HepG2 and Huh7 cell lines were obtained from the American Type Culture Collection and analyzed with short tandem repeat profiling semiannually after the first recovery. HepG2 and Huh7 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (HyClone; Cytiva), D-glucose (5.5 mM) and 1% penicillin/streptomycin at 37˚C in a 5% CO2 incubator. To model hepatic steatosis, the cells were plated in 6-well plates overnight, followed by a 4-h treatment in serum-free DMEM that was subsequently supplemented with 100 mM D-glucose for an additional 48 h at 37˚C and the control group was treated with DMSO.

HepG2 and Huh7 cells (1x10⁶ cells/per well) were seeded into 24-well plates, 4% formalin-fixed at room temperature for 1 h and subsequently stained for 15 min at room temperature with Oil Red O solution (cat. no. ab223796; Abcam) at room temperature to detect intracellular lipids upon microscopic analysis. In addition, Oil Red O staining quantification was performed by treating samples with isopropanol to solubilize the dye, and the absorbance of the isopropanol solution was subsequently quantified at 510 nm with a microplate reader.

TG levels were measured as in a previous study (19). HepG2 and Huh7 cells (1x10⁶ cells/per well) were seeded into 24-well plates and were lysed in NP-40 for 1 h at room temperature. Subsequently, lysates were warmed for 5 min to 100˚C and cooled to room temperature. This procedure was repeated one additional time to fully solubilize lipids within these samples. Samples were then centrifuged, and TG levels were measured using an enzymatic TG assay kit (cat. no. K614-100; Biovision, Inc.) according to the manufacturer's protocols, with total protein levels in each sample used to normalize the TG content.

miR-506-3p mimics (50 nM), inhibitor (50 nM) and corresponding controls (50 nM) were purchased from Shanghai GenePharma Co., Ltd. Polyethylenimine (PEI; Sigma-Aldrich; Merck KGaA) was used to transfect all cells in the present study. For transfection, 1x10⁵ HepG2 and Huh7 cells were plated for 24 h at 37˚C in serum- and antibiotic-free DMEM, following which PEI-miRNA complexes in Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) were added. After 4 h, fresh DMEM containing 10% FBS was added, and the cells were incubated for an additional 48 h. miR-505-3p mimics (5'-GGGAGCCAGGAACGAUUGAUGU-3'), inhibitor (5'-ACUACUGAGCCGCAGUAGA-3'), mimics-NC (5'-UUCUCCGAACGUGUCACGUTT-3') and inhibitor-NC (5'-UUCUCCGAACGUGUCACGUTT-3') were used.

Total RNA was extracted from HepG2 and Huh7 cells using TRIzol^® Reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Total RNA was reverse transcribed into cDNA using the HiFiScipt cDNA Synthesis kit (CoWin Biosciences) at 16˚C for 30 min, 42˚C for 30 min and 85˚C for 5 min. qPCR was performed using SYBR^® Premix Ex Taq™ (Takara Biotechnology Co., Ltd.) and the ABI PRISM 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. qPCR reaction was performed using the following conditions: Pre-denaturation at 95˚C for 1 min, 40 cycles of denaturation at 95˚C for 30 sec, annealing at 67˚C for 30 sec and extension at 72˚C for 30 sec, followed by a final extension step at 72˚C for 5 min. The sequences of the RT-qPCR primers were listed in Table I. miRNA and mRNA expression levels were quantified using the 2^-∆∆Cq method (20) and normalized to the internal reference genes U6 and GAPDH, respectively.

Huh7 and HepG2 cells were harvested and lysed in RIPA lysis buffer supplemented with protease inhibitors (Bio Basic Inc.). A bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.) was used to quantify protein levels in each sample, following which 30 µg protein/sample were separated via 10% SDS-PAGE prior to transfer onto PVDF membranes (Millipore Sigma). The blots were then blocked using either 5% BSA (Gibco; Thermo Fisher Scientific, Inc.) for AMKP and phosphorylated (p)-AMPK or non-fat milk in TBST (for other proteins), followed by overnight incubation with antibodies specific to SIRT1 (1:1,000; cat. no. 8469), AMPK (1:1,000; cat. no. 5831), p-AMPK (Threonine 172; 1:1,000; cat. no. 8208) and GAPDH (1:1,000; cat. no. 5174; all from Cell Signaling Technology, Inc.) at 4˚C. The blots were subsequently probed for 1 h at 4˚C with an appropriate horseradish peroxidase-linked rabbit (1:10,000; cat. no. 7074) and anti-mouse IgG (1:10,000; cat. no. 7076; Cell Signaling Technology, Inc.) secondary antibodies followed by enhanced chemiluminescent substrate visualization (GE Healthcare). ImageJ Software version 1.46 (National Institutes of Health) was used for densitometric analyses.

SIRT1 activity in HepG2 and Huh7 cells transfected with either miR-506-3p mimics or inhibitor was quantified with a SIRT1 fluorometric assay kit (cat. no. CS1040; Sigma-Aldrich; Merck KGaA) using manufacturer's protocols. For this assay, a substrate that contained both a fluorophore and a quencher was used, so that following SIRT1-mediated deacetylation in the presence of NAD, this substrate was cleaved by a peptidase, leading to a fluorescent signal proportional to the degree of SIRT1 activity. Excitation and emission wavelengths at 350 and 460 nm, respectively, were applied. Recombinant SIRT1 and fluoro-deacetylated peptides were used as positive controls. NAD, samples or enzymes were omitted from the assays as negative controls.

The wild-type or mutated versions of SIRT1 3'-UTR were cloned downstream of the firefly gene in the psiCHECK2 plasmid (Promega Corporation), with the mutant construct containing mutations designed to disrupt miR-506-3p binding to its cognate target sequence. PEI transfection reagent (Sigma-Aldrich; Merck KGaA) was used to transfect HepG2 and Huh7 cells with either miR-506-3p mimics or inhibitor together with the vectors encompassing the wild-type or mutated versions of SIRT1 3'-UTR. Luciferase activity in these cells was analyzed at 48 h post-transfection using a Dual-Luciferase Reporter Assay System (Promega Corporation) based on the manufacturer's instructions, with Renilla activity used for normalization purposes.

GraphPad Prism 6 (GraphPad Software, Inc.) was used for all statistical analyses in the present study. Data are presented as the mean ± standard deviation from three experimental repeats and one-way ANOVA followed by Tukey's post hoc test was used to compare different groups. P<0.05 was considered to indicate a statistically significant difference.

Huh7 and HepG2 cells treated with high glucose concentration were used to model hepatic steatosis. Lipid accumulation within these cells was confirmed via Oil Red O staining (Fig. 1A). TG content was significantly increased in glucose-treated cells compared with control cells (Fig. 1B). miR-506-3p and SIRT1 expression level was examined in these cells, revealing that miR-506-3p expression level was significantly increased in glucose-treated cells (Fig. 1C), whereas SIRT1 expression was markedly decreased at the RNA and protein level in both cell lines following high glucose treatment (Fig. 1D-F).

To explore the functional relevance of miR-506-3p in the context of hepatic steatosis, HepG2 and Huh7 cells were transfected with miR-506-3p mimics or inhibitor (Fig. 2A and B). The analysis of TG levels in these cells following high glucose pretreatment revealed that transfection with miR-506-3p mimics led to a significant enhancement of glucose-induced TG accumulation compared with the control group, whereas miR-506-3p inhibitor transfection exhibited the opposite effect (Fig. 2C and D). miR-506-3p mimics also resulted in significant lipid accumulation within these cells compared with the control group, demonstrated via Oil Red O staining, whereas miR-506-3p inhibitor transfection had the opposite effect (Fig. 2E and F). Subsequently, the expression levels of major lipogenic genes, such as sterol regulatory element-binding protein 1 (SREBP1), fatty acid synthase (FASN), stearoyl-CoA desaturase-1 (SCD1) and acetyl-CoA carboxylase 1 (ACC1) were examined (21,22) in cells treated with high glucose concentration with or without transfection with miR-506-3p inhibitor or mimics. Treatment with high glucose increased the expression level of these genes, as indicated by RT-qPCR; however, their expression levels were decreased by miR-506-3p mimics (Fig. 3G) and increased by miR-506-3p inhibitor compared with non-transfected cells treated with high-glucose (Fig. 3H).

The association between SIRT1 and miR-506-3p in the cellular model of hepatic steatosis was further explored. miR-506-3p mimics transfection in cells resulted in a significant reduction in SIRT1 expression at the mRNA and protein level, whereas the opposite effect was observed following miR-506-3p inhibitor transfection (Fig. 3A-E). These data suggested that miR-506-3p may directly or indirectly regulate SIRT1 in this molecular context. To further explore this regulatory association, the potential complementarity between SIRT1 mRNA and miR-506-3p was examined, revealing a putative miR-506-3p binding site in the SIRT1 3'-UTR (Fig. 3F). Luciferase reporter assay revealed that miR-506-3p mimics significantly suppressed the luciferase activity of reporter constructs containing the wild-type but not a mutated version of the SIRT1 3'-UTR sequence (Fig. 3G and H). These results suggested that miR-506-3p could directly bind to the 3'-UTR of SIRT1 and suppress its expression.

The AMPK pathway has been indicated to be associated with the onset of NAFLD (23), and enhance intracellular NAD⁺ levels, thereby leading to enhanced SIRT1 activation (24). Similarly, SIRT1 overexpression has been demonstrated to result in increased AMPK activation, highlighting a regulatory feedback mechanism in this metabolic context (25). In the present study, SIRT1 deacetylase activity was measured in samples obtained from HepG2 and Huh7 cells that were not treated with high glucose transfected with miR-506-3p mimics or inhibitor using a fluorometric assay, revealing that miR-506-3p mimics transfection resulted in markedly reduced SIRT1 activity, whereas miR-506-3p inhibition exhibited the opposite effect (Fig. 4A and B). The role of the AMPK pathway as a mediator of SIRT1 activity in this cellular model of hepatic steatosis was subsequently examined by detecting AMPK phosphorylation via western blotting. The results revealed that lipid accumulation in high glucose-treated HepG2 and Huh7 cells was associated with a significant reduction in AMPK phosphorylation (Fig. 4C and D). In addition, miR-506-3p mimics transfection into these cells significantly decreased the level of AMPK phosphorylation, whereas miR-506-3p inhibitor transfection led to significantly enhanced AMPK phosphorylation compared with the control group (Fig. 4E and F).