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 27 weeks (n = 3 mice/group). (B) Heat map of Snhgs in livers of mice as indicated in (A) (n = 3 mice/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 40 weeks. (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 < 0.01 and ***p < 0.001 by Student’s t test.

Liver specific Snhg3 knockout alleviates hepatic steatosis in DIO mice. (A) The expression of Snhg3 was downregulated in the liver of Snhg3-HKO mice. n = 5/group. (B) Body weights of DIO Snhg3-Flox (n = 6) and Snhg3-HKO (n = 5) mice fed HFD for indicated time period. (C) ITT and GTT of DIO Snhg3-Flox (n = 6) and Snhg3-HKO (n = 5) mice fed HFD for 18 weeks were analyzed, (AUC, Area Under Curve). (D) Liver weight (left) and ratio (right) of liver weight/body weight of DIO Snhg3-Flox (n = 6) and Snhg3-HKO (n = 5) mice fed HFD for 21 weeks. (E) H&E and oil red O staining (left) and NASH score (right) of liver of DIO Snhg3-Flox and Snhg3-HKO mice as indicated in (D). Scare bars, 50 μm. (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 < 0.05 and **p < 0.01 by two-way ANOVA (B and C) and by Student’s t test (the others).

Liver specific Snhg3 overexpression aggravates hepatic steatosis in DIO mice. (A) The expression of Snhg3 was upregulated in the liver of Snhg3-HKI mice. N = 7/group. (B) Body weights of DIO WT mice (n = 6) and Snhg3-HKI mice (n = 7) fed HFD for indicated times. (C) ITT and GTT of DIO WT (n = 6) and Snhg3-HKI (n = 7) mice fed HFD for 11 weeks were analyzed. (D) Liver weight (left) and ratio (right) of liver weight/body weight of DIO WT (n = 6) and Snhg3-HKI (n = 7) mice fed HFD for 13 weeks. (E) Liver H&E and oil red O staining (left) and NASH score (right) of DIO WT and Snhg3-HKI mice as indicated in (D). Scare bars, 50 μm. (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 < 0.01 and ***p < 0.001 by two-way ANOVA (B and C) and by Student’s t test (the others).

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

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 & MS. (B) KEGG analysis of genes in specific Snhg3-binding proteins from RNA pull-down & MS. (C) Venn diagram of data from RNA pull-down & MS and bioinformatics predicted by RBPsuite (sjtu.edu.cn). (D) SND1 interacts with different fragments of Snhg3 predicted by bioinformatics using RBPsuite (sjtu.edu.cn). (E) RNA pull-down & 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 < 0.05 and ***p < 0.001 by two-way ANOVA (J) or Student’s t test (the others).

Snhg3 increases PPARγ expression through reducing H3K27me3 enrichment at Pparγ 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 DIO 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 DIO Snhg3-HKO mice. (F and G) ChIP result showed that Snhg3 affected H3K27me3 enrichment at Pparγ promoter in vivo (F) and in vitro (G). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA (C) or by Student’s t test (the others).

SND1 mediates Snhg3-induced PPARγ expression increase. (A) The mRNA levels of Pparγ were measured in the liver of DIO Snhg3-HKO (left) and Snhg3-HKI mice (right). (B) The protein level of PPARγ were measured in the liver of DIO Snhg3-Flox and Snhg3-HKO mice (up, western blotting; down, quantitative result). (C) The protein level of PPARγ were measured in the liver of DIO 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 Pparγ 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 Pparγ 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 < 0.01 and ***p < 0.001 by one-way ANOVA (G-I) or by Student’s t test (the others).

PPARγ mediates Snhg3-induced hepatic steatosis. (A and B) Body weights (A) and liver weight (B) of DIO Snhg3-HKI mice without (n = 6) or with (n = 7) T0070907 treatment for 8 weeks. (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. Scare bars, 100 μm. (E) T0070907 mitigated the hepatic Cd36 and Cidea/c increase in DIO 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 < 0.05 and ***p < 0.001 by two-way ANOVA (A) or by Student’s t test for the others.