miR-122 promotes hepatic lipogenesis via inhibiting the LKB1/AMPK pathway by targeting Sirt1 in non-alcoholic fatty liver disease

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is a common hepatic disease with an increasing prevalence but an unclear aetiology. This study aimed to investigate the functional implications of microRNA-122 (miR-122) in the pathogenesis of NAFLD and the possible molecular mechanisms.

Methods: Both in vitro and in vivo models of NAFLD were generated by treating HepG2 and Huh-7 cells with free fatty acids (FFA) and by feeding mice a high-fat diet (HFD), respectively. HE and Oil Red O staining were used to examine liver tissue morphology and lipid deposition, respectively. Immunohistochemical (IHC) staining was used to examine Sirt1 expression in liver tissues. qRT-PCR and Western blotting were employed to measure the expression of miR-122, Sirt1, and proteins involved in lipogenesis and the AMPK pathway. Enzyme-linked immunosorbent assay (ELISA) was used to quantify triglyceride (TG) levels in HepG2 and Huh-7 cells and in liver tissues. The interaction between miR-122 and the Sirt1 gene was further examined by a dual luciferase reporter assay and RNA-immunoprecipitation (RIP).

Results: NAFLD hepatic tissues and FFA-treated HepG2 and Huh-7 cells presented excess lipid production and TG secretion, accompanied by miR-122 upregulation, Sirt1 downregulation, and potentiated lipogenesis-related genes. miR-122 suppressed Sirt1 expression via binding to its 3'-untranslated region (UTR). Knockdown of miR-122 effectively mitigated excessive lipid production and suppressed the expression of lipogenic genes in FFA-treated HepG2 and Huh-7 cells via upregulating Sirt1. Furthermore, miR-122 knockdown activated the LKB1/AMPK signalling pathway.

Conclusion: The inhibition of miR-122 protects hepatocytes from lipid metabolic disorders such as NAFLD and suppresses lipogenesis via elevating Sirt1 and activating the AMPK pathway. These data support miR-122 as a promising biomarker and drug target for NAFLD.

Keywords: AMPK pathway; Lipogenesis; Non-alcoholic fatty liver disease; Sirt1; miR-122.

Fig. 1

Lipid pathology and altered gene expression in NAFLD mice. a HE staining (left) and Oil Red O staining (right) showing lipid accumulation in liver tissues. b Hepatic TG levels were assessed by ELISA. c The relative expression levels of miR-122, Sirt1 and lipogenesis-related genes in control diet- and high-fat diet (HFD)-fed mice were detected by qRT-PCR. d The expression level of Sirt1 was measured by Western blotting. e Quantification of the Western blotting results in d. f, g Representative images (f) and thepositive rate (g) of Sirt1 expression in the liver tissues of NAFLD mice as revealed by IHC staining. All the results are shown as the mean ± SD (n = 3), which are representative of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 2

Effects of FFA on lipogenesis and miR-122 expression in hepatocytes. HepG2 or Huh-7 cells were pre-treated with 1 mM FFA for 24 h. a Oil Red O staining for lipid droplets in HepG2 or Huh-7 cells. b The secretion of TG in HepG2 or Huh-7 cells was measured by ELISA. c, d The relative expression of miR-122 (c) and Sirt1 (d) in HepG2 or Huh-7 cells was determined by qRT-PCR. e The expression level of Sirt1 in HepG2 or Huh-7 cells was measured by Western blotting. f Quantification of the corresponding Western blotting results in e. All the results are shown as the mean ± SD (n = 3), which are representative of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 3

Direct interaction between miR-122 and Sirt1. a The relative expression levels of miR-122 and Sirt1 in HepG2 or Huh-7 cells transfected with miR-122 mimics or inhibitor. b Sequences of the putative binding sites of miR-122 in the 3′-UTR of Sirt1. c An RIP assay was performed to verify the interaction between miR-122 and Sirt1, and the Sirt1 mRNA level was measured by qRT-PCR. d A dual luciferase activity assay was implemented to confirm the direct association between miR-122 and the 3′-UTR of Sirt1. All the results are shown as the mean ± SD (n = 3), which are representative of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 4

Suppression of lipid accumulation by the miR-122 inhibitor via the elevation of Sirt1 expression. a The relative expression level of Sirt1 was assessed by qRT-PCR in HepG2 or Huh-7 cells transfected with shSirt1. b The protein level of Sirt1 was assessed by Western blotting in HepG2 or Huh-7 cells transfected with shSirt1. c Quantification of the corresponding Western blotting results in b. d Oil Red O staining was performed to examine lipid accumulation in HepG2 or Huh-7 cells transfected with miR-122 inhibitor and/or shSirt1. e TG secretion levels in all groups of HepG2 or Huh-7 cells were measured by ELISA. All the results are shown as the mean ± SD (n = 3), which are representative of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 5

The expression of lipogenesis-associated genes was regulated by miR-122 and Sirt1. HepG2 and Huh-7 cells were pre-transfected with miR-122 inhibitor and/or shSirt1 and were then treated with 1 mM FFA for 24 h. a-e The relative expression levels of SREBP1 (a), FASN (b), SCD1 (c), ACC1 (d) and ApoA5 (e) in HepG2 and Huh-7 cells were detected by qRT-PCR. All the results are shown as the mean ± SD (n = 3), which are representative of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01

Fig. 6

Downregulation of the LKB1/AMPK signalling pathway by the miR-122-Sirt1 axis. a The expression of Sirt1, LKB1, AMPK and the phosphorylation of AMPK (p-AMPK) were measured by Western blotting in HepG2 or Huh-7 cells that were transfected with miR-122 inhibitor and/or shSirt1. b Quantification of the corresponding Western blotting results in a. All the results are shown as the mean ± SD (n = 3), which are representative of three independent experiments performed in triplicate. *p < 0.05 and **p < 0.01