LncRNA Snhg3 aggravates hepatic steatosis via PPARγ signaling

Abstract

LncRNAs are involved in modulating the individual risk and the severity of progression in metabolic dysfunction-associated fatty liver disease (MASLD), but their precise roles remain largely unknown. This study aimed to investigate the role of lncRNA Snhg3 in the development and progression of MASLD, along with the underlying mechanisms. The result showed that Snhg3 was significantly downregulated in the liver of high-fat diet-induced obesity (DIO) mice. Notably, palmitic acid promoted the expression of Snhg3 and overexpression of Snhg3 increased lipid accumulation in primary hepatocytes. Furthermore, hepatocyte-specific Snhg3 deficiency decreased body and liver weight, alleviated hepatic steatosis and promoted hepatic fatty acid metabolism in DIO mice, whereas overexpression induced the opposite effect. Mechanistically, Snhg3 promoted the expression, stability and nuclear localization of SND1 protein via interacting with SND1, thereby inducing K63-linked ubiquitination modification of SND1. Moreover, Snhg3 decreased the H3K27me3 level and induced SND1-mediated chromatin loose remodeling, thus reducing H3K27me3 enrichment at the Pparg promoter and enhancing PPARγ expression. The administration of PPARγ antagonist T0070907 improved Snhg3-aggravated hepatic steatosis. Our study revealed a new signaling pathway, Snhg3/SND1/H3K27me3/PPARγ, responsible for mice MASLD and indicates that lncRNA-mediated epigenetic modification has a crucial role in the pathology of MASLD.

Keywords: PPARγ; Snhg3; chromosomes; gene expression; medicine; metabolic dysfunction-associated fatty liver disease; mouse.

Figure 1.. The expression of hepatic lncRNA-Snhg3 is downregulated in DIO mice.

(A) Differentially expressed lncRNAs in livers of 6~8-week-old littermate male mice that were fed an HFD and control diet for 27weeks (n=3mice/group). (B) Heat map of Snhgs in livers of mice as indicated in (A) (n=3mice/group). (C) Expression levels of Snhg3 in the liver of 6~8-week-old littermate male mice that were fed an HFD and control diet for indicated time period 11, 27, and 40weeks. (D) Relative Snhg3 expression levels in nuclear and cytosolic fractions of mouse primary hepatocytes. Nuclear controls: Neat1 and Xist; Cytosolic control: Gapdh. (E) PA promotes the expression of Snhg3 in primary hepatocytes. (F and G) Overexpression of Snhg3 (F) induces lipid accumulation (G) left, Oil red O staining; right, quantitative analysis) in primary hepatocytes with PA treatment. Data are represented as mean ± SEM. *p<0.05, **p<0.01and ***p<0.001 by Student’s t test.

Figure 2.. Hepatocyte-specific Snhg3 knockout alleviates hepatic steatosis in DIO mice.

(A) The expression of Snhg3 was downregulated in the liver of Snhg3-HKO mice. Snhg3-Flox (n=6) and Snhg3-HKO (n=5). (B) Body weights of Snhg3-Flox (n=6) and Snhg3-HKO (n=5) mice fed HFD for indicated time period. (C) ITT (n=5/group) and GTT (n=6/group) of Snhg3-Flox and Snhg3-HKO mice fed HFD for 18weeks were analyzed, (AUC, Area Under Curve). (D) Liver weight (left) and ratio (right) of liver weight/body weight of Snhg3-Flox (n=6) and Snhg3-HKO (n=5) mice fed HFD for 21weeks. (E) H&E and oil red O staining (left) and NASH score (right) of liver of Snhg3-Flox and Snhg3-HKO mice as indicated in (D). Scale bars, 50μm. (F) Hepatic TG and TC contents of mice as indicated in (D). (G) Serum ALT and AST concentrations of mice as indicated in (D). (H) Serum FFAs, TG and TC concentrations of mice as indicated in (D). Data are represented as mean ± SEM. *p<0.05and **p<0.01 by two-way ANOVA (B and C) and by Student’s t test (the others).

Figure 2—figure supplement 1.. Hepatocyte-specific Snhg3 knockout alleviates hepatic steatosis in DIO mice.

(A) The genome mapping of mouse Snhg3 (https://www.ncbi.nlm.nih.gov/gene/). (B) Schematic diagram for the creation of hepatocyte-specific Snhg3 knock-out (Snhg3-HKO) mice. (C) Heat production, total oxygen consumption and carbon dioxide production, and RER of Snhg3-Flox (n=6) and Snhg3-HKO (n=6) mice fed HFD for 16weeks were measured by CLAMS. (D) Liver fibrosis Snhg3-Flox and Snhg3-HKO mice fed HFD for 16weeks was visualized using Picro Sirius Red Stain. Scale bars, 50μm. (E) iWAT weight (left) and ratio (right) of iWAT weight/body weight of mice as indicated in Snhg3-Flox (n=6) and Snhg3-HKO (n=5) mice fed HFD. (F) Serum insulin concentration of mice as indicated in Snhg3-Flox (n=6) and Snhg3-HKO (n=5) mice fed HFD. Data are represented as mean ± SEM. ***p<0.001 by Student’s t test (the others).

Figure 3.. Hepatocyte-specific Snhg3 overexpression aggravates hepatic steatosis in DIO mice.

(A) The expression of Snhg3 was upregulated in the liver of Snhg3-HKI mice. WT (n=6) and Snhg3-HKI (n=7). (B) Body weights of WT mice (n=6) and Snhg3-HKI mice (n=7) fed HFD for indicated times. (C) ITT and GTT of WT (n=6) and Snhg3-HKI (n=7) mice fed HFD for 11weeks were analyzed. (D) Liver weight (left) and ratio (right) of liver weight/body weight of WT (n=6) and Snhg3-HKI (n=7) mice fed HFD for 13weeks. (E) Liver H&E and oil red O staining (left) and NASH score (right) of WT and Snhg3-HKI mice as indicated in (D). Scale bars, 50μm. (F) Hepatic TG and TC contents of mice as indicated in (D). (G) Serum ALT and AST concentrations of mice as indicated in (D). (H) Serum FFAs, TG and TG concentrations of mice as indicated in (D). Data are represented as mean ± SEM. *p<0.05, **p<0.01and ***p<0.001 by two-way ANOVA (B and C) and by Student’s t test (the others).

Figure 3—figure supplement 1.. Hepatocyte-specific Snhg3 overexpression aggravates hepatic steatosis in DIO mice.

(A) Schematic diagram for the creation of hepatocyte-specific Snhg3 knock-in (Snhg3-HKI) mice. (B) iWAT weight (left) and ratio (right) of iWAT weight/body weight of mice as indicated in WT (n=6) and Snhg3-HKI (n=7) mice fed HFD for 9weeks. (C) Heat production, total oxygen consumption and carbon dioxide production, and RER of WT (n=4) and Snhg3-HKI (n=4) mice fed HFD for 9weeks were measured by CLAMS. (D) Liver fibrosis in WT and Snhg3-HKI mice fed HFD for 9weeks was visualized using Picro Sirius Red Stain. Scale bars, 50μm. (E) Serum insulin concentration of mice as indicated in WT (n=6) and Snhg3-HKI (n=7) mice fed HFD for 9weeks. Data are represented as mean ± SEM. ***p<0.001 by Student’s t test.

Figure 4.. Snhg3 promotes hepatic steatosis through regulating chromatin remodeling.

(A) Differentially expressed genes in livers of Snhg3-HKI and WT mice (n=3mice/group). (B) GSEA showing the enrichment of PPAR signaling pathway (up) and fatty acid metabolism (down) (KEGG pathway database) in livers of Snhg3-HKI and WT mice (n=3mice/group). (C) Relative hepatic mRNA levels of fatty acid metabolism were measured in Snhg3-HKO (up) mice and Snhg3-HKI mice (down) compared to the controls. (D) Genome distribution ratio of the differentially accessible regions in the liver between WT and Snhg3-HKI mice by ATAC-Seq. (E and F) The transcription factors analysis in the accessible regions of the liver of Snhg3-HKI mice by HOMER (E) and CREMA (F). (G) Integrated ATAC-Seq data with RNA-Seq data. (H) Chromatin accessibility at Cd36 and Cidea/c genes. Data are represented as mean ± SD. *p<0.05and **p<0.01 by Student’s t test.

Figure 4—figure supplement 1.. Snhg3 influences the expression of profibrotic genes, not pro-inflammatory factors.

The mRNA levels of liver fibrosis and inflammation in DIO Snhg3-HKO mice (A) and Snhg3-HKI mice (B), compared to the controls. Data are represented as mean ± SEM.*p<0.05 and **p<0.01 by Student’s t test.

Figure 5.. Snhg3 induces SND1 expression and enhances the stability of SND1 protein through physiologically interacting with SND1.

(A) Venn diagram of data from RNA pull-down and MS. (B) KEGG analysis of genes in specific Snhg3-binding proteins from RNA pull-down and MS. (C) Venn diagram of data from RNA pull-down and MS and bioinformatics predicted by RBPsuite. (D) SND1 interacts with different fragments of Snhg3 predicted by bioinformatics using RBPsuite. (E) RNA pull-down and western blotting confirms Snhg3 interacting with SND1. (F) RIP confirms SND1 interacting with Snhg3. (G and H) Relative protein (G, up, western blotting; down, quantitative result) and RNA (H) levels of Snd1 were measured in the liver. (I) Snhg3 enhanced the protein level of SND1 in Hepa1-6 cells (up, western blotting; down, quantitative result). (J) Snhg3 promoted the stability of SND1 protein in Hepa1-6 cells (up, western blotting; down, quantitative result). (K and L) Snhg3 promoted the ubiquitination of endogenous (K) and exogenous (L) SND1 protein in Hepa1-6 cells. (M and N) Snhg3 increased the K63-linked, not K48-linked and K33-linked, ubiquitination modification of endogenous (M) and exogenous (N) SND1 protein. (O) Snhg3 induced the nuclear localization of SND1 in Hepa1-6 cells (up, western blotting; down, quantitative result). Data are represented as mean ± SEM. *p<0.05and ***p<0.001 by two-way ANOVA (J) or Student’s t test (the others).

Figure 6.. Snhg3 increases PPARγ expression through reducing H3K27me3 enrichment at Pparg promoter.

(A) Overexpression of Snhg3 or SND1 reduced the H3K27me3 level in Hepa1-6 cells with PA treatment (up, western blotting; down, quantitative result). (B) The expression of SND1 was disrupted with siRNA (up, western blotting; down, quantitative result). (C) Disruption SND1 expression reversed the Snhg3-induced decrease in H3K27me3 in primary hepatocytes (up, western blotting; down, quantitative result). (D) The H3K27me3 levels were measured in the liver of Snhg3-HKO and Snhg3-HKI mice (up, western blotting; down, quantitative result). (E) Genome distribution ratio of H3K27me3 enrichment genetic sequence in the liver of Snhg3-HKO mice. (F and G) ChIP result showed that Snhg3 affected H3K27me3 enrichment at Pparg promoter in vivo (F) and in vitro. (G) Data are represented as mean ± SEM. *p<0.05, **p<0.01and ***p<0.001 by one-way ANOVA (C) or by Student’s t test (the others).

Figure 7.. SND1 mediates Snhg3-induced PPARγ upregulation.

(A) The mRNA level of Pparg was measured in the liver of Snhg3-HKO (left) and Snhg3-HKI mice (right). (B) The protein level of PPARγ was measured in the liver of Snhg3-Flox and Snhg3-HKO mice (up, western blotting; down, quantitative result). (C) The protein level of PPARγ were measured in the liver of WT and Snhg3-HKI mice (up, western blotting; down, quantitative result). (D and E) Overexpression of Snhg3 (D) and SND1 (E) promoted the mRNA expression of Pparg and Cd36 in primary hepatocytes. (F) Overexpression of Snhg3 and SND1 increased the protein expression of PPARγ in Hepa1-6 cells (up, western blotting; down, quantitative result). (G) Disruption SND1 expression alleviated Snhg3-induced increase in the protein level of PPARγ in Hepa1-6 cells (left) and mouse primary hepatocytes (MPH, right) with PA treatment (up, western blotting; down, quantitative result). (H) Disruption SND1 expression alleviated Snhg3-induced increase in the mRNA levels of Pparg and Cd36 in Hepa1-6 cells with PA treatment. (I) Disruption SND1 expression alleviated Snhg3-induced increase in lipid accumulation (left, oil red O staining; right, quantitative result) in MPH with PA treatment. Data are represented as mean ± SEM. *p<0.05, **p<0.01and ***p<0.001 by one-way ANOVA (G–I) or by Student’s t test (the others).

Figure 7—figure supplement 1.. Sngh3-induced changes in PPARγ and SND1 are independent on U17 snoRNA.

(A) Hepatic U17 snoRNA expression in DIO Snhg3-HKO mice and Snhg3-HKI mice compared to the controls. (B and C) Overexpression U17 snoRNA has no effect on the mRNA (B) and protein (C) levels of PPARγ and SND1 (left, western blotting; right, quantitative result). Data are represented as mean ± SEM. *p<0.05and ***p<0.001 by Student’s t test.

Figure 8.. PPARγ mediates Snhg3-induced hepatic steatosis.

(A and B) Body weight (A) and liver weight (B) of Snhg3-HKI mice without (n=6) or with (n=7) T0070907 treatment for 8weeks. (C) Serum FFAs, TG and TG concentrations of mice as indicated in (A). (D) Hepatic H&E and oil red O staining (left) and NASH score (right) of mice as indicated in A. Scale bars, 100μm. (E) T0070907 mitigated the hepatic Cd36 and Cidea/c increase in Snhg3-HKI mice. (F) T0070907 disrupted Snhg3- and SND1-induced Cd36 increase in Hepa1-6 cells. (G) Model of how Snhg3 and SND1 interacting and influencing chromatin remodeling via H3K27me3, and promoting PPARγ expression thereby resulting in hepatic steatosis. Data are represented as mean ± SEM. *p<0.05and ***p<0.001 by two-way ANOVA (A) or by Student’s t test for the others.

Figure 8—figure supplement 1.. Fat weight of Snhg3-HKI mice without (n=6) or with (n=7) T0070907 treatment for 8weeks.

Data are represented as mean ± SEM and are analyzed by Student’s t test.