LncRNA-H19 promotes hepatic lipogenesis by directly regulating miR-130a/PPARγ axis in non-alcoholic fatty liver disease

Figures

• 

  Figure 1. The expression level of H19 and hepatic lipogenesisis in NAFLD mouse model (A) H&E staining showed general tissue morphology of mouse livers between control and HFD groups. (B) Oil-Red O staining showed prominent lipid deposition in HFD mice. (C) TG levels were assessed by ELISA assay in liver tissues. (D) qRT-PCR was used to detect the level of H19, miR-130a and molecular marker of NAFLD (PPARγ, ACC1, SCD1, FASN and SREBP1) in NAFLD mouse liver tissues. β-actin or U6 were used for normalization. (E) Western blot analysis of PPARγ level in NAFLD mouse liver tissues. GAPDH was used for normalization. (F) IHC detection of PPARγ in NAFLD mouse liver tissues. All the results were shown as mean ± SD (n=3), which were three separate experiments performed in triplicate. *P<0.05 and **P<0.01.

• 

  Figure 2. FFA induced hepatocyte lipid degeneration and increased H19 expression in hepatocytes (A) Oil-Red O staining for lipid droplet in HepG2 and Huh-7 cells treated with FFA. (B) TG levels were detected by ELISA assay in HepG2 or Huh-7 cells treated with FFA. Relative expression level of H19 (C), miR-130a (D) and PPARγ (E) was detected by qRT-PCR in HepG2 or Huh-7 cells treated with FFA. (F) Protein level of PPARγ was evaluated by Western blotting in HepG2 or Huh-7 cells treated with FFA. GAPDH was used for normalization. All the results were shown as mean ± SD (n=3), which were three separate experiments performed in triplicate. *P<0.05 and **P< 0.01.

• 

  Figure 3. H19 could directly regulate expression of miR-130a Relative expression level of H19 (A) and miR-130a (B) was measured by qRT-PCR in HepG2 and Huh-7 cells transfected with shH19. (C) Predicted binding site between miR-130a and H19 by TargetScan software. (D) The luciferase activity of the H19-WT and H19-MUT in HepG2 and Huh-7 cells transfected with miR-130a mimics and inhibitor. (E) PCR was used to detect H19 in the samples pulled down by biotinylated miR-130a. All the results were shown as mean ± SD (n=3), which were three separate experiments performed in triplicate. *P<0.05 and **P<0.01.

• 

  Figure 4. Knockdown of H19 inhibited steatosis and TG secretion in FFA-induced hepatocytes (A) Oil-Red O staining for lipid droplet in FFA-challenged HepG2 and Huh-7 cells transfected with shH19. (B) TG levels were detected by ELISA assay in FFA-challenged HepG2 and Huh-7 cells transfected with shH19. All the results were shown as mean ± SD (n=3), which were three separate experiments performed in triplicate. *P<0.05 and **P<0.01.

• 

  Figure 5. miR-130a inhibited the expression of PPARγ by binding with its 3′-UTR Relative expression level of miR-130a (A) and PPARγ (B) was measured by qRT-PCR in HepG2 and Huh-7 cells transfected with miR-130a mimics and inhibitor. (C) Predicted binding site between miR-130a and PPARγ by TargetScan software. (D) The luciferase activity of the PPARγ-WT and PPARγ-MUT in HepG2 and Huh-7 cells transfected with miR-130a mimics and inhibitor. All the results were shown as mean ± SD (n=3), which were three separate experiments performed in triplicate. *P<0.05 and **P<0.01.

• 

  Figure 6. Overexpression of miR-130a inhibited steatosis and TG secretion by directly targeting PPARγ in FFA-induced hepatocytes (A) Oil-Red O staining for lipid droplet in FFA-challenged HepG2 and Huh-7 cells treated with miR-NC, miR-130a mimics, GW9662 and miR-130a mimics with GW1929. (B) TG levels were detected by ELISA assay in FFA-challenged HepG2 and Huh-7 cells treated with miR-NC, miR-130a mimics, GW9662 and miR-130a mimics with GW1929. All the results were shown as mean ± SD (n=3), which were three separate experiments performed in triplicate. *P<0.05 and **P<0.01.

• 

  Figure 7. miR-130a inhibited expression of NAFLD-related genes Relative expression level of PPARγ (A), SREBP1 (B), SCD1 (C), ACC1 (D) and FASN (E) was measured by qRT-PCR in FFA-challenged HepG2 and Huh-7 cells transfected with miR-130a mimics and inhibitor. (F) Relative protein level of lipogenesis-related genes was evaluated by Western blotting in FFA-challenged HepG2 and Huh-7 cells transfected with miR-130a mimics and inhibitor, GAPDH was used for normalization. All the results were shown as mean ± SD (n=3), which were three separate experiments performed in triplicate. *P<0.05 and **P<0.01.