MicroRNA-506-3p targets SIRT1 and suppresses AMPK pathway activation to promote hepatic steatosis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a complex type of liver disease that represents an important global health threat. The mechanistic basis of this disease remains incompletely understood. The present study sought to explore whether microRNA (miR)-506-3p served a functional role in the onset and/or progression of NAFLD. To that end, high levels of glucose were used to treat liver cancer cell lines (HepG2 and Huh7) to model hepatic steatosis, and the expression levels of miR-506-3p and its downstream target genes were assessed. The cells of this hepatic steatosis model were transfected with miR-506-3p mimic molecules to explore the effect of miR-506-3p overexpression on cell viability, target gene expression and AMP-activated protein kinase (AMPK) phosphorylation. Via bioinformatics approaches, sirtuin 1 (SIRT1) was identified as a potential miR-506-3p target gene with relevance in NAFLD, and this interaction was confirmed via luciferase reporter assay. In the hepatic steatosis model of the present study, miR-506-3p expression level was significantly increased, whereas SIRT1 mRNA/protein levels and AMPK phosphorylation levels were markedly decreased. Transfection of the cells with miR-506-3p mimics led to significant SIRT1 downregulation, while miR-506-3p inhibitor molecules exhibited the opposite effect, with similar trends observed in the phosphorylation status of AMPK. These results suggested that miR-506-3p can inhibit SIRT1 expression and associated AMPK phosphorylation in HepG2 and Huh7 cells in an in vitro hepatic steatosis model system. These data indicated that the miR-506-3p/SIRT1/AMPK axis may be valuable as a therapeutic target in patients affected by NAFLD.

Keywords: AMP-activated protein kinase; liver; microRNA-506-3p; nonalcoholic fatty liver disease; sirtuin 1.

Figure 1

miR-506-3p and SIRT1 expression is altered in a model of hepatic steatosis. (A) Left panel: Oil Red O staining of HepG2 and Huh7 cells after incubation with 100 mM glucose. Right panel: Quantification via spectrophotometry. Scale bars, 100 µm. (B) Triglyceride levels were assessed in HepG2 and Huh7 cells after incubation with 100 mM glucose and control cells. Reverse transcription-quantitative PCR was used to measure (C) miR-506-3p and (D) SIRT1 mRNA levels in high-glucose treated cells. (E) SIRT1 protein levels were assessed in these cells via western blotting, (F) with densitometric quantification performed using ImageJ. Data are presented as the mean ± SD (n=3). ^*P<0.05; ^**P<0.01; ^***P<0.001. miR, microRNA; SIRT1, sirtuin 1; OD, optical density.

Figure 2

Overexpression of miR-506-3p induces lipid accumulation in HepG2 and Huh7 cells. (A and B) Following transfection with miR-506-3p mimics or inhibitor, miR-506-3p expression in HepG2 and Huh7 cells was quantified via RT-qPCR. (C and D) HepG2 and Huh7 cells were treated with high glucose prior to transfection with miR-506-3p mimics or inhibitor or with corresponding NC constructs, and an enzymatic method was used to measure intracellular triglyceride levels. (E and F) HepG2 and Huh7 cells were treated with high glucose prior to transfection with miR-506-3p mimics or inhibitor or with corresponding NC constructs. Oil Red O staining and quantification via spectrophotometry are presented. Scale bars, 100 µm. (G and H) The relative expression levels of SREBP1, FASN, SCD1 and ACC1 in high-glucose treated HepG2 and Huh7 cells were detected by RT-qPCR. Data are presented as the mean ± SD (n=3). ^*P<0.05; ^**P<0.01; ^***P<0.001. miR, microRNA; SIRT1, sirtuin 1; NC, negative control; RT-qPCR, reverse transcription quantitative-PCR; SREBP1, sterol regulatory element-binding protein 1; FASN, fatty acid synthase; SCD1, stearoyl-CoA desaturase-1; ACC1, acetyl-CoA carboxylase 1; OD, optical density.

Figure 3

miR-506-3p binds to the 3'-UTR of SIRT1 and suppresses its expression. HepG2 and Huh7 cells were transfected with miR-506-3p mimics or inhibitor, and SIRT1 levels were quantified via (A and B) reverse transcription quantitative-PCR and (C-E) western blotting. (F) Putative miR-506-3p binding site in the SIRT1 3'-UTR, with the mutant sequence additionally presented in red font. (G and H) The interaction between miR-506-3p and SIRT1 3'-UTR was confirmed via dual luciferase reporter assay. Data are presented as the mean ± SD (n=3). ^*P<0.05; ^**P<0.01; ^***P<0.001. miR, microRNA; SIRT1, sirtuin 1; NC, negative control; UTR, untranslated region; WT, wild-type; MUT, mutant; ns, not significant.

Figure 4

miR-506-3p modulates AMPK phosphorylation in an in vitro model of hepatic steatosis. (A and B) A fluorometric assay was used to measure SIRT1 activity in HepG2 and Huh7 cells without high glucose treatment transfected with miR-506-3p mimics or inhibitor. (C and D) AMPK phosphorylation was examined via western blotting in HepG2 and Huh7 cells treated with high glucose, and densitometry was used for quantification purposes. (E and F) Western blotting was performed to observe the effect of miR-506-3p mimics or inhibitor transfection on AMPK phosphorylation in HepG2 and Huh7 cells not treated with high glucose. Data are presented as the mean ± SD (n=3). ^*P<0.05; ^**P<0.01; ^***P<0.001. miR, microRNA; AMPK, AMP-activated protein kinase, SIRT1, sirtuin 1; NC, negative control; p, phosphorylated.