Hepatic transcriptome analysis from HFD-fed mice defines a long noncoding RNA regulating cellular cholesterol levels

Figures

• 

  Fig. 1.. Nonalcoholic steatohepatitis with insulin resistance induced by a HFD. A: The body weights of mice fed a HFD were significantly increased as compared with control mice after 3 months (males, n = 12, ***P < 0.001). B, C: AUC measurements of glucose tolerance and insulin sensitivity tests show an impaired insulin sensitivity in mice fed a HFD as compared with controls (P < 0.001). D: Phosphorylation of Akt (Ser473 and Thr308) was significantly decreased in livers from mice fed a HFD when compared with control mice (n = 3). E: HFD mice showed remarkable liver fat accumulation as observed by H&E staining (n = 3). F: Liver triglyceride and cholesterol levels were elevated in the HFD mice as compared with control mice (n = 12, *P < 0.05). G: There was no significant difference in the plasma triglyceride levels between HFD and control mice (P > 0.05). Total cholesterol (H), HDL (I), and LDL (J) levels were elevated in HFD mice as compared with controls (n = 12, ***P < 0.001).

• 

  Fig. 2.. Bioinformatics analysis pipeline and classification of identified transcripts. A: The pipeline employed in the identification and function annotation of protein-coding and noncoding RNAs. B: Classification and statistics of protein-coding and noncoding genes identified using the CNCI utility (potential novel genes were additionally annotated in the right smaller pie). C: Distribution by transcript number per gene for identified known genes and novel lncRNA genes. D: Distribution by exon number per transcript for identified known genes and novel lncRNA genes.

• 

  Fig. 3.. Classification and functional annotation of DEGs. A: Classification of DEGs in the livers of HFD and control mice. B: Cluster analysis for differentially expressed protein-coding genes. C: Enrichment in biological processes of differentially expressed protein-coding genes by GO analysis. D: KEGG pathway analysis of the differentially expressed protein-coding genes.

• 

  Fig. 4.. lncRNA and protein-coding gene pairs based on genomic juxtaposition and coexpression.

• 

  Fig. 5.. Expression of selected genes validated by qPCR and cellular assays. The upregulation of selected lncRNAs (A) and protein-coding genes (B) in HFD livers was validated by qPCR (n = 10; *P < 0.05, **P < 0.01, ***P < 0.001). Treatment of AML12 cells with PA (700 μM) for 16 h resulted in the upregulation of selected lncRNAs (C) and protein-coding genes Elovl5, Elovl6, and Mgll (n = 3, **P < 0.01, ***P < 0.001), as well as the downregulation of Lamb3 (n = 3, **P < 0.01) (D). Treatment of AML12 cells with WY14643 (300 μM) for 12 h resulted in the upregulation of four selected lncRNAs (n = 3; *P < 0.05, ***P < 0.001) (E) and three protein-coding genes Elovl5, Elovl6, and Mgll (*P < 0.05, **P < 0.01, ***P < 0.001) (F).

• 

  Fig. 6.. Characterization of NONMMUG027912. A: Prediction of the NONMMUG027912 secondary structure, which indicated the peculiar stem-loop structures identified on the RNAfold WebServer (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). B: Tissue-specific expression of NONMMUG027912, as quantified by qPCR. The liver was the main site of NONMMUG027912 expression. C: FISH showed that NONMMUG027912 was localized predominantly in the nucleus of Hepa1-6 cells. D: Nuclear/cytoplasm RNA fractionation verified the nuclear localization of NONMMUG027912.

• 

  Fig. 7.. Effect of NONMMUG027912 on hepatic lipid metabolism. A: NONMMUG027912 was efficiently knocked down by 70% in Hepa1-6 cell lines using siRNA oligonucleotides (10 μmol/l) (n = 3, *P < 0.05). B: Total cellular cholesterol levels were significantly elevated in Hepa1-6 cells and culture medium following the knockdown of NONMMUG027912 (n = 3, **P < 0.01). C: Expression of cholesterol biosynthesis-related genes Mvk, Fdft1, and Idi1 was significantly increased via the knockdown of NONMMUG027912 in Hepa1-6 cells (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). D, E: Protein levels of cholesterol synthesis-related proteins Mvk, Fdft1, and Idi1 increased significantly after NONMMUG027912 knockdown (n = 3, *P < 0.05, **P < 0.01). F: HMG-CoA reductase activity significantly increased in the NONMMUG027912 knockdown group as compared with the control group (n = 3, *P < 0.05).