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METTL3-Mediated m6A Modification of lncRNA-0949 Drives Microglial Inflammation in an vitro Model of Sepsis-Associated
Xing Zeng1, Xiao-Juan Luo1, Zhen-Ze Zhang1
1Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 330006, People's Republic of China.
Introduction:
Sepsis-associated encephalopathy (SAE) is a severe complication of sepsis with limited therapeutic options. Although neuroinflammation driven by microglial activation is central to SAE pathogenesis, the underlying epitranscriptional regulatory mechanisms remain poorly defined. Here, we investigated the role of the m6A methyltransferase METTL3 in regulating microglial inflammation using lipopolysaccharide (LPS)-stimulated HMO6 microglial cells as an in vitro SAE model.
Methods:
HMO6 cells were stimulated with LPS (1 μg/mL) for 0-24 h to establish an SAE model. METTL3 expression was assessed by Western blotting and immunofluorescence. MeRIP-qPCR was used to detect m6A deposition on lncRNA-0949. METTL3 was inhibited pharmacologically (3-DAA) or by siRNA knockdown. lncRNA-0949 stability was evaluated by actinomycin D chase assay. Luciferase reporters containing wild-type or m6A site-mutated lncRNA-0949 3'UTR were constructed to identify functional m6A sites. Wild-type and m6A site-mutated lncRNA-0949 overexpression vectors were employed to assess modification-dependent pro-inflammatory function. Cytokine mRNA (qRT-PCR) and protein (ELISA) levels were measured.
Results:
LPS stimulation time-dependently increased oxidative stress (MDA), pro-inflammatory cytokines (MIP-2, IL-1β, TNF-α, IL-6), and METTL3 protein expression (2.99-fold at 24 h, P < 0.001). METTL3 catalyzed m6A deposition on lncRNA-0949, with enrichment reaching 21.3-fold at 24 h post-LPS (P < 0.001). METTL3 knockdown abolished this modification and reduced lncRNA-0949 stability, decreasing its half-life from ~30 h to 11 h (P < 0.001). The m6A site within the 3'UTR (RRACH motif) was essential for LPS-induced reporter activity (7.7-fold increase for WT vs. no response for Mut, P < 0.001). Overexpression of wild-type lncRNA-0949 amplified LPS-triggered cytokine release (eg, TNF-α increased by additional 33%, P < 0.001), whereas the m6A site mutant had no effect. Conversely, METTL3 knockdown attenuated LPS-induced inflammatory responses, with mRNA levels reduced by 35-58% and protein levels by 30-63% (all P < 0.001).
Conclusion:
Together, these findings define a METTL3-m6A-lncRNA-0949 regulatory axis that amplifies microglial inflammation in an in vitro SAE model. This study provides the first evidence that METTL3-driven m6A modification of lncRNA-0949 contributes to neuroinflammation, offering a new mechanistic perspective and highlighting the need for in vivo validation to assess therapeutic potential.
Insights
The m6A methyltransferase METTL3 amplifies microglial inflammation in sepsis-associated encephalopathy (SAE) by modifying lncRNA-0949. This METTL3-m6A-lncRNA-0949 axis offers new therapeutic targets for SAE neuroinflammation.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Sepsis-associated encephalopathy (SAE) is a severe complication of sepsis with limited treatment options.
- Neuroinflammation, driven by microglial activation, is central to SAE pathogenesis.
- Epitranscriptional regulation of microglial inflammation in SAE is not well understood.
Purpose of the Study:
- To investigate the role of the m6A methyltransferase METTL3 in regulating microglial inflammation in an in vitro SAE model.
- To elucidate the epitranscriptional mechanisms underlying neuroinflammation in SAE.
Main Methods:
- Established an in vitro SAE model using lipopolysaccharide (LPS)-stimulated HMO6 microglial cells.
- Assessed METTL3 expression and m6A deposition on lncRNA-0949 using Western blotting, immunofluorescence, and MeRIP-qPCR.
- Investigated the functional role of METTL3 and lncRNA-0949 in inflammation via pharmacological inhibition, siRNA knockdown, and overexpression studies.
Main Results:
- LPS stimulation increased oxidative stress, pro-inflammatory cytokines, and METTL3 protein expression.
- METTL3 catalyzed m6A deposition on lncRNA-0949, enhancing its stability and promoting cytokine release.
- METTL3 knockdown or lncRNA-0949 m6A site mutation attenuated LPS-induced inflammatory responses.
Conclusions:
- A METTL3-m6A-lncRNA-0949 regulatory axis amplifies microglial inflammation in an in vitro SAE model.
- METTL3-driven m6A modification of lncRNA-0949 contributes to neuroinflammation in SAE.
- This pathway presents a novel mechanistic insight and potential therapeutic target for SAE.
