
==== Front
Cell Mol Life Sci
Cell Mol Life Sci
Cellular and Molecular Life Sciences: CMLS
1420-682X
1420-9071
Springer International Publishing Cham

39261317
5434
10.1007/s00018-024-05434-6
Original Article
MicroRNA-411-5p alleviates lipid deposition in metabolic dysfunction-associated steatotic liver disease by targeting the EIF4G2/FOXO3 axis
Wan Zhiping 12
Liu Xiaoquan 12
Yang Xiaoan 12
Huang Zexuan 2
Chen Xiaoman 12
Feng Qingqing 12
Cao Hong caohong@mail.sysu.edu.cn

12
http://orcid.org/0000-0001-9164-7755
Deng Hong dhong@mail.sysu.edu.cn

12
1 https://ror.org/04tm3k558 grid.412558.f 0000 0004 1762 1794 Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510630 China
2 https://ror.org/04tm3k558 grid.412558.f 0000 0004 1762 1794 Guangdong Key Laboratory of Liver Disease Research, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510630 China
11 9 2024
11 9 2024
12 2024
81 1 3983 4 2024
12 8 2024
30 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Background

Abnormal lipid deposition is an important driver of the progression of metabolic dysfunction-associated steatotic liver disease (MASLD). MicroRNA-411-5p (miR-411-5p) and eukaryotic translation initiation factor 4γ2 (EIF4G2) are related to abnormal lipid deposition, but the specific mechanism is unknown.

Methods

A high-fat, high-cholesterol diet (HFHCD) and a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) and a high-fructose diet (HFrD) were used to establish MASLD rat and mouse models, respectively. MiR-411-5p agomir and mimic were used to upregulate the miR-411-5p in vivo and in vitro, respectively. Adeno-associated virus type 8 (AAV8) carrying EIF4G2 short hairpin RNA (shRNA) and small interfering RNA (siRNA) were used to downregulate the EIF4G2 expression in vivo and in vitro, respectively. Liver histopathological analysis, Biochemical analysis and other experiments were used to explore the functions of miR-411-5p and EIF4G2.

Results

MiR-411-5p was decreased in both MASLD rats and mice, and was negatively correlated with liver triglycerides and serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. Upregulation of miR-411-5p alleviated liver lipid deposition and hepatocellular steatosis. Moreover, miR-411-5p targeted and downregulated EIF4G2. Downregulation of EIF4G2 not only reduced liver triglycerides and serum ALT and AST levels in MASLD model, but also alleviated lipid deposition. Notably, upregulation of miR-411-5p and downregulation of EIF4G2 led to the reduction of forkhead box class O3 (FOXO3) and inhibited the expression of sterol regulatory-element binding protein 1 (SREBP1), acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), thereby reducing fatty acid synthesis.

Conclusions

Upregulation of miR-411-5p inhibits EIF4G2 to reduce the FOXO3 expression, thereby reducing fatty acid synthesis and alleviating abnormal lipid deposition in MASLD.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-024-05434-6.

Keywords

Fatty liver disease
Fat metabolism
MicroRNA
http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 81870597 Deng Hong http://dx.doi.org/10.13039/501100010256 Guangzhou Municipal Science and Technology Project 202103000060 Cao Hong issue-copyright-statement© Springer Nature Switzerland AG 2024
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pmcIntroduction

Metabolic dysfunction-associated steatotic liver disease (MASLD), the nomenclature for nonalcoholic fatty liver disease (NAFLD), refers to hepatic steatosis accompanied by cardiometabolic risk factors [1]. The prevalence of MASLD continues to grow, with approximately 32.4% of the global population affected by MASLD [2]. Hepatic steatosis and abnormal lipid deposition are prominent manifestations of liver pathology in patients with MASLD. Excessive lipid accumulation causes liver cell apoptosis or necrosis, triggers a liver inflammatory response, and damages the liver [3]. Hepatic steatosis and abnormal lipid deposition in MASLD are early stages of a severe form of liver disease that may evolve into metabolic dysfunction-associated steatohepatitis (MASH), even end-stage liver disease (cirrhosis, and hepatocellular carcinoma) [4]. Unfortunately, no specific drugs have been developed to treat patients with MASLD.

Lipid metabolism disorders in hepatocytes are important factors that lead to lipid deposition in MASLD [3]. Excessive lipid deposition damages hepatocytes through endoplasmic reticulum stress and mitochondrial dysfunction [3]. When hepatocytes are damaged, alanine aminotransferase (ALT) is released into the blood [5]. ALT is often used to assess the extent of liver damage [6]. Moreover, free fatty acids, triacylglycerols, and other lipids accumulate excessively in hepatocytes, triggering oxidative stress and leading to hepatocytes apoptosis and necrosis [4, 7]. Increased production of reactive oxygen species further activates inflammasomes and aggravates liver inflammation [8, 9]. Excess fatty acids stimulate macrophages to release inflammatory factors, promote inflammatory response, and activate hepatic stellate cells to induce liver fibrosis, thereby driving the progression of MASLD to MASH [4, 10, 11]. Improving abnormal lipid deposition is an important strategy for treating patients with MASLD and preventing the progression of MASLD to MASH.

When cells are in a pathological state, cap-dependent messenger RNA (mRNA) translation is inhibited, while cap-independent mRNA translation increases [12, 13]. Eukaryotic translation initiation factor 4γ2 (EIF4G2) is an important translation activator that promotes cap-independent mRNA translation during cellular stress [14, 15]. It has been reported that EIF4G2 regulates key genes expression in some liver diseases [16–18]. EIF4G2 mediates activation of the protein kinase B (PKB) pathway and the extracellular signal-regulated protein kinase (ERK) pathway [16, 19], with activation of these pathways being closely related to MASLD [20–23]. Importantly, EIF4G2 also regulates the translation of forkhead box class O3 (FOXO3), which upregulates sterol regulatory-element binding protein 1 (SREBP1) to promote lipid deposition [24, 25]. We speculated that EIF4G2 may regulate MASLD progression, but more investigations are warranted.

The EIF4G2 expression is affected by the regulation of microRNAs (miRNAs) [16, 26]. MicroRNA-411-5p (miR-411-5p), a highly conserved miRNA with a length of 21 nucleotides, is associated with various liver diseases [27–31]. A high-throughput sequencing study using a MASLD mouse model suggests that the miR-411-5p level is decreased in the liver of MASLD mice compared to that of normal mice [30]. In addition, our previous study showed that the miR-411-5p level is reduced not only in the blood exosomes of patients with MASH but also in MASH model livers [31]. However, the biological functions and regulatory effects of miR-411-5p on lipid deposition in MASLD remain unclear. Based on our previous findings, we speculated that miR-411-5p has the function of alleviating MASLD, the purpose of this study was to explore the roles of miR-411-5p and EIF4G2 and the underlying mechanisms in lipid deposition in MASLD.

Materials and methods

Animals

Sprague–Dawley (SD) rats (male, seven-week-old) and C57BL/6 mice (male, six-week-old or eleven-week-old) were provided by the Guangzhou University of Chinese Medicine (Guangzhou, China) and housed in a pathogen-free environment (temperature-controlled, 12-h light/dark cycle) with ad libitum access to food and water. These SD rats and C57BL/6 mice were divided into groups after a week of adaptation. Specifically, SD rats were assigned to the Normal group (N = 5) or MASLD group (N = 5) and fed a conventional diet or a high-fat, high-cholesterol diet (HFHCD) (#D10070802; Readydietech, Shenzhen, China), respectively, for 8 weeks. Weight of the rats was measured and recorded every two weeks. C57BL/6 mice were assigned to the Normal group (N = 5) or MASLD group (N = 5) and fed a conventional diet or a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) (#A06071302; Research Diets, New Brunswick, NJ, USA), respectively, for 9 weeks. For short hairpin RNA (shRNA) knock-down experiments, additional C57BL/6 mice were assigned to a non-targeting control shNC group (N = 5) and EIF4G2-targeting shEif4g2 group (N = 5) and fed a CDAHFD for 9 weeks. The mice were weighed weekly. During the fourth week of CDAHFD feeding, adeno-associated virus type 8 (AAV8, 2.5 × 1011 vg) carrying Eif4g2 shRNA (shEif4g2) or NC shRNA (shNC) was injected into the tail vein of the respective groups of mice.

For miR-411-5p intervention experiments, C57BL/6 mice (male, eleven-week-old) were divided into groups after a week of adaptation. These mice were assigned to the Normal group (N = 5), MASLD group (N = 5), MASLD + NC group (N = 5) or MASLD + miR-411-5p group (N = 5). The mice in the Normal group were fed a conventional diet, and the mice in the other three groups were fed a high-fructose diet (HFrD) (#D08040107; Research Diets, New Brunswick, NJ, USA), respectively, for 3 weeks. In addition, mice in MASLD + NC group and MASLD + miR-411-5p group were injected with negative control or miR-411-5p agomir (5 nmol, once every three days) through the tail vein.

After the above-noted interventions, the SD rats and C57BL/6 mice were euthanized. Blood was collected from each animal, and serum was prepared. Livers were removed, rinsed with normal saline, blotted dry, weighed, and snap frozen in liquid nitrogen. The serum and liver specimens were stored at − 80 °C until use. All animal experiments were approved by the Experimental Animal Ethics Committee (approval numbers: F2008032, F2308003 and 20,240,527,001).

Liver histopathological analysis

The livers of SD rats and C57BL/6 mice were embedded in paraffin or optimal cutting temperature (OCT) compound. A microtome and cryostat were used to prepare paraffin sections and frozen sections of the liver tissue specimens, respectively. For hematoxylin and eosin (H&E) staining, the paraffin sections of SD rat and C57BL/6 mouse liver tissue were stained with hematoxylin (#G1004; Servicebio, Wuhan, China) and eosin (#G1003, Servicebio) stains. For Oil Red O staining, the frozen sections of SD rat and C57BL/6 mouse liver tissue were stained with Oil Red O reagent (#G1016, Servicebio) and then stained with hematoxylin. A microscope (Leica, Wetzlar, Germany) was used to observe and take representative images. The lipid vacuole and droplet areas were analyzed by ImageJ software (NIH, Bethesda, MD, USA).

Liver fluorescence in situ hybridization analysis

Liver tissue sections from SD rats and C57BL/6 mice were deparaffinized using dewaxing clear solution (#G1128, Servicebio), repaired with citric acid, and digested with recombinant proteinase K (#G1234, Servicebio). The processed sections were then incubated with a hybridization solution containing the miR-411-5p probe and placed in an incubator (37 °C) overnight. Finally, the liver tissue sections were washed, and the nuclei stained with DAPI solution (#C1005; Beyotime, Shanghai, China). For combination fluorescence in situ hybridization / immunofluorescence staining, sections were blocked after incubation with probes and then incubated with Albumin primary antibody (1:500; #ab207327; Abcam, MA, USA) overnight (4 °C), followed by incubation with secondary antibodies at room temperature for 60 min. Finally, the nuclei were stained with DAPI. Microscope was used to capture images and ImageJ software was used for analysis.

Biochemical analysis and liver weight index calculation

Levels of alanine transaminase (ALT), aspartate transaminase (AST), high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) in the serum of SD rats and C57BL/6 mice were determined using ALT assay kits (#C009; Nanjing Jiancheng, Nanjing, China), AST assay kits (#C010, Nanjing Jiancheng), HDL-c assay kits (#A112, Nanjing Jiancheng), LDL-c assay kits (#A113, Nanjing Jiancheng), respectively. Levels of total cholesterol (TC) in the cells and serum of SD rats and C57BL/6 mice and were determined using TC assay kits (#A111, Nanjing Jiancheng). Levels of triglyceride (TG) in the livers of SD rats and C57BL/6 mice were measured using TG assay kits (#A110, Nanjing Jiancheng). All assays were performed according to the manufacturers’ instructions. Liver weight index = liver weight/body weight × 100%.

Cell culture and treatment

HepG2 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (#C11995500BT; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (# 10,270–106, Gibco), and placed in an incubator (5% CO2, 37 °C). The HepG2 cells were treated with 300 μM palmitic acid (PA) to steatosis hepatocytes. The cells were transfected with 100 nM miR-411-5p mimic (#miR1020; Ribo, Guangzhou, China) (the PA + miR-411-5p group) or transfected with a negative control miRNA (#miR1020; Ribo, Guangzhou, China) (the PA + NC group) using a transfection reagent (#C10511, Ribo) to upregulate the miR-411-5p expression. To downregulate the expression of EIF4G2 or FOXO3, cells were transfected with 150 nM small interfering RNA (siRNA) targeting EIF4G2 or FOXO3 (#siB10005, Ribo) and a negative control siRNA (#siB10005, Ribo) using transfection reagent.

The human liver stellate cells (LX2) were cultured in high-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (# 10,270–106, Gibco) and the human acute monocytic leukaemia cells (THP1) were cultured in RPMI-1640 (# C11875500BT, Gibco) containing 10% fetal bovine serum (# 10,270–106, Gibco). The THP1 cells were treated with 100 ng/mL PMA (# P8139, Sigma-Aldrich, MO, USA) for 24 h to induce differentiation into macrophages. The LX2 cells or macrophages were treated with 300μM PA for 24 h.

Liver parenchymal and nonparenchymal cells of rats and mice were isolated by a collagenase perfusion method [32, 33]. Briefly, the liver was fully perfused and digested with collagenase to obtain a single cell suspension. Parenchymal cells (hepatocytes) and non-parenchymal cells were separated from the single cell suspension by differential centrifugation. The mouse primary hepatocytes (MPH) were treated with 300μM PA to steatosis hepatocytes and were then transfected with 30 nM miR-411-5p mimic (#miR1020, Ribo).

Cell BODIPY staining analysis

For BODIPY staining, cells were treated with 4% paraformaldehyde and 0.1% Triton-X100 (#T9284, Sigma-Aldrich, Saint Louis, MO, USA) for 30 min and then treated with 5 μM BODIPY reagent (#HY-W090090; Med Chem Express, Monmouth Junction, NJ, USA) for 30 min. All images were captured using a microscope and analyzed using ImageJ software.

Prediction of target genes of miR-411-5p.

The target genes of miR-411-5p were predicted by three online tools: miRDB (http://www.mirdb.org/), TargetScan (http://www.targetscan.org) and miRTarBase (https://mirtarbase.cuhk.edu.cn/). Venn diagrams were used to analyze predicted common genes across the three databases.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis

RNA was extracted from rat and mouse liver tissues and cells using TRIzol reagent (#15,596,018; Invitrogen, Carlsbad, CA, USA). For mRNA analysis, first-strand complementary DNA was synthesized using Reverse Transcriptase Reagent (#RR036; Takara, Kyoto, Japan). For miRNA analysis, first-strand complementary DNA was synthesized using Mir-X miRNA First-Strand Synthesis Kit (#638,313, Takara). TB Green Premix Ex Taq II (#RR820A, Takara) was used for fluorescence quantification of the RT-qPCR product. The relative mRNA or miRNA levels were calculated based on the CT values relative to the β-actin or U6 expression, respectively. All primer sequences are listed in Table S1 of the Supplementary materials and methods.

Statistics

Data were expressed as mean ± standard deviation (SD). Student’s t-test or one-way analysis of variance (ANOVA) was used for statistical comparisons of two or more groups, and Spearman’s rank correlation test was used for correlation analysis. GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical analysis. P < 0.05 considered statistically significant. Figures were produced using GraphPad Prism 8.0 software and Adobe Illustrator CS6 software (Adobe Inc., San Jose, CA, USA).

Results

MiR-411-5p was decreased in the HFHCD-induced MASLD rat model

To explore the expression of miR-411-5p in MASLD, we fed SD rats a HFHCD to establish the MASLD model and recorded their body weight regularly (Fig. 1, a–b). The serum ALT, AST, TC and LDL-c levels of rats in the MASLD group were higher than those of the control group, while the HDL-c levels were lower than those of the control group (Fig. 1, c–g). Moreover, the liver weight index and triglyceride (TG) levels of MASLD rats also increased (Fig. 1, h–i). Importantly, liver histopathology revealed numerous instances of hepatocellular steatosis and lipid deposition in the livers of MASLD rats (Fig. 1, j).Fig. 1 MiR-411-5p was decreased in the HFHCD-induced MASLD rat model. a Schematic diagram showing the establishment of the MASLD rat model. b Rat body weight curve during the modeling period. c-i Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in rats at the end of modeling. (N = 5/group). j H&E staining and Oil Red O staining of rat liver tissue sections and quantitative analysis. (N = 5/group). k Fluorescence in situ hybridization for miR-411-5p in rat liver tissue sections. l Quantification of fluorescence in situ hybridization signal intensity for miR-411-5p in rat liver tissue sections. (N = 5/group). m Relative expression levels of miR-411-5p in rat liver determined by RT-qPCR analysis. (N = 5/group). n Spearman rank correlation analysis of relative expression levels of miR-411-5p and ALT, AST, liver weight index or TG levels, respectively. *P < 0.05, **P < 0.01, ***P < 0.001

Next, we detected significant reduction of miR-411-5p in the liver of MASLD rats (Fig. 1, k-l). Same findings were validated by RT-qPCR. Particularly, hepatic miR-411-5p in the liver of MASLD rats was about 40% lower than that of normal rats (Fig. 1, m). In addition, we found that miR-411-5p was negatively correlated with serum ALT content and AST content, and was also negatively correlated with liver triglyceride levels (Fig. 1, n). Taken together, miR-411-5p was decreased during MASLD, which was related to liver lesions and damage in MASLD.

MiR-411-5p was decreased in the CDAHFD-induced MASLD mouse model

To further clarify the miR-411-5p expression during MASLD, we also established a MASLD mouse model using a CDAHFD and recorded their body weight weekly (Fig. 2, a–b). The serum ALT, AST and LDL-c levels of mice in the MASLD group were higher than those of the control group, while the TC and HDL-c levels were lower than those of the control group (Fig. 2, c–g). Moreover, MASLD mice had a greater liver index and liver TG levels than the control mice (Fig. 2, h–i). Liver histopathology showed numerous steatotic hepatocytes and lipid deposits in the livers of the MASLD group of mice (Fig. 2, j).Fig. 2 MiR-411-5p was decreased in the CDAHFD-induced MASLD mouse model. a Schematic diagram showing the establishment of the MASLD mouse model. b Mouse body weight curve during the modeling period. c-i Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in mice at the end of modeling. (N = 5/group). j H&E staining and Oil Red O staining of mouse liver tissue sections and quantitative analysis. (N = 5/group). k Fluorescence in situ hybridization for miR-411-5p in mouse liver tissue sections. l Quantification of fluorescence in situ hybridization signal intensity for miR-411-5p in mouse liver tissue sections. (N = 5/group). m Relative expression levels of miR-411-5p in mouse liver determined by RT-qPCR analysis. (N = 5/group). n Spearman rank correlation analysis of relative expression levels of miR-411-5p and ALT, AST, liver weight index or TG levels, respectively. *P < 0.05, **P < 0.01, ***P < 0.001

Importantly, compared with normal mice, miR-411-5p expression was reduced in the livers of MASLD mice (Fig. 2, k-m). In addition, miR-411-5p level was negatively correlated with serum ALT, AST, liver weight index, and liver triglyceride levels in MASLD mice (Fig. 2, n). These data further confirmed that miR-411-5p is decreased in MASLD models.

MiR-411-5p alleviated lipid deposition in hepatocytes

Albumin (ALB) is a hepatocyte-specific marker. In fluorescence in situ hybridization analysis combined with immunofluorescence experiments showed that ALB-labeled cells have a signal of miR-411-5p (Fig. 3, a). We further found that the expression of miR-411-5p in rat liver parenchymal cells in the MASLD group was significantly lower than that in the Normal group, but there was no difference in the expression of miR-411-5p in rat liver non-parenchymal cells in the MASLD group and the Normal group (Fig. 3, b). Similar findings were found in mice (Fig. 3, c). In addition, after PA treatment, the expression of miR-411-5p in HepG2 cells decreased, while the expression of miR-411-5p in macrophages and hepatic stellate cells did not change (Fig. 3, d). The miR-411-5p level in the PA + miR-411-5p group was hundreds of times higher than that in the PA + NC group, indicating that we successfully up-regulated the expression of miR-411-5p (Fig. 3, e). Subsequent BODIPY staining analysis revealed that compared with the PA + NC group, the lipid deposition in the PA + miR-411-5p group was significantly reduced (Fig. 3, f-g).Fig. 3 MiR-411-5p alleviated lipid deposition in hepatocytes. a Fluorescence in situ hybridization combined with immunofluorescence experiment for miR-411-5p (red) and ALB (green) in rat or mouse liver tissue sections (DAPI, blue). b-c Relative miR-411-5p levels in parenchymal cells and non-parenchymal cells of rat or mouse liver determined by RT-qPCR analysis. (N = 5/group, normalized to U6). d Relative miR-411-5p levels in HepG2 cells, macrophages and LX2 cells determined by RT-qPCR analysis. (N = 5/group, normalized to U6). e Relative expression levels of miR-411-5p in PA-induced steatosis hepatocytes determined by RT-qPCR analysis. (N = 6/group, normalized to U6). f BODIPY staining of PA-induced steatosis hepatocytes. g Quantification of BODIPY staining signal intensity in PA-induced steatosis hepatocytes. (N = 3/group). h–k Relative expression levels of lipid uptake-related genes (CD36, FATP1, FATP2), lipid synthesis-related genes (ACC1, FASN, SREBP1c), lipid oxidation-related genes (ACOX1, CPT1A, PPARα) and lipid transport-related genes (APOB, APOC, APOE) in PA-induced steatosis hepatocytes determined by RT-qPCR analysis. (N = 6/group, normalized to β-actin). l Protein levels of ACC1, FASN, and SREBP1 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group, normalized to Tubulin protein). m TC levels in PA-induced steatosis hepatocytes determined by ELISA analysis. (N = 6/group). *P < 0.05, **P < 0.01, ***P < 0.001

Next, we examined expression changes in the genes involved in lipid uptake, synthesis, oxidation, and transport. MiR-411-5p inhibited the expression of CD36, fatty acid transport protein 2 (FATP2) and apolipoprotein B (APOB), but increased the expression of fatty acid transport protein 1 (FATP1) and apolipoprotein C (APOC) (Fig. 3, h, k). We found that the expression levels of three lipid synthesis-related genes, acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and SREBP1c, were all inhibited by miR-411-5p (Fig. 3, i). MiR-411-5p promoted the expression of peroxisome proliferator-activated receptor alpha (PPARα), but had no effect on the expression of acyl-CoA oxidase 1 (ACOX1) or carnitine palmitoyltransferase 1 (CPT1) (Fig. 3, j). Besides, compared with the PA + NC group, the protein levels of SREBP1, ACC1 and FASN in the PA + miR-411-5p group were decreased (Fig. 3, l). MiR-411-5p also reduced the content of TC in PA-HepG2 cells (Fig. 3, m).

EIF4G2 was a direct target of miR-411-5p

After demonstrating that miR-411-5p could reduce lipid deposition, we further explored the underlying mechanism. Using three prediction databases (TargetScan, miRDB, and miRTarBase), we found that four genes (EIF4G2, CDH2, KPNA2, SF3B3) were most likely directly regulated by miR-411-5p (Fig. 4, a). With the upregulation of miR-411-5p, only the EIF4G2 mRNA expression was inhibited (Fig. 4, b-e). In addition, the EIF4G2 protein expression was reduced by the miR-411-5p mimic (Fig. 4, f). In the livers of rats and mice, the expression of EIF4G2 in the MASLD group was higher than that in the control group (Fig. 4, g-h). Sequence alignment analysis suggested that there is a binding site between miR-411-5p and the EIF4G2 3′ untranslated region (Fig. 4, i). Importantly, miR-411-5p mimic effectively reduced the luciferase activity of EIF4G2 in the WT group, but had no effect in the MUT group (Fig. 4, j-k). Therefore, EIF4G2 is the gene directly regulated by miR-411-5p.Fig. 4 EIF4G2 was a direct target of miR-411-5p. a Three miRNA-target gene prediction databases (miRDB, TargetScan, miRTarBase) were used to identify potential miR-411-5p targets. b-e Relative expression levels of potential miR-411-5p targets (EIF4G2, CDH2, KPNA2, SF3B3) determined by RT-qPCR analysis. (N = 6/group). f Protein levels of EIF4G2 determined by western blot analysis. (N = 6/group). g Protein levels of EIF4G2 in rat liver determined by western blot analysis. (N = 5/group). h Protein levels of EIF4G2 in mouse liver determined by western blot analysis. (N = 5/group). i The binding of the EIF4G2 3′UTR with miR-411-5p. j Dual-luciferase reporter assay to evaluate the effect of miR-411-5p mimic or inhibitor on wild-type EIF4G2 3′UTR. (N = 3/group). k Dual-luciferase reporter assay to evaluate the effect of miR-411-5p mimic or inhibitor on mutant EIF4G2 3′UTR. (N = 3/group). *P < 0.05, **P < 0.01, ***P < 0.001

Down-regulation of EIF4G2 alleviated lipid deposition in hepatocytes

RT-qPCR analysis revealed that compared with the PA + NC group, the EIF4G2 mRNA levels in the PA + siEIF4G2 group decreased by more than 80% (Fig. 5, a). Moreover, the EIF4G2 protein level decreased by more than 70% under the influence of siEIF4G2 (Fig. 5, b). BODIPY staining revealed that compared with the PA + NC group, the degree of lipid deposition in the PA + siEIF4G2 group was significantly weaker (Fig. 5, c-d). In addition, the expressions of ACC1, FASN, and SREBP1 were significantly reduced under the influence of siEIF4G2 (Fig. 5, e). Knockdown of EIF4G2 also reduced the content of TC in PA-HepG2 cells (Fig. 5, f). These results demonstrate that the downregulation of EIF4G2 alleviates lipid deposition in PA-induced steatosis in hepatocytes.Fig. 5 Down-regulation of EIF4G2 alleviated lipid deposition in hepatocytes. a Relative expression levels of EIF4G2 in PA-induced steatosis hepatocytes determined by RT-qPCR analysis. (N = 6/group). b Protein levels of EIF4G2 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). c-d BODIPY staining of PA-induced steatosis hepatocytes and quantification of BODIPY staining signal intensity. (N = 3/group). e Protein levels of ACC1, FASN, and SREBP1 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). f TC levels in PA-induced steatosis hepatocytes determined by ELISA analysis. (N = 6/group). *P < 0.05, **P < 0.01, ***P < 0.001

Down-regulation of EIF4G2 alleviated lipid deposition in MASLD mice

As in vitro experiments revealed that the downregulation of EIF4G2 improved lipid deposition, we further investigated the effect of EIF4G2 in MASLD mice. We injected the virus-based AAV8 vectors carrying shEif4g2 or shNC into MASLD mice and recorded their body weight weekly (Fig. 6, a–b). Under the influence of AAV8 carrying shEif4g2, serum ALT, AST and LDL-c levels decreased significantly in the shEif4g2 group of MASLD mice compared with the shNC group (Fig. 6, c–g). In addition, compared with MASLD mice in the shNC group, the liver weight index and liver TG levels of MASLD mice in the shEif4g2 group decreased (Fig. 6, h–i). Importantly, H&E staining revealed that the number of fat vacuoles in the liver tissue of MASLD mice in the shEif4g2 group was reduced compared to that in the shNC group (Fig. 6, j, l). Oil Red O staining also showed that lipid deposition in liver tissue has improved in the shEif4g2 group (Fig. 6, k, m). Subsequent western blotting analysis showed that the EIF4G2 protein level in the livers of MASLD mice in the shEif4g2 group was reduced under the influence of AAV8 carrying shEif4g2 (Fig. 6, n). Downregulation of EIF4G2 also resulted in decreased expression of ACC1, FASN, and SREBP1 in the livers of MASLD mice (Fig. 6, o). Overall, the downregulation of EIF4G2 expression effectively reduced fatty acid synthesis and lipid deposition in the livers of MASLD mice.Fig. 6 Down-regulation of EIF4G2 alleviated lipid deposition in MASLD mice. a Schematic diagram showing the intervention of MASLD with AAV8 carrying shEif4g2 or negative control in the MASLD mouse model. b Mouse body weight curve during the intervention period. c-i Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in mice at the end of intervention. (N = 5/group). j-m H&E staining and Oil Red O staining of mouse liver tissue sections and quantitative analysis. (N = 5/group). n Protein levels of EIF4G2 in mouse liver tissue determined by western blot analysis. (N = 5/group). o Protein levels of ACC1, FASN, and SREBP1 in mouse liver tissue determined by western blot analysis. (N = 5/group). *P < 0.05, **P < 0.01, ***P < 0.001

MiR-411-5p targeted EIF4G2 to reduce FOXO3 expression to alleviate lipid deposition

Immunofluorescence staining of the liver tissue showed that the fluorescence signal of FOXO3 in the livers of MASLD mice in the shEif4g2 group was weaker than that in the livers of MASLD mice in the shNC group (Fig. 7, a). Moreover, compared with MASLD mice in the shNC group, the FOXO3 protein levels in the shEif4g2 group were reduced (Fig. 7, b). In addition, compared with the control group, the fluorescence signal and protein levels of FOXO3 in the miR-411-5p and siEIF4G2 groups were decreased (Fig. 7, c-d).Fig. 7 MiR-411-5p targeted EIF4G2 to reduce FOXO3 expression to alleviate lipid deposition. a Expression of FOXO3 in mouse liver tissue determined by immunofluorescence. b Protein levels of FOXO3 in mouse liver tissue determined by western blot analysis. (N = 5/group). c Expression of FOXO3 in PA-induced steatosis hepatocytes determined by immunofluorescence. d Protein levels of FOXO3 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). e Protein levels of FOXO3 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). f-g BODIPY staining of PA-induced steatosis hepatocytes and quantification of BODIPY staining signal intensity. (N = 3/group). h Protein levels of ACC1, FASN, and SREBP1 in PA-induced steatosis hepatocytes determined by western blot analysis. (N = 6/group). i TC levels in PA-induced steatosis hepatocytes determined by ELISA analysis. (N = 6/group). *P < 0.05, **P < 0.01, ***P < 0.001

Next, we used FOXO3-siRNA to successfully downregulate the FOXO3 expression in steatosis hepatocytes (Fig. 7, e). BODIPY staining revealed that down-regulation of FOXO3 effectively reduces lipid deposition in hepatocytes (Fig. 7, f-g). Besides, the levels of ACC1, FASN, and SREBP1 were significantly reduced when FOXO3 was downregulated (Fig. 7, h). Knockdown of FOXO3 also reduced the content of TC in PA-HepG2 cells (Fig. 7, i).

MiR-411-5p alleviated lipid deposition in the HFrD-induced MASLD mouse model

We established a MASLD mouse model using a HFrD and injected the agomir to upregulate miR-411-5p, and recorded their body weight regularly (Fig. 8, a–b). RT-qPCR results showed that miR-411-5p was successfully up-regulated in the liver of MASLD mice (Fig. 8, c). Under the influence miR-411-5p, serum ALT, AST, TC and LDL-c levels decreased significantly in the MASLD mice (Fig. 8, d–h). In addition, compared with MASLD mice in the NC group, the liver weight index and liver TG levels of MASLD mice in the miR-411-5p group decreased (Fig. 8, i–j). Importantly, H&E staining and Oil Red O staining showed that lipid deposition in liver tissue has improved in the miR-411-5p group (Fig. 8, k). Western blotting analysis showed that the EIF4G2 and FOXO3 protein level in the livers of MASLD mice in the miR-411-5p group was reduced (Fig. 8, l). Upregulation of miR-411-5p also resulted in decreased expression of ACC1, FASN, and SREBP1 in the livers of MASLD mice (Fig. 8, m).Fig. 8 MiR-411-5p alleviated lipid deposition in the HFrD-induced MASLD mouse model. a Schematic diagram showing the establishment of the MASLD and intervention. b Mouse body weight curve during the intervention period. c Relative expression levels of miR-411-5p in mouse liver determined by RT-qPCR analysis. (N = 5/group). d-j Serum ALT, AST, TC, HDL-c, LDL-c levels and liver weight index and liver TG levels in mice at the end of intervention. (N = 5/group). k H&E staining and Oil Red O staining of mouse liver tissue sections and quantitative analysis. (N = 5/group). l Protein levels of EIF4G2 or FOXO3 in mouse liver tissue determined by western blot analysis. (N = 5/group). m Protein levels of ACC1, FASN, and SREBP1 in mouse liver tissue determined by western blot analysis. (N = 5/group). *P < 0.05, **P < 0.01, ***P < 0.001

MiR-411-5p inhibited EIF4G2/FOXO3 axis to reduce fatty acid synthesis in mouse primary hepatocytes

After PA treatment, the expression of miR-411-5p in mouse primary hepatocytes decreased (Fig. 9, a). RT-qPCR results showed that miR-411-5p mimic significantly increased the expression of miR-411-5p in PA-treated mouse primary hepatocytes (Fig. 9, b). With increased expression of miR-411-5p, the EIF4G2 and FOXO3 protein levels in PA-treated mouse primary hepatocytes were reduced (Fig. 9, c). In addition, the protein levels of SREBP1, ACC1 and FASN in the PA + miR-411-5p group were significantly lower than those in the PA + NC group (Fig. 9, d). MiR-411-5p also reduced the content of TC in PA-treated mouse primary hepatocytes (Fig. 9, e).Fig. 9 MiR-411-5p inhibited EIF4G2/FOXO3 axis to reduce fatty acid synthesis in mouse primary hepatocytes. a-b Relative miR-411-5p levels in mouse primary hepatocytes determined by RT-qPCR analysis. (N = 5/group, normalized to U6). c Protein levels of EIF4G2 or FOXO3 in mouse primary hepatocytes determined by western blot analysis. (N = 5/group). d Protein levels of ACC1, FASN, and SREBP1 in mouse primary hepatocytes determined by western blot analysis. (N = 5/group). e TC levels in mouse primary hepatocytes determined by ELISA analysis. (N = 5/group). f The expressions of miR-411-5p reduces during the progression from normal liver to MASLD. The reduction of miR-411-5p leads to an increase in the expression of EIF4G2, which upregulates FOXO3 and promotes lipid synthesis in hepatocytes. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

Hepatic steatosis and excess lipid deposition are drivers of MASLD progression, and there is a need for treatments to improve these lesions [3]. Obstacles in lipid uptake, synthesis, oxidation, and transport can lead to lipid metabolism disorders and trigger MASLD lipid deposition [34]. In the current study, we successfully constructed MASLD rat and MASLD mouse models through 8-week HFHCD intervention and 9-week CDAHFD intervention, respectively. Besides, MASLD mouse model was successfully constructed after 3 weeks HFrD. We found that miR-411-5p was decreased in the livers of animals for both models when MASLD was induced. Reduction of miR-411-5p expression was closely related to the damage of MASLD liver lesions. In vitro and in vivo experiments show that the upregulation of miR-411-5p can reduce de novo fatty acid synthesis, thereby alleviating lipid deposition. Our current research demonstrated that miR-411-5p targets EIF4G2. This targeting reduced lipid synthesis and improved lipid deposition. Notably, targeting EIF4G2 inhibited FOXO3 expression and alleviated liver lipid deposition and liver injury in MASLD mice (Fig. 9, f). Our findings indicate that upregulating miR-411-5p or targeting EIF4G2 may be strategies to treat lipid deposition in MASLD.

MiR-411-5p is a conserved miRNA, and studies have suggested that it plays a regulatory role in various liver diseases, including liver fibrosis and hepatocellular carcinoma [27, 31, 35–37]. MiR-411-5p regulates the progression of hepatocellular carcinoma by targeting both karyopherin subunit alpha 2 (KPNA2) and signal transducer and activator of transcription 3 (STAT3) [27, 35]. The level of miR-411-5p in serum exosomes of MASLD patients is lower than that of healthy subjects [31]. In addition, miR-411-5p expression is downregulated during NASH, hepatocyte ballooning, and liver fibrosis [36]. Upregulation of miR-411-5p inhibits hepatic stellate cell activation to improve MASH fibrosis [31]. In addition, miR-411-5p downregulates dynamin-related protein 1 (DRP1) to inhibit PM2.5-induced hepatic stellate cell activation [37]. These findings suggest that miR-411-5p has the potential to ameliorate liver disease. Consistent with previous studies, the current study found that miR-411-5p was decreased in both MASLD models. Up-regulation of miR-411-5p reduced the expression of CD36, FATP2 and APOB, but increased the expression of FATP1 and APOC. This suggests that miR-411-5p may not alleviate lipid deposition through lipid uptake or transport. Importantly, miR-411-5p inhibited the mRNA and protein levels of ACC1, FASN and SREBP1, indicating that miR-411-5p improved lipid deposition in MASLD by reducing fatty acid synthesis. In addition, miR-411-5p also improved liver lipid deposition and liver damage in HFrD-induced MASLD mice. Thus, miR-411-5p may play a role in the early stages of MASLD, thereby preventing its progression.

In the current study, EIF4G2 was confirmed as a target gene of miR-411-5p. In pathological states, cap-dependent translation is often inhibited, resulting in EIF4G2 promoting cap-independent translation to increase protein expression [12–15]. It has been reported that EIF4G2-mediated cap-independent translation also promotes hepatocellular carcinoma progression and colon cancer liver metastasis [16, 18]. To the best of our knowledge, the role of EIF4G2 in MASLD have not yet been reported. In our study, the expression of EIF4G2 increased under palmitic acid stimulation, leading to an increase in SREBP1, ACC1, and FASN, thus promoting lipid deposition. The mRNA and protein levels of EIF4G2 were significantly reduced under the action of siEIF4G2, indicating that EIF4G2 was successfully knocked down in steatosis hepatocytes. After the knockdown of EIF4G2, the fatty acid synthesis-related protein levels were reduced, and lipid deposition was alleviated. Importantly, the downregulation of EIF4G2 also alleviated lipid deposition and liver damage in MASLD mice. Therefore, targeting EIF4G2 may be a viable approach to help control the progression of MASLD in its early stages.

EIF4G2-mediated cap-independent translation affects FOXO3 expression [24]. In our current study, knockdown of EIF4G2 inhibited the expression of FOXO3 in both in vivo and in vitro experiments. These results confirmed that EIF4G2 was able to effectively regulate the expression of FOXO3. FOXO3 is a transcription factor that regulates various cellular stress responses, such as oxidative stress [38, 39]. After treating cells with palmitic acid, the expression of FOXO3 increases [25, 40]. FOXO3 is also elevated in the livers of patients with MASLD and the livers of mice of multiple MASLD models [25]. It has been demonstrated that FOXO3 promotes lipid deposition by upregulating SREBP1 [25]. In the current study, we found in vitro and in vivo experiments that up-regulation of miR-411-5p and knockdown of EIF4G2 both resulted in reduced expression of FOXO3. These results indicated that miR-411-5p downregulates EIF4G2 to reduce FOXO3 expression. In PA-induced steatosis hepatocytes, FOXO3 knockdown also inhibited the expression of ACC1, FASN and SREBP1. These results indicate that the knockdown of FOXO3 also alleviates lipid deposition by reducing fatty acid synthesis. Therefore, EIF4G2 mediates cap-independent translation to promote FOXO3 expression, which is important for excessive lipid deposition in MASLD. Meanwhile, miR-411-5p improves lipid deposition via the EIF4G2/FOXO3 axis.

In conclusion, the strengths of this study are to use multiple animal models to prove that the expression of miR-411-5p is reduced during MASLD, and to prove that miR-411-5p reduces lipid deposition in MASLD through in vivo and in vitro experiments. Mechanistically, miR-411-5p targets EIF4G2 to downregulate the FOXO3 expression, thereby reducing the levels of SREBP1 and fatty acid synthesis to alleviate lipid deposition. EIF4G2-mediated cap-independent translation promoting FOXO3 expression may be a mechanism by which increased fatty acid synthesis leads to excessive lipid deposition during the development and progression of MASLD. This study did not explore the expression of miR-411-5p and EIF4G2 in human liver tissue. Overall, our findings further our understanding of lipid deposition and suggest that miR-411-5p has therapeutic potential to treat lipid deposition in MASLD. Furthermore, EIF4G2 may be a potential target for treating the early stages of MASLD.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 20 KB)

Acknowledgements

We thank the members of our department and laboratory for their support and technical help.

Author contributions

Zhiping Wan: Conceptualization, Investigation, Methodology, Data curation, Writing – original draft, Visualization. Xiaoquan Liu: Conceptualization, Investigation, Methodology, Data curation, Writing – original draft, Visualization. Xiaoan Yang: Conceptualization, Investigation, Methodology, Data curation, Writing – original draft, Visualization. Zexuan Huang: Methodology, Software. Xiaoman Chen: Investigation, Visualization. Qingqing Feng: Visualization, Validation. Hong Cao: Funding acquisition, Project administration, Supervision, Writing – review & editing. Hong Deng: Funding acquisition, Project administration, Supervision, Writing – review & editing.

Funding

This study was supported by National Natural Science Foundation of China (No. 81870597), and the Guangzhou Municipal Science and Technology Project (No. 202103000060).

Data availability

Data will be made available on request.

Declarations

Conflict of interest

All authors declare no conflict of interest.

Ethical approval

All animal experiments were approved by the Animal Ethics Committee of Guangzhou Forevergen Medical Experimental Animal Center (approval numbers: F2008032, F2308003 and 20240527001).

Consent for publication

All authors approved the publication of this manuscript.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zhiping Wan, Xiaoquan Liu, and Xiaoan Yang contributed equally to this work.
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