Mettl3介导m6A甲基化修饰对过氧化氢诱导的脊髓神经元氧化应激和凋亡的影响

Effects of Mettl3-mediated m6A methylation modification on oxidative stress and apoptosis induced by hydrogen peroxide in spinal cord neurons

中文关键词:  脊髓损伤  m6A甲基化修饰  Mettl3  细胞凋亡  氧化应激

英文关键词:Spinal cord injury  m6A modification  Mettl3  Apoptosis  Oxidative stress

中文摘要:

  【摘要】 目的：探讨Mettl3介导m6A甲基化修饰对过氧化氢（H2O2）诱导的脊髓神经元氧化应激和凋亡的影响。方法：分离新生（24h内）SD大鼠脊髓，经消化、离心后重悬沉淀，差速贴壁30min后收集未贴壁细胞培养于Neurobasal培养基中，经免疫荧光鉴定呈β3-tubulin阳性，确认为脊髓神经元。将脊髓神经元随机分为6组：空白组，细胞不予任何处理，正常培养；转染对照组，细胞转染阴性对照siRNA（si-NC）；转染组，细胞转染Mettl3 siRNA（si-Mettl3）；诱导组，细胞采用300μmol/L的H2O2处理；转染对照诱导组，细胞先转染si-NC，然后采用300μmol/L H2O2处理；转染诱导组，细胞先转染si-Mettl3，然后采用300μmol/L H2O2处理。实时荧光定量聚合酶链反应（RT-qPCR）检测各组细胞Mettl3的mRNA表达水平，Western blot检测Mettl3、Bax、Bcl-2和cleaved Caspase-3的蛋白表达水平，免疫荧光检测Mettl3表达，酶标仪检测各组整体m6A水平，并检测白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平，流式细胞仪检测细胞凋亡。结果：与空白组相比，诱导组的m6A甲基化修饰水平显著提高（P<0.05），Mettl3的mRNA和蛋白表达水平显著性上调（P<0.05），IL-1β（73.39±8.82ng/L vs 125.58±15.31ng/L）、IL-6（63.34±7.12ng/L vs 101.28±12.49ng/L）、TNF-α（103.29±12.19ng/L vs 204.37±23.65ng/L）和MDA（4.01±0.67pmol/L vs 9.23±1.05pmol/L）水平显著性升高（P<0.05），SOD（28.37±3.72U/mg vs 17.23±2.05U/mg）和GSH-Px（158.19±19.26U/mg vs 83.35±9.05U/mg）水平显著性降低（P<0.05），且Bax和cleaved Caspase-3的蛋白表达水平显著性上调（P<0.05），Bcl-2的蛋白表达水平显著性下调（P<0.05），细胞凋亡率显著升高[（8.30±0.68）% vs （34.29±3.16）%，P<0.05]；与诱导组相比，转染诱导组的m6A甲基化修饰水平显著性降低（P<0.05），Mettl3的mRNA和蛋白表达水平显著性下调（P<0.05），IL-1β（125.58±15.31ng/L vs 96.28±11.27ng/L）、IL-6（101.28±12.49ng/L vs 84.56±10.24ng/L）、TNF-α（204.37±23.65ng/L vs 147.15±18.46ng/L）和MDA（9.23±1.05pmol/L vs 7.28±0.96pmol/L）水平显著性降低（P<0.05），SOD（17.23±2.05U/mg vs 24.01±2.76U/mg）和GSH-Px（83.35±9.05U/mg vs 121.48±15.47U/mg）水平显著升高（P<0.05），且Bax和cleaved Caspase-3的蛋白表达水平显著性下调（P<0.05），Bcl-2的蛋白表达水平显著性上调（P<0.05），细胞凋亡率显著性降低[（34.29±3.16）% vs （23.57±2.01）%，P<0.05]。与空白组相比，转染组的炎性因子、氧化应激和凋亡水平均无显著性差异（P>0.05）。结论：H2O2可上调脊髓神经元中m6A甲基化修饰水平，并可诱导氧化应激和细胞凋亡；抑制Mettl3表达能够降低H2O2诱导的脊髓神经元m6A甲基化修饰水平，进而缓解氧化应激和细胞凋亡。

英文摘要:

  【Abstract】 Objectives: To investigate the effects of Mettl3-mediated m6A methylation modification on the oxidative stress and apoptosis of spinal cord neurons induced by hydrogen peroxide(H2O2). Methods: The spinal cord of neonatal SD rats(within 24h) were isolated, and resuspended precipitated after digestion and centrifugation, and the non-adherent cells were collected and cultured in Neurobasal medium after 30min of differential adhesion, which was β3-tublin positive by immunofluorescence and confirmed to be spinal cord neurons. Then the spinal cord neurons were randomly divided into 6 groups: blank group, cells were cultured without any treatment; Transfection control group, cells were transfected with negative control siRNA(si-NC); Transfection group, cells were transfected with Mettl3 siRNA(si-Mettl3); Induction group, cells were treated with 300μmol/L H2O2; Transfection control-induction group, cells were transfected with si-NC and then treated with 300μmol/L H2O2. Transfection-induction group, cells were transfected with si-Mettl3 and then treated with 300μmol/L H2O2. Real-time quantitative polymerase chain reaction(RT-qPCR) was used to detect the expression level of Mettl3 mRNA. The protein expression levels of Mettl3, Bax, Bcl-2 and cleaved Caspase-3 were detected by Western blot. Immunofluorescence was used to determine Mettl3 expression. The level of m6A and the levels of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF-α), superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), and malondialdehyde(MDA) were detected by microplate reader. Cell apoptosis was assessed by flow cytometry. Results: Compared with the blank group, of the induction group, the level of m6A methylation modification, as well as the Mettl3 mRNA and protein expression were significantly up-regulated(P<0.05), the levels of IL-1β(73.39±8.82ng/L vs 125.58±15.31ng/L), IL-6(63.34±7.12ng/L vs 101.28±12.49ng/L), TNF-α(103.29±12.19ng/L vs 204.37±23.65ng/L) and MDA(4.01±0.67pmol/L vs 9.23±1.05pmol/L) were markedly increased(P<0.05), while the levels of SOD(28.37±3.72U/mg vs 17.23±2.05U/mg) and GSH-Px (158.19±19.26U/mg vs 83.35±9.05U/mg) were significantly decreased(P<0.05), the protein expression levels of Bax and cleaved Caspase-3 were significantly up-regulated(P<0.05), and the protein expression level of Bcl-2 was significantly down-regulated(P<0.05), meanwhile the apoptosis rate was significantly increased[(8.30±0.68)% vs (34.29±3.16)%, P<0.05]. Compared with the induction group, of the transfection-induction group, the m6A methylation modification level was significantly decreased(P<0.05), the mRNA and protein expression of Mettl3 were significantly down-regulated(P<0.05), the levels of IL-1β(125.58±15.31ng/L vs 96.28±11.27ng/L), IL-6(101.28±12.49ng/L vs 84.56±10.24ng/L), TNF-α (204.37±23.65ng/L vs 147.15±18.46ng/L) and MDA (9.23±1.05pmol/L vs 7.28±0.96pmol/L) were significantly decreased(P<0.05), while the levels of SOD(17.23±2.05U/mg vs 24.01±2.76U/mg) and GSH-Px(83.35±9.05U/mg vs 121.48±15.47U/mg) were significantly increased(P<0.05), the protein expression levels of Bax and cleaved Caspase-3 were significantly down-regulated(P<0.05), and the protein expression level of Bcl-2 was significantly up-regulated(P<0.05), the apoptosis rate was markedly decreased[(34.29±3.16)% vs (23.57±2.01)%, P<0.05]. Compared with the blank group, there were no significant differences in the levels of pro-inflammatory factors, oxidative stress, and apoptosis in the transfection group(P>0.05). Conclusions: H2O2 could elevate the level of m6A methylation modification, oxidative stress, and cell apoptosis in spinal neurons; Inhibition of Mettl3 expression could reduce the level of m6A methylation modification induced by H2O2, thereby alleviating oxidative stress and cell apoptosis in spinal neurons.