郑婧婧, 赵钊, 张佳靖, 王素华, 卜宁, 史雪敏, 赵宇航. 砷致人胚肺成纤维细胞氧化应激过程中m6A修饰的变化[J]. 环境与职业医学, 2022, 39(5): 499-505, 511. DOI: 10.11836/JEOM21566
引用本文: 郑婧婧, 赵钊, 张佳靖, 王素华, 卜宁, 史雪敏, 赵宇航. 砷致人胚肺成纤维细胞氧化应激过程中m6A修饰的变化[J]. 环境与职业医学, 2022, 39(5): 499-505, 511. DOI: 10.11836/JEOM21566
ZHENG Jingjing, ZHAO Zhao, ZHANG Jiajing, WANG Suhua, BU Ning, SHI Xuemin, ZHAO Yuhang. Changes of m6A modification in arsenic-induced oxidative stress of human embryonic lung fibroblasts[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 499-505, 511. DOI: 10.11836/JEOM21566
Citation: ZHENG Jingjing, ZHAO Zhao, ZHANG Jiajing, WANG Suhua, BU Ning, SHI Xuemin, ZHAO Yuhang. Changes of m6A modification in arsenic-induced oxidative stress of human embryonic lung fibroblasts[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 499-505, 511. DOI: 10.11836/JEOM21566

砷致人胚肺成纤维细胞氧化应激过程中m6A修饰的变化

Changes of m6A modification in arsenic-induced oxidative stress of human embryonic lung fibroblasts

  • 摘要: 背景 砷可以通过触发氧化应激对机体产生毒作用,这一过程伴随表观遗传修饰的改变。

    目的 探讨亚砷酸钠(NaAsO2)引起人胚肺成纤维细胞(HELF)氧化应激过程中的N6-甲基腺苷(m6A)修饰变化。

    方法 使用不同浓度的NaAsO2(0、2.5、5、10、20 μmol·L−1)处理HELF细胞48 h,采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTS)法检测细胞活力;使用相应试剂盒检测氧化应激因子总超氧化物歧化酶(T-SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性以及丙二醛(MDA)的含量;酶联免疫吸附法检测细胞总RNA中的m6A甲基化水平;实时荧光定量PCR法检测m6A修饰酶:甲基转移酶样3(METTL3)、甲基转移酶样14(METTL14)、肾母细胞瘤1关联蛋白(WTAP)、脂肪量与肥胖相关蛋白(FTO)、Fe(II)和α-KG依赖的双加氧酶AlkB家族成员5(ALKBH5)、YTH结构域蛋白2(YTHDC2)、YTH结构域家族蛋白2(YTHDF2)、YTH结构域家族蛋白3(YTHDF3)的mRNA表达情况;Western blotting法检测METTL3、FTO、YTHDC2、YTHDF3、核因子E2相关因子2(NRF2)的蛋白表达情况。采用RNA甲基化免疫共沉淀-实时荧光定量PCR法(MeRIP-qPCR)检测NRF2 mRNA中m6A的富集情况。

    结果 使用0、2.5、5、10、20 μmol·L−1 NaAsO2处理细胞后,MTS实验结果显示,相较于对照组,20 μmol·L−1组的细胞活力降低至84%(P<0.05)。比色法检测结果显示,相较于对照组,10、20 μmol·L−1组T-SOD活性降低(均P<0.05);2.5、10 μmol·L−1组GSH-Px活性降低(均P<0.05);10、20μmol·L−1组MDA含量升高(均P<0.05)。酶联免疫吸附法结果显示,0、2.5、5、10、20 μmol·L−1 组总体m6A甲基化水平依次为(0.193±0.023)%、(0.247±0.021)%、(0.253±0.006)%、(0.233±0.006)%、(0.262±0.010)%,相较于对照组,各组m6A甲基化水平均升高(均P<0.05)。实时荧光定量PCR结果显示,相较于对照组,METTL3的mRNA相对表达量在NaAsO2浓度为2.5、10、20 μmol·L−1时降低(均P<0.05),FTO的mRNA相对表达量在NaAsO2浓度为20 μmol·L−1时降低(P<0.05),YTHDC2的mRNA相对表达量在NaAsO2浓度为10、20 μmol·L−1时升高(均P<0.05),YTHDF3的mRNA相对表达量在NaAsO2浓度为2.5、10、20 μmol·L−1时升高(均P<0.05)。Western blotting结果显示,相较于对照组,METTL3的蛋白相对表达量在NaAsO2浓度为10、20 μmol·L−1时降低(均P<0.05),FTO的蛋白相对表达量在NaAsO2浓度为5、20 μmol·L−1时降低(均P<0.05),YTHDC2的蛋白相对表达量在NaAsO2浓度为20 μmol·L−1时降低(P<0.05),NRF2的胞核蛋白相对表达量在NaAsO2浓度为10、20 μmol·L−1时降低(均P<0.05)。MeRIP-qPCR结果显示,相较于对照组,20 μmol·L−1 NaAsO2暴露组的m6A富集比例明显增加(P<0.05)。过表达FTO后,FTO组FTO的mRNA及蛋白相对表达量较对照组升高,NRF2的胞核蛋白相对表达量较对照组升高(均P<0.05);NaAsO2+FTO组的FTO的mRNA及蛋白相对表达量较NaAsO2组明显升高(均P<0.05),NRF2的胞核蛋白相对表达量较NaAsO2组升高(P<0.05)。

    结论 NaAsO2引起HELF细胞发生氧化应激过程中,总体m6A甲基化程度、m6A修饰酶、NRF2 mRNA的m6A修饰以及NRF2的表达发生变化。

     

    Abstract: Background Arsenic can be toxic to human by triggering oxidative stress, which is companied by epigenetic modifications.

    Objective To investigate the modification of N6-methyladenosine (m6A) in human embryonic lung fibroblasts (HELF) during oxidative stress induced by sodium arsenite (NaAsO2).

    Methods HELF cells were treated by designed concentrations of NaAsO2 (0, 2.5, 5, 10, and 20 μmol·L−1) for 48 h. Cell viability was detected by 3-(4,5-dimethylthia zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) method; the activities of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) as well as the content of malondialdehyde (MDA) were detected with corresponding kits; the level of m6A methylation in total RNA was detected by enzyme-linked immunosorbent assay; the mRNA expressions of m6A modified enzymes were detected by real-time fluorescence quantitative PCR, including methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms' tumor 1-associated protein (WTAP), fat mass and obesity-associated protein (FTO), alkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases 5 (ALKBH5), YTH domain containing protein 2 (YTHDC2), YTH domain family protein 2 (YTHDF2), and YTH domain family protein 3 (YTHDF3); the protein expressions of METTL3, FTO, YTHDC2, YTHDF3, and nuclear factor erythroid 2-related factor 2 (NRF2) were detected by Western blotting. The enrichment of m6A in NRF2 mRNA was detected by RNA methylated immunoprecipitation combined with real-time fluorescence quantitative PCR (MeRIP-qPCR).

    Results After the 0, 2.5, 5, 10, and 20 μmol·L−1 NaAsO2 treatment, the MTS results showed that compared with the control group, the cell viability of the 20 μmol·L−1 group decreased to 84% (P<0.05). The colorimetry results showed that compared with the control group, the activities of T-SOD in the 10 and 20 μmol·L−1 groups decreased (P<0.05); the activities of GSH-Px in the 2.5 and 10 μmol·L−1 groups decreased (P<0.05); the contents of MDA in the 10 and 20 μmol·L−1 groups increased. The results of enzyme-linked immunosorbent assay showed that the overall m6A methylation levels in the 0, 2.5, 5, 10, and 20 μmol·L−1 groups were (0.193 ± 0.023)%, (0.247 ± 0.021)%, (0.253 ± 0.006)%, (0.233 ± 0.006)%, and (0.262 ± 0.010)%, respectively, and compared with the control group, the m6A methylation levels in all the NaAsO2 treated groups increased (P<0.05). The real-time fluorescence quantitative PCR results showed that compared with the control group, the mRNA relative expression level ofMETTL3 decreased in the 2.5, 10, and 20 μmol·L−1 groups (P<0.05); the mRNA relative expression level ofFTO decreased in the 20 μmol·L−1 group; the mRNA relative expression level of YTHDC2 increased in the 10 and 20 μmol·L−1 groups (P<0.05); the mRNA relative expression level ofYTHDF3 increased in the 2.5, 10, and 20 μmol·L−1 groups (P<0.05). The Western blotting results showed that compared with the control group, the relative protein expression of METTL3 decreased in the 10 and 20 μmol·L−1 groups; the relative protein expression of FTO decreased in the 5 and 20 μmol·L−1 groups; the relative protein expression of YTHDC2 decreased in the 20 μmol·L−1 group (P<0.05); the relative nuclear protein expression of NRF2 decreased in the 10 and 20 μmol·L−1 groups (P<0.05). The MeRIP-qPCR results showed that m6A enrichment was significantly increased in the 20 μmol·L−1 NaAsO2 exposure group compared with the control group (P<0.05). After over-expression of FTO, the mRNA and protein relative expression levels ofFTO and the relative expression level of nuclear protein of NRF2 in the FTO group were higher than those in the control group (P<0.05); the mRNA and protein relative expression levels ofFTO in the NaAsO2 + FTO group and the nuclear protein expression level of NRF2 were higher than those in the NaAsO2 group (P<0.05).

    Conclusion In the process of oxidative stress induced by NaAsO2, m6A methylation level, m6A modified enzymes, m6A modification of NRF2 mRNA, and NRF2 expression could change in HELF cells.

     

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