affected by RNF13 in vitro.
- To explore the role of RNF13 in lipid metabolism and cell viability.
Main Methods:
- Lentivirus-mediated RNF13 knockdown in ARPE-19 cells.
- Tandem mass tag (TMT) proteomics to analyze differentially expressed proteins (DEPs).
- Gene Ontology (GO) and KEGG pathway analyses.
- Validation using Western blotting, qRT-PCR, and parallel reaction monitoring (PRM).
- siRNA-mediated knockdown and overexpression studies for SCD.
Main Results:
- RNF13 knockdown altered expression of 340 proteins, primarily involved in membrane components, cellular, and metabolic processes.
- KEGG analysis indicated significant disruption of metabolic pathways, notably the PPAR signaling pathway.
- SCD (Stearoyl-CoA Desaturase) expression was downregulated in RNF13-knockdown cells.
- SCD knockdown impaired lipid metabolism and reduced cell proliferation.
- SCD overexpression rescued lipid metabolism and cell viability defects caused by RNF13 knockdown.
Conclusions:
- RNF13 influences lipid metabolism in ARPE-19 cells, potentially through the PPAR signaling pathway.
- SCD acts as a potential mediator in the RNF13-regulated lipid metabolism pathway.
- These findings highlight RNF13 and SCD as potential targets for managing RPE cell dysfunction.
