METTL3-mediated N6-methyladenosine modification governs pericyte dysfunction during diabetes-induced retinal vascular
Long Suo1,2,3, Chang Liu2,3, Qiu-Yang Zhang2,3
1Eye Institute, Eye & ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, China.
Abstract:
Rationale: Microvascular complication is a major cause of morbidity and mortality among the patients with diabetes. Pericyte dysfunction is the predominant pathological manifestation of microvascular complication. N6-methyladenosine (m6A) serves as the most prevalent modification in eukaryotic mRNAs. However, the role of m6A RNA modification in pericyte dysfunction is still unclear. Methods: Quantitative polymerase chain reactions and western blots were conducted to detect the change of m6A RNA modification in pericytes and mouse retinas following diabetic stress. MTT assay, transwell migration assay, caspase 3/7 activity assay, calcein-AM/propidium iodide (PI) staining, and TUNEL staining were conducted to determine the role of METTL3 in pericyte biology in vitro. Retinal trypsin digestion, vascular permeability assay, and IB4-NG2 double immunofluorescent staining were conducted to determine the role of METTL3 in retinal pericyte dysfunction and vascular complication. RNA sequencing, RNA pull-down assays and immunoblots were conducted to clarify the mechanism of METTL3-mediated pericyte dysfunction and vascular complication. Results: The levels of m6A RNA methylation were significantly up-regulated in pericytes and mouse retinas following diabetic stress, which were caused by increased expression of METTL3. METTL3 regulated the viability, proliferation, and differentiation of pericytes in vitro. Specific depletion of METTL3 in pericytes suppressed diabetes-induced pericyte dysfunction and vascular complication in vivo. METTL3 overexpression impaired pericyte function by repressing PKC-η, FAT4, and PDGFRA expression, which was mediated by YTHDF2-dependent mRNA decay. Conclusion: METTL3-mediated m6A methylation epigenetically regulates diabetes-induced pericyte dysfunction. METTL3-YTHDF2-PKC-η/FAT4/PDGFRA signaling axis could be therapeutically targeted for treating microvascular complications.
Insights
Diabetic microvascular complications involve pericyte dysfunction. METTL3-mediated m6A RNA methylation epigenetically regulates this dysfunction, offering a potential therapeutic target.
Area of Science:
- Biochemistry
- Molecular Biology
- Diabetology
Background:
- Microvascular complications are a leading cause of death in diabetic patients.
- Pericyte dysfunction is a key pathological feature of diabetic microvascular complications.
- The role of N6-methyladenosine (m6A) RNA modification in pericyte dysfunction remains largely unknown.
Purpose of the Study:
- To investigate the role of m6A RNA modification and METTL3 in diabetic pericyte dysfunction.
- To elucidate the underlying molecular mechanisms of METTL3-mediated pericyte dysfunction.
Main Methods:
- Quantitative PCR and Western blots to assess m6A levels and METTL3 expression.
- In vitro assays (MTT, Transwell, caspase activity, cell viability staining) to evaluate pericyte function.
- In vivo studies (retinal trypsin digestion, vascular permeability, immunofluorescence) to assess retinal pericyte dysfunction and vascular complications.
- RNA sequencing, RNA pull-down, and immunoblotting to identify molecular targets.
Main Results:
- m6A RNA methylation and METTL3 expression were significantly upregulated in diabetic pericytes and retinas.
- METTL3 depletion in pericytes ameliorated diabetes-induced pericyte dysfunction and vascular complications.
- METTL3 overexpression impaired pericyte function by repressing PKC-η, FAT4, and PDGFRA via YTHDF2-dependent mRNA decay.
Conclusions:
- METTL3-mediated m6A methylation epigenetically regulates diabetes-induced pericyte dysfunction.
- The METTL3-YTHDF2-PKC-η/FAT4/PDGFRA signaling pathway represents a potential therapeutic target for diabetic microvascular complications.
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