LncRNA-Snhg3 Aggravates Hepatic Steatosis by Regulating PPARγ via SND1/H3K27me3

Department of Biochemistry & Molecular Biology, State Key Laboratory of Common Mechanism Research for Major Diseases, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China
Department of Microbiology and Immunology, Shanxi Medical University, Taiyuan 030001, China
Department of Pathophysiology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences & School of Basic Medicine Peking Union Medical College, Beijing 100005, China
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai, 201203, China
Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, Guangdong Province, China
State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

https://doi.org/10.7554/eLife.96988.1

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Figures and data

The expression of hepatic lncRNA-Snhg3 is downregulated in high-fat diet-induced obesity 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. Data are represented as mean ± SEM. *p < 0.05 and ***p < 0.001 by Student’s t test.

Liver specific Snhg3 knockout improves high-fat diet-induced hepatic steatosis.
(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 - E) Heat production (C), total oxygen consumption and carbon dioxide production (D), and RER (E) of DIO Snhg3-Flox (n = 6) and Snhg3-HKO (n = 5) mice fed HFD for 16 weeks were measured by CLAMS. (F) 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). (G) 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. (H) 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 (G). Scare bars, 50 μm. (I) Hepatic TG and TC contents of mice as indicated in (G). (J) Serum ALT and AST concentrations of mice as indicated in (G). (K) Serum FFAs, TG and TG concentrations of mice as indicated in (G). (L) iWAT weight (left) and ratio (right) of iWAT weight/body weight of mice as indicated in (G). (M) Serum insulin concentration of mice as indicated in (G). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 by two-way ANOVA (B and F) and by Student’s t test (the others).

Liver specific Snhg3 overexpression aggravates high-fat diet-induced hepatic steatosis.
(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 - E), Heat production (C), total oxygen consumption and carbon dioxide production (D) and RER (E) of DIO WT (n = 6) and Snhg3-HKI (n = 7) mice fed HFD for 9 weeks were measured by CLAMS. (F) ITT and GTT of DIO WT (n = 6) and Snhg3-HKI (n = 7) mice fed HFD for 11 weeks were analyzed. (G) 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. (H) Liver H&E and oil red O staining (left) and NASH score (right) of DIO WT and Snhg3-HKI mice as indicated in (G). Scare bars, 50 μm. (I) Hepatic TG and TC contents of mice as indicated in (G). (J) Serum ALT and AST concentrations of mice as indicated in (G). (K) Serum FFAs, TG and TG concentrations of mice as indicated in (G). (L) iWAT weight (left) and ratio (right) of iWAT weight/body weight of mice as indicated in (G). (M) Serum insulin concentration of mice as indicated in (G). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 by two-way ANOVA (B and F) 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 enrichment plot of PPAR signaling pathway set (KEGG pathway database) in livers of DIO Snhg3-HKI and WT mice (n = 3 mice/group). (C) Genome distribution ratio of the differentially accessible regions in the liver between DIO WT and DIO Snhg3-HKI mice by ATAC-Seq. (D) The transcription factors analysis in the accessible regions of the liver of DIO Snhg3-HKI mice by CREMA. (E) Integrated ATAC-Seq data with RNA-Seq data of DIO male mice and the control mice. (F) Chromatin accessibility at Cd36 gene. (G) The heatmap of Cd36 in livers of DIO male mice and the control mice (n = 3 mice/group). (H and I) Relative RNA (H) and protein (I, up, western blotting; down, quantitative result) levels of Cd36 were measured in the liver. 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 & mass spectrometry (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 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 and B) The mRNA (A) and protein (B, up, western blotting; down, quantitative result) expression level of PPARγ were measured in vivo. (C and D) Overexpression of Snhg3 (C) and SND1 (D) promoted the mRNA expression of Pparγ and Cd36 in primary hepatocytes. (E) Overexpression of Snhg3 and SND1 increased the protein expression of PPARγ in Hepa1-6 cells (up, western blotting; down, quantitative result). (F - H) Disruption SND1 expression alleviated Snhg3-induced increase in the protein (F) and mRNA (G) levels of PPARγ and CD36, and lipid accumulation (H, left, oil red O staining; right, quantitative result) in primary hepatocytes with PA treatment. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA (G and H) or by Student’s t test (the others).

PPARγ mediates Snhg3-induced hepatic steatosis. (A - C) Body weights (A), liver weight (B) and adipose tissue weight (C) of DIO Snhg3-HKI mice without (n = 6) or with (n = 7) T0070907 treatment for 8 weeks. (D) Serum FFAs, TG and TG concentrations of mice as indicated in (A). (E) Hepatic H&E and oil red O staining (left) and NASH score (right) of mice as indicated in A. Scare bars, 100 μm. (F) T0070907 decreased hepatic Cd36 expression in DIO Snhg3-HKI mice. (G) T0070907 disrupted Snhg3- and SND1-induced Cd36 increase in Hepa1-6 cells. (H) 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.

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