杨倩, 张祎凡, 韩致超, 刘如洁, 李宇星, 申炜垌, 梁宇芬, 张佳晨, 李双婧, 周涵, 雷立健. 镉暴露大鼠肾脏m6A甲基转移酶的表达及其与微小RNA-21和转化生长因子-β1的关系[J]. 环境与职业医学, 2022, 39(8): 902-907. DOI: 10.11836/JEOM21495
引用本文: 杨倩, 张祎凡, 韩致超, 刘如洁, 李宇星, 申炜垌, 梁宇芬, 张佳晨, 李双婧, 周涵, 雷立健. 镉暴露大鼠肾脏m6A甲基转移酶的表达及其与微小RNA-21和转化生长因子-β1的关系[J]. 环境与职业医学, 2022, 39(8): 902-907. DOI: 10.11836/JEOM21495
YANG Qian, ZHANG Yifan, HAN Zhichao, LIU Rujie, LI Yuxing, SHEN Weitong, LIANG Yufen, ZHANG Jiachen, LI Shuangjing, ZHOU Han, LEI Lijian. Expressions of m6A methyltransferases and their associations with microR-21 and transforming growth factor-β1 in kidney of rats exposed to cadmium[J]. Journal of Environmental and Occupational Medicine, 2022, 39(8): 902-907. DOI: 10.11836/JEOM21495
Citation: YANG Qian, ZHANG Yifan, HAN Zhichao, LIU Rujie, LI Yuxing, SHEN Weitong, LIANG Yufen, ZHANG Jiachen, LI Shuangjing, ZHOU Han, LEI Lijian. Expressions of m6A methyltransferases and their associations with microR-21 and transforming growth factor-β1 in kidney of rats exposed to cadmium[J]. Journal of Environmental and Occupational Medicine, 2022, 39(8): 902-907. DOI: 10.11836/JEOM21495

镉暴露大鼠肾脏m6A甲基转移酶的表达及其与微小RNA-21和转化生长因子-β1的关系

Expressions of m6A methyltransferases and their associations with microR-21 and transforming growth factor-β1 in kidney of rats exposed to cadmium

  • 摘要: 背景

    环境污染物会导致机体N6-甲基腺苷(m6A)水平发生变化,但镉介导肾脏m6A水平如何变化及肾损伤分子机制还需进一步研究。

    目的

    探索镉暴露大鼠肾脏m6A修饰、(去)甲基转移酶与微小RNA-21(miR-21)和转化生长因子-β1(TGF-β1)表达水平的关系。

    方法

    24只SPF级雄性SD大鼠随机分为4组,每组6只,分别皮下注射2.0、1.0、0.5 mg·kg−1氯化镉溶液和等体积生理盐水2周,每周7 d。采用酶联免疫吸附试验检测大鼠尿液中N-乙酰-β-D-葡萄糖苷酶(UNAG)和尿白蛋白(UALB)、肾脏TGF-β1和m6A修饰水平。采用脲酶法测定大鼠血浆尿素氮(BUN)水平。分别采用硫代巴比妥酸法、水溶性四唑盐法和比色法检测肾脏氧化应激指标丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的水平。采用逆转录聚合酶链反应测定肾组织纤维化因子TGF-β1 mRNA和m6A(去)甲基转移酶水平。采用荧光定量聚合酶链反应检测肾组织miR-21的表达水平。

    结果

    2周染毒结束后,2.0、1.0 mg·kg−1氯化镉组的大鼠体重减轻,2.0 mg·kg−1氯化镉组肾脏系数、BUN、UNAG及TGF-β1的mRNA和蛋白水平均升高(P<0.05)。肾脏m6A修饰和甲基转移酶METTL3、METTL14、Wilms肿瘤1-相关蛋白(WTAP)及miR-21表达水平在2.0、1.0 mg·kg−1氯化镉组升高,与对照组差异具有统计学意义(P<0.05)。相关性分析显示m6A修饰水平与SOD(r=−0.4489)和GSH-Px(r=−0.4874)呈负相关(P<0.05),METTL3与MDA呈负相关(r=−0.5158,P<0.05),而FTO与GSH-Px之间具有正相关关系(r=0.4802,P<0.05)。此外,miR-21与METTL3(r=0.7491)、METTL14(r=0.6157)和WTAP(r=0.6660)表达水平呈正相关(均P<0.05),TGF-β1与METTL3表达水平也具有正相关关系(r=0.5025,P<0.05),但与FTO呈负相关关系(r=−0.5634,P<0.05)。

    结论

    镉可致大鼠肾脏组织m6A修饰和METTL3、METTL14、WTAP及miR-21表达水平上调,提示m6A和miR-21可能与镉诱导的肾纤维化有关。

     

    Abstract: Background

    Environmental pollutants can affect N6-methyladenosine (m6A) level in the body, but the change of m6A level in kidney after being exposed to cadmium (Cd) and the molecular mechanism of renal injury need to be further studied.

    Objective

    To analyze the associations of m6A modification and methyltransferases/demethylases with microRNA-21 (miR-21) and transforming growth factor- β1 (TGF - β1) in kidney of rats exposed to Cd.

    Methods

    Twenty-four SPF male SD rats were divided into 4 groups, with 6 rats in each group, and were exposed to Cd by subcutaneous injection of 2.0, 1.0, and 0.5 mg·kg−1 cadmium chloride (CdCl2) and equal volume of normal saline for 2 weeks, 7 d a week, respectively. The levels of N-acetyl-β-D-glucosidase (UNAG) and albumin (UALB) in urine, and the levels of m6A methylation and TGF-β1 in kidney were detected by enzyme-linked immunosorbent assay (ELISA). The level of blood urea nitrogen (BUN) was measured by urease method. The levels of renal oxidative stress indicators such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were detected by total bile acid method, water-soluble tetrazolium asssay, and colorimetric method respectively. The relative levels of TGF-β1, methyltransferases, and demethylases in kidney were measured by reverse transcription-polymerase chain reaction. The expression of miR-21 in kidney was detected by fluorescent quantitative polymerase chain reaction.

    Results

    After 2 weeks of exposure to Cd, the body weights of rats in the 2.0 and 1.0 mg·kg−1 cadmium chloride groups decreased, and the ratio of kidney/body weight and the levels of BUN, UNAG, and TGF-β1 mRNA and protein increased in the 2.0 mg·kg−1 cadmium chloride group (P<0.05). The expression levels of m6A modification, methyltransferases METTL3, METTL14, Wilms’ tumor 1-associated protein (WTAP), and miR-21 were increased both in the 2.0 and 1.0 mg·kg−1 cadmium chloride groups, with significant differences compared with the control group (P<0.05). The results of correlation analysis showed that the m6A modification level was negatively correlated with SOD (r=−0.4489, P<0.05) and GSH-Px (r=−0.4874, P<0.05), METTL3 was negatively correlated with MDA (r=−0.5158, P<0.05), while there was a positive correlation between FTO and GSH-Px (r=0.4802, P<0.05). In addition, miR-21 was positively correlated with METTL3 (r=0.7491), METTL14 (r=0.6157), and WTAP (r=0.6660) (P<0.05), TGF-β1 was positively correlated with METTL3 (r=0.5025, P<0.05) but negatively correlated with FTO (r=−0.5634, P<0.05) .

    Conclusion

    Cd can induce m6A methylation and up-regulation of METTL3, METTL14, WTAP, and miR-21 expression levels in rat kidney tissues, indicating that m6A and miR-21 may be associated with Cd-induced renal fibrosis.

     

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