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METTL3 modification of circStk4 affects mouse glomerular messangial cell autophagy, proliferation and apotosis by
Xiujuan Qin1, Huiyu Chen2, Wenjia Zheng2
1Department of Pharmacy, The First Affiliated Hospital of Anhui University of Chinese Medicine, 117 Meishan Road, Hefei, China; Anhui Province Key Laboratory of Chinese Medicinal Formula, Hefei, Anhui 230012, China.
Objective:
The study aimed to explore the impact of N6-methyladenosine (m6A) modification in circStk4 on glomerular mesangial cells (GMCs) autophagy, proliferation and apoptosis.
Methods:
The interactions between circStk4 and miR-133a-3p, miR-133a-3p and C1 were demonstrated through luciferase reporter assays. The circStk4 localization was analyzed using fluorescence in situ hybridization and nuclear/cytosol fractionation assays. Colorimetric assays, MeRIP-qPCR, and western blot (WB) were employed to confirm the m6A modification of circStk4 and identify the key methylation enzyme. RT-qPCR was conducted to determine the impact of METTL3 on the circStk4 RNA expression. Additionally, CCK-8, flow cytometry, transmission electron microscopy, immunofluorescence, WB and RT-qPCR were employed to investigate the effects of METTL3 or circStk4 on the proliferation, autophagy and apoptosis of GMCs. Enzyme-linked immunosorbent assay was utilized to assess the inflammatory factors.
Results:
m6A modifications were found in circStk4 and METTL3 was a key methylating enzyme. Furthermore, it was observed that circStk4 competitively bound miR-133a-3p and increased C1 levels. Silencing circStk4 resulted in decreased GMCs proliferation, increased autophagy and apoptosis, and reduced inflammation levels. Additionally, METTL3 played a role in inhibiting GMCs proliferation and promoting autophagy and apoptosis by regulating the circStk4 expression. On verifying the interplay between autophagy, proliferation and apoptosis, and found that the inhibition of autophagy led to an increase in cell proliferation and a decrease in apoptosis.
Conclusion:
m6A modification of circStk4 mediated by METTL3 influenced circStk4 expression and impacted autophagy, proliferation and apoptosis in GMCs via the miR-133a-3p/C1 axis. This discovery introduces a novel therapeutic approach for CGN treatment.
Insights
N6-methyladenosine (m6A) modification of circStk4 by METTL3 impacts glomerular mesangial cell autophagy, proliferation, and apoptosis. This pathway offers a new therapeutic strategy for chronic glomerulonephritis (CGN).
Area of Science:
- Molecular Biology
- Cell Biology
- Epigenetics
Background:
- N6-methyladenosine (m6A) is the most prevalent internal mRNA modification.
- Circular RNAs (circRNAs) play crucial roles in various biological processes.
- Dysregulation of circRNAs is implicated in kidney diseases.
Purpose of the Study:
- To investigate the role of m6A modification in circStk4.
- To elucidate the impact of circStk4 on glomerular mesangial cell (GMC) functions.
- To explore the therapeutic potential for chronic glomerulonephritis (CGN).
Main Methods:
- Luciferase reporter assays to confirm molecular interactions.
- MeRIP-qPCR and Western blot to analyze m6A modification and protein expression.
- Cell proliferation, autophagy, and apoptosis assays (CCK-8, flow cytometry, TEM, immunofluorescence).
Main Results:
- METTL3 mediates m6A modification of circStk4, influencing its expression.
- circStk4 interacts with miR-133a-3p, affecting C1 levels.
- Silencing circStk4 or METTL3 modulates GMC proliferation, autophagy, and apoptosis.
Conclusions:
- m6A modification of circStk4 by METTL3 regulates GMC autophagy, proliferation, and apoptosis via the miR-133a-3p/C1 axis.
- This mechanism presents a potential therapeutic target for CGN.
- Interplay between autophagy, proliferation, and apoptosis is critical in GMC function.
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