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. 2024 Nov 8;19(11):e0313185.
doi: 10.1371/journal.pone.0313185. eCollection 2024.

Inhibition of the miR-1914-5p increases the oxidative metabolism in cellular model of steatosis by modulating the Sirt1-PGC-1α pathway and systemic cellular activity

Affiliations

Inhibition of the miR-1914-5p increases the oxidative metabolism in cellular model of steatosis by modulating the Sirt1-PGC-1α pathway and systemic cellular activity

Thais Porto-Barbosa et al. PLoS One. .

Abstract

Metabolic associated fatty liver disease (MAFLD) is considered an indicator of metabolic syndrome, which affects millions of people around the world and no effective treatment is currently available. MAFLD involves a wide spectrum of liver damage, that initiates from steatosis (fatty live) and may progress to more complex pathophysiology. Then, details in lipid metabolism controlling should be explored aiming to control the fatty liver. In this context, the miR-1914-5p can be considered a potential biotechnology tool to control lipid metabolism in hepatic cells. This miRNA finds potential mRNA binding sequences in more than 100 molecules correlated with energy production and lipid metabolism pointed in bioinformatic platforms. The present study addressed the miR-1914-5p effects in hepatic HepG2/LX-2 co-cultured cells in a in vitro steatotic environment stablished by the addition of 400 μM of a mixture of oleic and palmitic acids. The analyses demonstrated that the inhibition of the miRNA reduced energetic metabolites such as total lipids, triglycerides, cholesterol and even glucose. In addition, the miR-inhibitor-transfected cells did not present any deleterious effect in cellular environment by controlling reactive oxygen species production (ROS), mitochondrial membrane potential (ΔΨm) and even the pro-inflammatory environment. Moreover, the functional effect of the investigated miR, suggested its close connection to the modulation of Sirt-1-PGC1-α pathway, a master switch metabolic route that controlls cellular energetic metabolism. Our assays also suggested a synergistic effect of this miR-1914-5p in cell metabolism, which should be considered as a strong candidate to control steatotic environment in future clinical trials.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Establishing in vitro steatotic cellular model.
A) HepG2 and LX-2 cells were seeded at the proportion rate of 7:3 units and cultivated for 24 h under regular conditions. Next, different concentrations of FA mixture were added to the cultures to simulate in vitro steatotic condition. After 24 h cell viability was investigated by MTT analyses; B) Triglycerides and cholesterol levels measurement after FA were added to the cellular co-culture mode. The graphs represent the mean values of at least three independent experiments (*p<0.05).
Fig 2
Fig 2. Molecular and biochemical effects of miR-1914-5p on lipid metabolism.
A) microscopy analyses of hepatic co-cultures transfected with the miRNA-1914-5p; (B) qRT-PCR of miRNA expression levels in different group of cells. C) Biochemical measurements of triglycerides, cholesterol and total lipids in different groups of cells. D) Gas chromatography/ mass spectrometry (GC/MS) analyses of fatty acids in investigated group of cells. E) qRT-PCR of transcript levels of CD36. The graphs and images present mean values of the average from at least three independent experiments. ANOVA testing showed significant differences (*p< 0.05) in the assays. Nc-miRNA = negative control (Scramble miRNAs).
Fig 3
Fig 3. Heatmaps of the mRNA expressed between investigated cellular groups in q-RT-PCR analyses.
Each column represents an individual cellular group, and each row represents an individual mRNA. Colors of the heatmap represent the Z-score: Higher–blue/ green/red, lower–white. The heatmap was packed using Graph Pad Prism® 8 software. ANOVA testing showed significant differences between the cellular groups, and significance was set at *p<0.05. The Figure represents mRNAs found in A) lipid biosynthesis and packing; B) transcription factors and central molecules on bioenergetic routes; C) glycolytic enzymes. Legends (C = control; FA = fatty acid; M = miR-1914-5p mimics; I = miR-1914-5p inhibitor; Nc-miRNA = negative control, Scramble miRNAs, mirVana™).
Fig 4
Fig 4. Effects of the miRNA-1914-5p mimics or inhibitor in co-culture of hepatic cells cultivated or not in the presence of a mixture of FA.
A) Fluorescent FA pulse-chase. The images present BODIPY 558/568 C12 and the overlap between the lipid droplets and the cell nucleus for different treatments. B) The quantification of LD was calculated and is presented in the graph representing Person’s coefficient. C) mRNA and protein levels of PGC1-α in different groups of cells. Graphs represent the mean ± SD from at least three independent experiments and the statistics was performed using one-way analysis of variance (ANOVA), followed by Tukey’s test, between the investigated groups. The significance level at the statistical analyses was set at p<0.01 (*).
Fig 5
Fig 5. Functional activities of miR-1914-5p in relevant elements that control bioenergetic metabolism in co-culture of hepatic cells.
A) Sirt1 and FOXO1 mRNA and protein levels in different groups of hepatic co-cultivated cells. B) In addition, the acetylated FOXO1 protein was measured. C) Representative western blotting from investigated cellular groups; β-actin was used as the loading control. The graphs and images present mean values of the average from at least three independent experiments. ANOVA testing showed significant differences (*p< 0.05) in the assays.
Fig 6
Fig 6. Overexpression of Sirt1 and PGC-1α in co-culture of hepatic cells to validate their functional activity on lipid metabolism in a in vitro cellular model of steatosis.
A) Sirt1 and PGC-1α mRNA and protein levels in different groups of hepatic co-cultivated cells. B) Biochemical analyses of molecules correlated with lipid metabolism in different groups of cells. C) Beta-oxidation analyses to validate the effect of Sirt1 and PGC-1α overexpression in hepatic cells. The graphs and images present mean values of the average from at least three independent experiments. ANOVA testing showed significant differences (*p< 0.05) in the assays.
Fig 7
Fig 7. Schematic representation of multisystemic effects of miR-1914-5p in bioenergetic metabolism in co-culture of hepatic cells.

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