METTL3-m6A-Rubicon axis inhibits autophagy in nonalcoholic fatty liver disease

Abstract

N6-methyladenosine (m⁶A) mRNA modification plays critical roles in various biological events and is involved in multiple complex diseases. However, the role of m⁶A modification in autophagy in nonalcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we report that m⁶A modification was increased in livers of NAFLD mouse models and in free fatty acid (FFA)-treated hepatocytes, and the abnormal m⁶A modification was attributed to the upregulation of methyltransferase like 3 (METTL3) induced by lipotoxicity. Knockdown of METTL3 promoted hepatic autophagic flux and clearance of lipid droplets (LDs), while overexpression of METTL3 inhibited these processes. Mechanistically, METTL3 directly bound to Rubicon mRNA and mediated the m⁶A modification, while YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), as a partner of METTL3, interacted with the m⁶A-marked Rubicon mRNA and promoted its stability. Subsequently, RUBICON inhibited autophagosome-lysosome fusion and further blocked clearance of LDs. Taken together, our results showed a critical role of METTL3 and YTHDF1 in regulating lipid metabolism via the autophagy pathway and provided a novel insight into m⁶A mRNA methylation in NAFLD.

Keywords: METTL3; NAFLD; YTHDF1; autophagy; m(6)A modification.

Graphical abstract

Figure 1

Lipotoxicity upregulates m⁶A mRNA modification in vivo and in vitro (A) Representative macroscopic images of liver morphology (scale bars, 5 mm). (B) Representative microscopic images of liver sections stained with oil red O and H&E (scale bars, 20 μm). (C) Liver weights of each group (n = 6). (D) Liver index was calculated as the ratio of liver weight to body weight (%) (n = 6). (E and F) Liver TG and TC content of each group (n = 6). (G) m⁶A mRNA dot blot assays of mouse livers of CD or HFD group. Methylene blue staining served as a loading control. (H) HepG2 and Hepa1-6 cells were treated with BSA or FFAs for 24 h, and then the deposition of LDs was evaluated by oil red O staining (scale bars, 20 μm). (I–L) TG and TC content of HepG2 cells (I and J) and Hepa1-6 cells (K and L) in each group. (M and N) m⁶A mRNA dot blot assays of HepG2 (M) and Hepa1-6 cells (N) treated with BSA or FFAs for 24 h. Methylene blue staining served as a loading control. The data in (C)–(F) and (I)–(L) are presented as the means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. All experiments were repeated at least three times. CD, standard chow diet; HFD, high-fat diet; H&E, hematoxylin-eosin; LDs, lipid droplets; TG, triglyceride; TC, total cholesterol; BSA, bovine serum albumin; FFAs, free fatty acids.

Figure 2

METTL3 and YTHDF1 contribute to m⁶A modification in NAFLD (A and B) qRT-PCR analysis of the mRNA expression levels of m⁶A methylation-associated genes in mouse livers (n = 8) (A) and in HepG2 cells (n = 3) (B) of each group; Gapdh was used as an internal control. (C and D) Western blotting (C) and quantitative analysis (D) of the protein expression levels of METTL3, FTO, and YTHDF1 in livers from each group; GAPDH was used as a loading control (n = 6). (E and F) The protein expression levels of METTL3, FTO, and YTHDF1 in HepG2 cells of each group were determined by western blot (E) and quantification (F). (G and H) The overexpression levels of METTL3 in HepG2 cells were determined by western blot (G) and quantification (H). (I) m⁶A mRNA dot blot assays in control and METTL3-overexpressing HepG2 cells. (J) m⁶A mRNA dot blot assays in control and METTL3-silencing HepG2 cells treated with FFAs for 24 h. Methylene blue staining served as a loading control. The data in (A), (B), (D), (F) and (H) are presented as the means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. All experiments were repeated at least three times. OE-METTL3, overexpression of METTL3; siMETTL3, knockdown of METTL3 with siRNA.

Figure 3

Lipotoxicity suppresses hepatic autophagy and lipid metabolism (A and B) Western blotting and quantitative analysis of the protein expression levels of LC3B and p62 in livers of CD or HFD mice (n = 6). (C) TEM analysis of autophagosomes and lysosomes in CD and HFD mouse livers (n = 6). Red and blue arrows indicate autophagosomes and lysosomes, respectively. Scale bars, 5 μm (left panel) and 1 μm (right panel). (D) Quantification of autophagosomes in liver tissues by TEM. (E–H) Western blotting analysis and quantification of the levels of LC3B and p62 in HepG2 cells (E and F) and Hepa1-6 cells (G and H) treated with BSA or FFAs for 24 h, which were incubated with or without 100 nM Baf A1 for 5 h. (I) HepG2 cells were infected with recombinant adenovirus expressing tandem fluorescent mCherry-GFP-LC3B for 24 h and then subjected to BSA or FFAs for an additional 24 h. The fluorescence was detected with a confocal microscope (scale bars, 10 μm). Microscopic images show red fluorescent autolysosomes or yellow fluorescent autophagosomes in merged images. (J) Semiquantitative analysis of autolysosomes (red puncta) and autophagosomes (yellow puncta) in the cells (n = 30). (K) LD clearance assays in Hepa1-6 cells. The cells were treated with DMEM, EBSS for 2 h, Rap (1 μmol/L, 5 h), Baf A1 (100 nmol/L, 5 h), or 3-MA (5 mmol/L, 5 h) and then cultured in either BSA or FFAs for 24 h. Images of cells were observed by fluorescence microscopy (scale bars, 10 μm). Green, lipid probes BODIPY 493/503; blue, DAPI. (L) Semi-quantification of BODIPY 493/503 fluorescence intensity in the LD clearance assays. The data in (B), (D), (F), (H), (J), and (L) are presented as the means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. All experiments were repeated at least three times. TEM, transmission electron microscopy; DMEM, Dulbecco’s modified Eagle’s medium; EBSS, Earle’s balanced salt solution; Rap, rapamycin; Baf A1, bafilomycin A1; 3-MA, 3-methyladenine.

Figure 4

METTL3 suppresses autophagic flux and accelerates accumulation of LDs (A–D) Western blotting and quantitative analysis of the protein expression levels of METTL3, LC3B, and p62 in METTL3-silencing (A and B) and METTL3-overexpressing HepG2 cells (C and D), with or without 100 nM Baf A1 for 5 h. (E–H) METTL3-overexpressing AML-12 (F) and HepG2 (G) cells were infected with adenovirus expressing mCherry-GFP-LC3B for 48 h. The fluorescence of autolysosomes (red puncta) and autophagosomes (yellow puncta) was detected with confocal microscopy (scale bars, 10 μm) and were semi-quantified in the cells (n = 30) (E and H). (I–L) LD clearance assays in METTL3-silencing and -overexpressing HepG2 cells that were treated with Baf A1 (100 nmol/L, 5 h), 3-MA (5 mmol/L, 5 h), EBSS for 2 h, or Rap (1 μmol/L, 5 h) and then cultured in FFAs for 24 h. Images of cells were observed by fluorescence microscopy (scale bars, 10 μm). Green, lipid probes BODIPY 493/503; blue, DAPI. Semi-quantification of BODIPY 493/503 fluorescence intensity in the LD clearance assays is shown in (J) and (L). The data in (B), (D), (F), (H), (J), and (L) are presented as the means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. All experiments were repeated at least three times.

Figure 5

METTL3-mediated m⁶A modification regulates Rubicon expression by targeting its mRNA (A) qRT-PCR analysis of the mRNA expression levels of genes involved in the process of autophagosome-lysosomes fusion in livers of each group (n = 8). (B and C) The protein level of RUBICON in mouse livers of each group was determined by western blot and quantitative analysis (n = 6). (D–F) qRT-PCR, western blotting, and quantitative analysis of the expression level of Rubicon in METTL3-silencing HepG2 cells. (G–J) RIP-qPCR analysis showed the enrichment of METTL3 on Rubicon mRNA and MeRIP-qPCR analysis detected the m⁶A modification levels on the mRNA in mouse livers (n = 6) (G and H) and METTL3-overexpressing Hepa1-6 cells (n = 3) (I and J). The data were normalized to input. (K–M) qRT-PCR, western blotting, and quantitative analysis of the expression levels of Rubicon in HepG2 cells transfected with vector, wild-type METTL3, or catalytic mutant METTL3 plasmid. (N) Rescue assays in HepG2 cells. Western blotting analysis of the levels of METTL3, RUBICON, LC3B, and p62 in control and METTL3-silencing cells followed by being transfected with or without RUBICON-overexpressing plasmid. (O) Quantitative analysis of the levels of METTL3, RUBICON, LC3B, and p62 in the rescue assays. The data in (A), (C), (D), (F)–(K), (M), and (O) are presented as the means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant. All experiments were repeated at least three times. METTL3-WT, wild-type METTL3; METTL3-Mut, catalytic mutant METTL3; OE-RUBICON, overexpression of RUBICON; RIP-qPCR, RNA immunoprecipitation combined with qRT-PCR; MeRIP-qPCR, methylated RNA immunoprecipitation combined with qRT-PCR.

Figure 6

YTHDF1 promoting Rubicon mRNA stability contributes to the blockade of autophagic flux and accumulation of LDs (A and B) Rescue assays in HepG2 cells. Western blotting and quantitative analysis of the levels of METTL3, YTHDF1, LC3B, and p62 in control and METTL3-silencing cells transfected with or without YTHDF1-expressing plasmid. (C and D) The effect of YTHDF1 on autophagic flux in HepG2 cells. The activity of autophagic flux in YTHDF1-overexpressing HepG2 cells was detected using tandem fluorescence mCherry-GFP-LC3B and confocal microscope (scale bars, 10 μm), and the amount of autolysosomes (red puncta) and autophagosomes (yellow puncta) were semi-quantified in the cells (n = 30). (E and F) The levels of YTHDF1, RUBICON, LC3B, and p62 in control or YTHDF1-overexpressing HepG2 cells were determined by western blot and quantification. (G) Confirmation of YTHDF1 binding to Rubicon mRNA. RIP-qPCR using YTHDF1 antibody analyzed the enrichment of YTHDF1 upon the mRNA of Rubicon in Hepa1-6 cells. The data were normalized to input. (H and I) mRNA stability assays showed the lifetime of Rubicon mRNA in YTHDF1-silencing (H) and -overexpressing (I) HepG2 cells. (J–M) LD clearance assay in YTHDF1-silencing and -overexpressing HepG2 cells transfected with or without RUBICON-expressing plasmid (J) and siRNA targeted to Rubicon mRNA (L), respectively. Images of cells were observed by fluorescence microscopy (scale bars, 10 μm). Additionally, the fluorescence intensity of BODIPY 493/503 in the assay was semi-quantified (K and M). The data in (B), (D), (F)–(I), (K), and (M) are presented as the means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. All experiments were repeated at least three times. siRUBICON, knockdown of RUBICON with siRNA; OE-YTHDF1, overexpression of YTHDF1; shYTHDF1, knockdown YTHDF1 with shRNA.