镉暴露大鼠肾脏m^(6)A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 m^(6)A methyltransferases and their associations with microR-21 and transforming growth factor-β1 in kidney of rats exposed to cadmium

[背景]环境污染物会导致机体N6-甲基腺苷(m^(6)A)水平发生变化,但镉介导肾脏m^(6)A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m^(6)A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m^(6)A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m^(6)A(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m^(6)A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m^(6)A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m^(6)A修饰和METTL3、METTL14、WTAP及miR-21表达水平上调,提示m^(6)A和miR-21可能与镉诱导的肾纤维化有关。

[Background]Environmental pollutants can affect N6-methyladenosine(m^(6)A)level in the body,but the change of m^(6)A 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 m^(6)A 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 Nacetyl-β-D-glucosidase(UNAG)and albumin(UALB)in urine,and the levels of m^(6)A 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 transcriptionpolymerase 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 m^(6)A 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 m^(6)A 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 m^(6)A methylation and up-regulation of METTL3,METTL14,WTAP,and miR-21 expression levels in rat kidney tissues,indicating that m^(6)A and miR-21 may be associated with Cd-induced renal fibrosis.