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Published on: May 3, 2024
The METTL3/m6A Reader Protein YTHDF1 Regulates Endothelial Cell Pyroptosis by Enhancing NLRP3 Expression to Affect
1Department of Orthopedics, Xiangtan Central Hospital, Xiangtan, 411100, People's Republic of China.
Background:
Pyroptosis is inflammation-associated programmed cell death triggered by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, which plays a crucial role in acute soft tissue injury (ASTI). This study aimed to explore whether methyltransferase-like 3 (METTL3) can regulate NLRP3 expression through N6-methyladenosine (m6A) modification to mediate endothelial cell pyroptosis and thus affect soft tissue injury.
Methods:
An experimental ASTI rat model was created by inducing muscle injury through striking the rat muscle. In vitro, an ASTI cell model was established using human umbilical vein endothelial cells (HUVECs) stimulated with lipopolysaccharide (LPS) and ATP. The severity of ASTI in rats was evaluated using H&E staining. To assess protein levels, Western blot and Immunohistochemistry (IHC) analyses were performed, focusing on METTL3, pyroptosis-associated proteins, and m6A reader proteins. Immunofluorescence (IF) assay was conducted to examine the expression of NLRP3 and CD31. The levels of inflammatory cytokines were measured using an ELISA assay, while flow cytometry was used to detect levels of ROS and cellular pyroptosis. The m6A levels in cells were analyzed by RNA m6A colorimetry. The interactions between METTL3 and NLRP3, and YTHDF1 and NLRP3 were analyzed using RIP and RNA pull-down assays, respectively.
Results:
METTL3 and YTHDF1 were significantly upregulated in ASTI rats and LPS-ATP-induced HUVECs. Knockdown of METTL3 ameliorated ASTI and inhibited cellular pyroptosis. Knockdown of METTL3 reduced the levels of total m6A and NLRP3 m6A in HUVECs and suppressed NLRP3 expression. Meanwhile, knockdown of YTHDF1 decreased NLRP3 protein expression without affecting NLRP3 mRNA levels. In addition, overexpression of NLRP3 was able to reverse the effect of METTL3 on LPS-ATP-induced endothelial cell pyroptosis.
Conclusion:
The METTL3/m6A reader protein YTHDF1 regulates endothelial cell pyroptosis by enhancing NLRP3 expression to affect soft tissue injury.
Insights
Methyltransferase-like 3 (METTL3) regulates N6-methyladenosine (m6A) modification of NLRP3, promoting pyroptosis in endothelial cells and exacerbating acute soft tissue injury (ASTI). Inhibiting METTL3 ameliorates ASTI by reducing pyroptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Inflammation Research
Background:
- Pyroptosis, an inflammation-associated programmed cell death, is crucial in acute soft tissue injury (ASTI).
- The NOD-like receptor protein 3 (NLRP3) inflammasome activation is a key driver of pyroptosis in ASTI.
- Understanding the regulatory mechanisms of NLRP3 in ASTI is critical for developing therapeutic strategies.
Purpose of the Study:
- To investigate the role of methyltransferase-like 3 (METTL3) in regulating NLRP3 expression via N6-methyladenosine (m6A) modification.
- To determine if METTL3-mediated pyroptosis in endothelial cells contributes to ASTI.
- To elucidate the molecular pathway involving METTL3, m6A, and NLRP3 in the context of soft tissue injury.
Main Methods:
- Establishment of experimental acute soft tissue injury (ASTI) rat and human umbilical vein endothelial cell (HUVEC) models.
- Assessment of ASTI severity, protein and cytokine levels, ROS, and pyroptosis using H&E staining, Western blot, IHC, ELISA, and flow cytometry.
- Analysis of m6A modification, and molecular interactions using RNA m6A colorimetry, RIP, and RNA pull-down assays.
Main Results:
- METTL3 and YTHDF1 were upregulated in ASTI models.
- METTL3 knockdown ameliorated ASTI and inhibited endothelial cell pyroptosis by reducing m6A modification of NLRP3 and suppressing its expression.
- YTHDF1 knockdown decreased NLRP3 protein levels, and NLRP3 overexpression reversed METTL3's effect on pyroptosis.
Conclusions:
- METTL3, through m6A modification of NLRP3 via the reader protein YTHDF1, promotes endothelial cell pyroptosis.
- This METTL3/m6A/NLRP3 pathway plays a significant role in the pathogenesis of acute soft tissue injury.
- Targeting METTL3 may offer a therapeutic approach for managing ASTI.
