RNF2 Modulates Lipid Metabolism and Inflammation in Alcohol-associated Liver Disease by Interacting with USP7

Figures

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  Figure 1. RNF2 expression was increased in ALD. A. H&E staining of ALD patients and healthy control donors.B. Immunohistochemistry (IHC) staining of RNF2 in the livers of ALD patients and healthy control donors. C. Western blotting and quantitative analysis of RNF2 expression in the livers of ALD patients and healthy control donors. D. Schematic diagram establishing the ALD model. E. Appearance of mouse livers in pair group and EtOH group. F. Liver-to-body weight ratio of mice in pair group and EtOH group (n=6/group). G, H, I. Determination of serum ALT, AST and hepatic TG levels in the pair group and EtOH group. J. H&E staining of liver tissue in the two groups. Scale bar:50 μm. K. IHC staining of RNF2 in EtOH-induced mouse livers. Scale bar:50 μm. L. RNF2 expression levels in the livers of mice in pair group and EtOH group.

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  Figure 2. RNF2 was upregulated in EtOH-induced AML-12 cells. A. AML-12 cells were pretreated with different concentrations of EtOH (0, 25, 50, 100, 150, or 200 mM), after which RNF2 protein expression was measured. B. RNF2 protein expression peaked after stimulation for 24 h with 100 mM EtOH. C. Oil Red O staining of EtOH-induced AML-12 cells. The cells were treated with EtOH (100 mM) for 24 h. D, E. RNF2 mRNA and protein expression levels. All the results were obtained from three independent experiments.

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  Figure 3. RNF2 silencing reduced lipid accumulation in EtOH-induced AML-12 cells. A-D. The mRNA and protein expression levels of PPAR-α, ACOX-1 and SREBP-1c in pEGFP-C2- or pEGFP-C2-RNF2-transfected and EtOH-induced AML-12 cells. E. Oil Red O staining of EtOH-induced AML-12 cells with RNF2 overexpression. F-I. The protein and mRNA expression levels of PPAR-α, ACOX-1 and SREBP-1c were measured in NC-shRNA- or RNF2-shRNA-infected and EtOH-induced AML-12 cells. J. Oil Red O staining of EtOH-induced AML-12 cells with RNF2 knockdown.

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  Figure 4. RNF2 promoted inflammatory response in EtOH-induced RAW264.7 cells. A-B. Western blotting and quantitative analysis of TNF-α, IL-6 and IL-1β expression in pEGFP-C2- or pEGFP-C2-RNF2-transfected and EtOH-induced RAW264.7 cells. C-E. The mRNA expression levels of TNF-α, IL-6 and IL-1β. F-G. Western blotting and quantitative analysis of TNF-α, IL-6 and IL-1β expression in NC-shRNA- or RNF2-shRNA-infected and EtOH-induced RAW264.7 cells. H-J. The mRNA and expression levels of TNF-α, IL-6 and IL-1β.

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  Figure 5. RNF2 regulated lipid accumulation and the inflammatory response via the PI3K/AKT signaling pathway. A. KEGG enrichment analysis of DEGs in NC-shRNA- or RNF2-shRNA-infected AML-12 cells treated with EtOH. B-C. The protein expression of p-PI3K and p-AKT in pEGFP-C2- or pEGFP-C2-RNF2-transfected and EtOH-induced AML-12 cells. D-E. The protein expression of p-PI3K and p-AKT in NC-shRNA or RNF2-shRNA infected and EtOH-induced AML-12 cells. F-G. AML-12 cells were incubated with LY294002 (0, 5, 10, 20, 40) μM for 24 h, and protein levels of p-PI3K and p-AKT were detected to determine the appropriate concentration. H-J. The relative protein expression levels of PPAR-α, ACOX-1, SREBP-1c, IL-1β, TNF-α, and IL-6 were detected by Western blotting in pEGFP-C2- or pEGFP-C2-RNF2-transfected AML-12 cells, with or without LY294002 treatment. K-M.The relative protein expression of PPAR-α, ACOX-1, SREBP-1c, IL-1β, TNF-α, and IL-6 were detected by Western blotting in NC-shRNA- or RNF2-shRNA-infected AML-12 cells, with or without LY294002 treatment.

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  Figure 6. RNF2 interacted with USP7 and activated the PI3K/AKT signaling pathway. A. The prediction of RNF2-binding proteins in the STING database. B. Double-labeling immunofluorescence revealed a nuclear co-localization of RNF2 and USP7 proteins in AML-12 cells. C. The interaction between RNF2 and USP7 was tested by co-IP. D. The interaction between RNF2 and USP7 was tested by GST pull-down, and the purified GST protein was used as the control. E-H. Effect of RNF2 overexpression or knockdown on USP7 expression. I-J. Verification of the utility of USP7-siRNA. K-L. Effect of USP7-siRNA on the PI3K/AKT signaling pathway. M-N. Effect of USP7-siRNA on the expression of lipid metabolism-related and inflammation-related genes.

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  Figure 7. Function of USP7 in ALD model mice. A. Analysis of small animal imaging. B-C. Western blotting and quantitative analysis of USP7. D. Liver-to-body weight ratios of mice in the shNC EtOH group and shUSP7 EtOH group (n=6/group). E-G. Determination of the serum ALT, AST and hepatic TG levels in the shNC EtOH group and shUSP7 EtOH group. H. H&E staining of liver tissues from the two groups. Scale bar:50 μm. I. Oil Red O staining. J. The relative protein expression levels of TNF-α, IL-6 and IL-1β were detected by Western blotting in liver tissues of shNC EtOH group and shUSP7 EtOH group. K. The relative protein expression levels of PPAR-α, ACOX-1 and SREBP-1c in liver tissues from the shNC EtOH group and shUSP7 EtOH group.

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  Figure 8. RNF2 knockdown attenuated liver injury in ALD model mice. A.The mRNA and protein expression levels of RNF2. B. Liver-to-body weight ratios of mice in the shNC EtOH group and shRNF2 EtOH group (n=6/group). C-E. Determination of the serum ALT, AST and hepatic TG levels in the shNC EtOH group and shRNF2 EtOH group. F. H&E staining of liver tissues from the two groups. Scale bar:50 μm. G. Oil Red O staining. H-J. Serum levels of TNF-α, IL-6 and IL-1β in the shNC EtOH group and shRNF2 EtOH group. K. IHC staining of F4/80 and Ly6G in the livers of mice in shNC EtOH group and shRNF2 EtOH group. L. The relative protein expression levels of TNF-α, IL-6 and IL-1β were detected by Western blotting in liver tissues of shNC EtOH group and shRNF2 EtOH group. M. The changes in intracellular structure were observed via TEM. The arrow points to lipid droplets. N. The relative protein expression levels of PPAR-α, ACOX-1 and SREBP-1c in liver tissues from the shNC EtOH group and shRNF2 EtOH group.