SRSF3-TRIM28-MDC1 prevents DNA damage caused by R-loops in fatty liver disease in mice
Panyisha Wu, Manasi Das, Yanting Wang, Yichun Ji, Yuli Wu, Deepak Kumar, Lily J. Jih, Nicholas J.G. Webster
Panyisha Wu, Manasi Das, Yanting Wang, Yichun Ji, Yuli Wu, Deepak Kumar, Lily J. Jih, Nicholas J.G. Webster
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Research Article Endocrinology Hepatology

SRSF3-TRIM28-MDC1 prevents DNA damage caused by R-loops in fatty liver disease in mice

Abstract

Serine-rich splicing factor 3 (SRSF3) is crucial for the metabolic functions of the liver. The genetic deletion of SRSF3 in mouse hepatocytes impairs hepatic lipid and glucose metabolism and leads to fibrosis and formation of hepatocellular adenoma that progresses to hepatocellular carcinoma. SRSF3 protein is proteosomally degraded in metabolic-dysfunction associated fatty liver disease (MAFLD) and metabolic-dysfunction-associated steatohepatitis (MASH). We show here that depleting SRSF3 protein in hepatocytes promoted R-loop accumulation and increased DNA damage in the liver. Prevention of SRSF3 degradation in vivo protected hepatocytes from DNA double-strand breaks in mice with MASH. This protection extended to other DNA-damaging agents such as camptothecin, palmitic acid, or hydrogen peroxide when tested on HepG2 cells in vitro. SRSF3 interacted with TRIM28 and MDC1, which are components of the ATM DNA-damage repair complex, and knockdown of any of these 3 proteins reduced the expression of the other 2 proteins, suggesting they form a functional complex. Lastly, by preventing degradation of SRSF3, we were able to reduce tumors in a diethyl-nitrosamine–induced (DEN-induced) model of cirrhotic HCC. These findings suggest that maintenance of SRSF3 protein stability is crucial for preventing DNA damage and protecting liver from early metabolic liver disease and progression to HCC.

Authors

Panyisha Wu, Manasi Das, Yanting Wang, Yichun Ji, Yuli Wu, Deepak Kumar, Lily J. Jih, Nicholas J.G. Webster

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Figure 9

Preventing SRSF3 degradation reduces HCC.

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Preventing SRSF3 degradation reduces HCC. (A) Schematic of the chemicall...
(A) Schematic of the chemically induced mouse HCC model. DEN (25 mpk, i.p.) was administered to mice at 2 weeks of age, mice were placed on Western diet starting at 4 weeks of age and injected with TAA (300 mpk, i.p.) twice a week for a further 20 weeks. AAV8 infection was performed by tail vein injection on week 6. (B) Whole liver images and H&E and Sirius red–stained sections from lean mice (control), HCC mice (DEN+TAA+NASH), HCC mice infected with AAV8-GFP (AAV8-GFP), and HCC mice infected with AAV8-SRSF3-K11R (AAV8-K11R). Scale bar for liver pictures: 1 cm. Scale bar for sections: 250 μm. Graphs show liver to body weight ratio (n = 4–7/group) and quantification of fibrosis by Sirius red staining (n = 4/group). Control mice are shown in white, DEN+TAA+MASH-treated (DEN) in yellow, GFP-infected in green, and K11R-infected in red. (C) Sirius red–stained whole liver sections from mice infected with AAV8-GFP or AAV8-K11R (n = 4/group) showing pale steatotic tumors (arrows). Graph shows quantification of tumor number. (D) Representative reticulin staining of liver sections from AAV8-GFP– or AAV8-K11R–infected mice. T, tumor; NL, adjacent nontumor liver. Scale bar for reticulin-stained sections: 250 μm. Scale bar for magnified sections: 50 μm. (E) Immunohistochemical staining for γH2ax (brown) and quantification of positive nuclei/field (n = 3/group). Black arrows indicate representative positive cells. Scale bar: 250 μm. All quantified results are presented as mean ± SD; *P < 0.05, **P < 0.01, ****P < 0.0001 by 1-way ANOVA or 2-tailed t test.