METTL3调控miR-126-5p对人肾小球系膜细胞焦亡的影响及机制研究

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

目的:研究甲基转移酶样蛋白3(METTL3)调控miR-126-5p对人肾小球系膜细胞(HRMC)焦亡的影响并探讨潜在机制。方法:将HRMC分为7组,即对照组、高糖处理组、pcD-null组、pcD-METTL3组、pcD-METTL3+miR-126-5p抑制剂(inhibitor)组、pcD-METTL3+inhibitor-NC组、pcD-METTL3+inhibitor+AKT抑制剂(AZD5363)组。用流式细胞仪检测细胞焦亡,实时荧光定量PCR(RT-qPCR)检测miR-126-5p表达,western blotting检测METTL3、Gasdermin D、NLRP3、磷酸化(p-)AKT、p-NF-κB表达。用RNA甲基化免疫沉淀技术(Me-RIP)测定miR-126-5p前体的m6A修饰水平。结果:与对照组比较较,高糖处理组细胞焦亡增多,miR-126-5p前体N6-甲基腺苷(m6A)修饰增加,p-AKT和p-NF-κB表达水平升高,METTL3和miR-126-5p表达水平降低(均P<0.05)。转染METTL3过表达载体后,高糖处理的细胞上述指标发生了显著逆转(均P<0.05)。而在过表达METTL3的基础上加入miR-126-5p inhibitor后,miR-126-5p表达下调,细胞焦亡增多,p-AKT、p-NF-κB表达上调(均P<0.05),但METTL3表达水平和miR-126-5p前体m6A修饰无显著变化(P>0.05)。在过表达METTL3的基础上加入AZD5363后,细胞焦亡减少(P<0.05),p-AKT、p-NF-κB表达下调,METTL3和miR-126-5p表达及miR-126-5p前体m6A修饰无显著改变(P>0.05)。结论:METTL3促进miR-126-5p的m6A甲基化,并通过抑制AKT/NF-κB信号通路减少高糖诱导的肾小球系膜细胞焦亡。

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-q PCR) 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/NF-κB signaling pathway.