METTL3-Mediated N6-Methyladenosine Modification of Trim59 mRNA Protects Against Sepsis-Induced Acute Respiratory Distress Syndrome

Abstract

N6-methyladenosine (m⁶A) RNA modification is a fundamental determinant of mRNA metabolism in eukaryotic cells and is involved in numerous physiological and pathological processes. However, the specific role of m⁶A modification in sepsis-induced acute respiratory distress syndrome(ARDS) remains unknown. Here, we show that the levels of m⁶A RNA were significantly decreased in septic lungs and that METTL3 was the main regulator involved in the absence of m⁶A RNA modification. Pulmonary endothelial barrier damage is a critical process in the pathogenesis of acute lung injury during sepsis. METTL3 regulated endothelial barrier dysfunction and inflammatory responses in sepsis-induced ARDS in vivo and in vitro. Furthermore, we identified tripartite motif-containing (Trim)59 as a key m⁶A effector and Trim59 deficiency exacerbated lung injury. Mechanistically, METTL3 inhibited endothelial injury in sepsis-induced ARDS through Trim59-associated NF-κB inactivation. Our findings revealed novel insights into epitranscriptional mechanisms in sepsis-induced ARDS via m⁶A modifications, which has important application value in the diagnosis, prognosis, and molecular-targeted therapy of sepsis-associated lung injury.

Keywords: METTL3; acute lung injury; endothelial barrier; epigenetic regulation; m6A; sepsis.

Figure 1

The METTL3-m⁶A pathway is downregulated in sepsis-induced ARDS. (A) ELISA was used to measure mRNA m⁶A levels in sham and CLP-24 h lungs. (B, C) RT-qPCR analysis showing the mRNA expression of m⁶A-related genes in mouse lungs and PMVECs. 18S rRNA was used as the internal control. (D, E) Immunoblot showing METTL3 protein expression in mouse lungs and PMVECs. GAPDH was used as the loading control. (F) Representative images of METTL3 immunostaining in sham lungs and CLP lungs. Scale bars, 100 mm. All data are expressed as the mean ± SD of three independent experiments (n=6). *p < 0.05; **p<0.01; ***p < 0.001; ****p < 0.0001; ns, no significance.

Figure 2

METTL3 deletion exacerbates lung endothelial injury in CLP models. (A) Hematoxylin and eosin (H&E)-stained lung sections from sham, CLP-NC siRNA and CLP-METTL3 siRNA mice. Scale bars, 100 μm. (B) Pathological lung injury scores of sham, CLP-NC siRNA and CLP-METTL3 siRNA mice. (C–E) Protein content (C), cell counts (D) and MPO activity (E) in the BALF of CLP-METTL3 siRNA mice compared with sham and CLP-NC siRNA mice. (F) Whole lung images and Evans blue levels in sham, CLP-NC siRNA and CLP-METTL3 siRNA mice. (G) Quantibody Mouse Inflammation Array analysis of circulating blood from sham, CLP-NC siRNA and CLP-METTL3 siRNA mice. (H) Survival analysis of CLP-METTL3 siRNA mice compared with sham and CLP-NC siRNA mice. All data are expressed as the mean ± SD of three independent experiments (n=6 for A-G and n=15 for H). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

METTL3 regulates vascular endothelial barrier function in vitro. (A, B) TEER was used to measure endothelial permeability in HULEC-5a cells transfected with scramble or METTL3 shRNA (A) and in HULEC-5a cells with or without METTL3 overexpression (B). (C, D) Immunoblot showing the protein expression of VE-Cadherin and ICAM-1 in HULEC-5a cells transfected with scramble or METTL3 shRNA (C) and in HULEC-5a cells with or without METTL3 overexpression (D). (E, F) RT-qPCR analysis showing inflammatory cytokine mRNA expression in HULEC-5a cells transfected with scramble or METTL3 shRNA (E) and in HULEC-5a cells with or without METTL3 overexpression (F). All data are expressed as the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

METTL3-mediated m⁶A modification of Trim59 mRNA maintains its YTHDF1-dependent stability (A) Heatmap showing the clusters with significant changes in both the RNA expression level (RNA-seq) and m⁶A level (MeRIP-seq) in scramble and METTL3 shRNA-treated HULEC-5a cells. (B) Number of m⁶A peaks in scramble and METTL3 shRNA-treated HULEC-5a cells. (C) Global profiling of m6A in HULEC-5a cells and the sequence motif identified from the top 1000 m⁶A peaks. (D) MeRIP-seq plotted the m⁶A abundances on Trim59 mRNA transcripts in HULEC-5a cells. (E) MeRIP-qPCR examined METTL3-mediated Trim59 mRNA m⁶A modifications. (F) RT-qPCR analysis showing the levels of Trim59 expression in METTL3-downregulated HULEC-5a cells treated with actinomycin D (2 μg/mL) at the indicated time points. (G) RNA immunoprecipitation (RIP)-qPCR assay showing the enrichment of YTHDF1 binding to Trim59 m⁶A modification sites. (H) RT-qPCR analysis showing the levels of Trim59 expression in YTHDF1-knockdown HULEC-5a cells. All data are expressed as the mean ± SD of three independent experiments. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 5

METTL3 regulates endothelial function by targeting Trim59. (A) RT-qPCR analysis showing the levels of Trim59 expression after siRNA transfection. (B) Immunoblot showing Trim59 protein expression after siRNA transfection. (C) TEER was used to measure endothelial permeability in METTL3-overexpressing HULEC-5a cells with or without Trim59 siRNA. (D) Immunoblot showing the protein expression of VE-Cadherin and ICAM-1 in METTL3-overexpressing HULEC-5a cells with or without Trim59 siRNA. (E) RT-qPCR analysis showing inflammatory cytokine mRNA expression in METTL3-overexpressing HULEC-5a cells with or without Trim59 siRNA. All data are expressed as the mean ± SD of three independent experiment. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

METTL3 inhibits endothelial injury in sepsis-induced ARDS through Trim59-associated NF-κB inactivation. (A, C) Immunoblot showing p65 and IκB phosphorylation in HULEC-5a cells treated with or without METTL3 overexpression (A) and METTL3-overexpressing HULEC-5a cells with or without Trim59 siRNA (C). (B) RT-qPCR analysis showing inflammatory cytokine and endothelial functional marker mRNA expression in METTL3-overexpressing HULEC-5a cells treated with or without the NF-κB inhibitor BAY. All data are expressed as the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001.