Effect and mechanism of METTL3 regulation of miR-126-5p on pyroptosis of human renal mesangial cells

Objective: To investigate the effect of methyltransferase-like protein 3 (METTL3) on pyroptosis of human renal mesangial cells (HRMC) by regulating miR-126-5p and explore the underlying mechanisms. Methods: HRMC was divided into 7 groups. Namely, control group, high-glucose treatment group, pcD-null group, pcD-METTL3 group, pcD-METTL3+miR-126-5p inhibitor (inhibitor) group, pcD-METTL3+inhibitor-NC group, and pcD-METTL3+inhibitor+AKT inhibition preparation (AZD5363) group. Cell pyroptosis was detected using flow cytometry, reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression of miR-126-5p, and western blotting was used to detect the expression of METTL3, Gasdermin D, NLRP3, phosphorylated (p-) AKT, and p-NF-κB. RNA methylation immunoprecipitation (Me-RIP) was used to determine the N6-methyladenosine (m6A) modification level of miR-126-5p precursor. Results: Compared with the control group, cell pyroptosis, m6A modification of miR-126-5p precursor, as well as the expression levels of p-Akt and P-NF-κB were increased, while METTL3 and miR-126-5p expression levels were decreased in the high-glucose treatment group (all P< 0.05). After transfecting METTL3 overexpression vector, the above indicators were significantly reversed in the cells treated with high glucose (all P< 0.05). When the inhibitor of miR-126-5p was added after overexpressing METTL3, the expression of miR-126-5p was down-regulated, cell pyroptosis was increased, and the expression of p-AKT and p-NF-κB was up-regulated (all P< 0.05), but the expression levels of METTL3 and m6A modification of miR-126-5p precursor had no significant changes (P> 0.05). Adding AKT inhibitor AZD5363 after overexpressing METTL3 resulted in reduced cell pyroptosis (P< 0.05), and down-regulated expression of p-AKT and p-NF-κB, but the expression of METTL3 and miR-126-5p, as well as the m6A modification of miR-126-5p precursor had no significant changes (P> 0.05). Conclusion: METTL3 promotes m6A methylation of miR-126-5p and reduces high glucose-induced pyroptosis of HRMC by inhibiting the AKT/NFκB signaling pathway.