Study On The Correlation Between N~6-methyladenosine And Oxidative Damage In Cadmium Induced Renal Injury

Objective（1）To detect the survival,apoptosis and oxidative damage of HK-2 cells after human renal tubular epithelial cells（HK-2）treated by cadmium sulfate（Cd SO₄）;（2）To understand the changes of N⁶-methyladenosine（m⁶A）methyltransferases,demethylases and methyl-binding proteins in the Cd SO₄-treated HK-2 cells and indicat whether m⁶A regulatory proteins are involved in HK-2 cell damage process induced by cadmium;（3）Analyzing the correlation between Nuclear factor-erythroid 2-related factor 2（Nrf2）and m⁶A catalytic enzymes（methyltransferases and demethylases）during the damage process of HK-2 cells induced by Cd SO₄;（4）Clarifying the molecular mechanisms of cadmium-induced kidney injury from the perspective of RNA epigenetics and providing theoretical basis for the prevention and diagnosis of cadmium-related diseasese.MethodsHK-2 cells were selected as subjects and treated with different concentrations of Cd SO₄for 24 hours,then the following experiments were carried out:（1）The cell viability were determined by CCK-8 assay;（2）Hoechst staining method was used to observe the apoptotic morphology of cell.Under the fluorescence microscope,the field of vision was randomly selected to take photos,then 200 cells were counted and the apoptosis rate was calculated;（3）Fluorescent probe was used to observe the fluorescence intensity under fluorescence microscopy,the field of vision was randomly selected and captured picturese,which analyzed by Image J software to get quantitative results and to calculate levels of reactive oxygen species（ROS）;（4）The activity of superoxide dismutase（SOD）in cells was detected by WST-8 method,the activity of glutathione peroxidase（GSH-PX）was obtained indirectly by measured the reduction of NADPH,and malondialdehyde（MDA）was determined by thiobarbituric acid colorimetry;（5）The m RNA levels of the following indices were detected by reverse transcription-polymerase chain reaction（RT-PCR）:Nrf2,heme oxygenase 1（HO-1）;m⁶A methyltransferases:methyltransferase like 3（METTL3）,methyltransferase like 14（METTL14）,methyltransferase like 16（METTL16）,wilms’tumor 1-associated protein（WTAP）;m⁶A demethylases:fat mass and obesity associated protein（FTO）,alk B family of nonheme Fe（II）/α-ketoglutarate（α-KG）-dependent dioxygenases 5（ALKBH5）;m⁶A methyl-binding proteins:YTH protein family members（YTHDF1,YTHDF2,YTHDF3,YTHDC1,YTHDC2）;（6）Nrf2,METTL3 and FTO protein expressions were detected by Western blotting assay,GAPDH was used as the internal parameter to adjust the gray value of the target band proteins and calculate the relative expressions of each protein.Results（1）Cell viability and apoptosis:the cell morphology was integrated or was not obvious with treatment of 0μM and 4μM under microscopic observation,but cells gradually lost their adhere ability and floated in the culture medium after exposed to 8 and 16μM Cd SO₄.CCK8 experimental results showed that the cell viability rates were 100%,103.01%,105.58%,114.55%,93.36%,65.48%,35.73%and 25.03%respectived after HK-2 cells was treated with 0,1,2,4,8,16,32 and 64μM Cd SO₄for 24 hours,indicating the cell viability first increased and then decreased（H=22.479,P=0.002）.In the determination of apoptosis rates,when Cd SO₄concentration at 0,4,8 and 16μM,the cell apoptosis rates were 6.83%,5.17%,21.17%,35.8%respectived,in which the apoptosis rates in the 8 and 16μM groups were higher than that in“0”group（P<0.01）;（2）Indicator related of Nrf2 signal pathway:m RNA expression levels of Nrf2 and HO-1by real-time PCR,Nrf2 m RNA expression levels increased at 8 and 16μM Cd SO₄treatment,in which 3.40 and 2.92 times of“0”group respectived（P<0.05）,Nrf2 protein expressions at 4 and 16μM Cd SO₄treatment were 1.13 and 1.26 times of“0”group and the differences were statistically significant（P=0.006;P<0.01）;HO-1 m RNA expression levels increased at 4,8 and 16μM Cd SO₄concentration,in which 1.41,4.90and 5.94 times of“0”group respectively（P<0.05）;（3）Indexes related of oxidative stress:results of ROS levels detected by fluorescent probes revealed that the content of ROS was 1.59 times higher than that of“0”group at the treatment with 16μM Cd SO₄（P=0.015）;When Cd SO₄concentration were 0,4,8and 16μM,SOD activity were 18.58±2.34,14.44±1.91,13.99±0.98 and 11.75±0.90U/mg protein respectived,the activity of SOD in each treatment group was decreased in comparison with“0”group（P=0.038;P=0.023;P=0.003）;the activity GSH-PX was decreased significantly when the Cd SO₄concentration at 8μM（8.74±3.91 m U/mg protein）,while treatment with 8 and 16μM Cd SO₄,the MDA contented increased significantly,of which were 94.30±14.33 and 96.76±6.08 nmol/mg protein,and the differences were statistically significant when compared to the“0”group（P=0.013;P=0.008）;（4）m⁶A related enzymes:the m RNA levels of METTL3 were increased in each Cd SO₄concentration group when compared with the“0”group（P<0.05）,and the protein expressions of METTL3 in Cd SO₄treatment groups were higher than that in the“0”group（P=0.005;P=0.001;P<0.01）,the m RNA levels of METTL16 were higher than that in the“0”group（P=0.004;P=0.032;P=0.046）,the m RNA levels of METTL14 and WTAP were higher than that of“0”group at 8μM Cd SO₄exposure（P=0.024;P<0.05）;when the Cd SO₄concentrated at 8 and 16μM,the m RNA levels of FTO were higher than that in“0”group（P=0.037;P=0.008）,and the protein expressions of FTO were lower than that in“0”group（P=0.013;P=0.003）;the m RNA levels of ALKBH5 at 4,8and 16μM Cd SO₄have no statistically significant when compared with the“0”group（F=1.811,P=0.223）;（5）m⁶A methyl-binding proteins:the m RNA level of YTHDF1 in the treatment with 4μM Cd SO₄was higher than that in the“0”group（P=0.009）;the m RNA levels of YTHDF2 were also higher in the treatment with 4 and 8μM Cd SO₄in comparison with the“0”group（P<0.05）;the m RNA levels of YTHDF3 were higher than that of the“0”group at 4 and 16μM Cd SO₄（P=0.013;P=0.045）,and YTHDC1 were also higher than that of the“0”group at 4 and 16μM Cd SO₄（P=0.025;P=0.003）;the difference of YTHDC2 m RNA levels at 4,8 and 16μM Cd SO₄were not statistically significant when compared with the“0”group（H=6.787,P=0.079）;（6）Analyzed the correlation between m⁶A related enzymes and Nrf2:Nrf2 m RNA levels positively correlated with the changes in m⁶A methyltransferases（METTL3,METTL14,METTL16 and WTAP）,the correlation coefficients were 0.3931,0.9398,0.3691 and0.2430 respectively;Nrf2 m RNA levels also positively correlated with changes in m⁶A demethylases（FTO and ALKBH5）,the correlation coefficients were 0.6112 and 0.1283respectively.Conclusions（1）Nrf2 signaling pathway was activated under Cd SO₄stimulation,then,it encourage antioxidant enzymes to exert their antioxidant capacity,with the increasing of Cd SO₄concentrations,the Nrf2 signaling pathway mediated antioxidant substances（SOD,GSH-PX）were continuously exhausted,implicating the development of intracellular oxidative damage,ultimately resulting the cell apoptosis;（2）The m RNA levels of m⁶A regulatory proteins（METTL3,METTL14,METTL16,WTAP,FTO,YTHDF1,YTHDF2,YTHDF3,YTHDC1）and the protein expression of m⁶A related enzymes（METTL3,FTO）were changed with Cd SO₄exposure,indicating that m⁶A regulatory proteins were maybe involved in the damage process of HK-2 cells induced by cadmium;（3）m RNA level of Nrf2 was positively correlated with the m⁶A related enzymes by Cd SO₄in HK-2 cells,suggesting that the changes of m⁶A related enzyme levels may affect the level of m⁶A modification in Nrf2 m RNA,then affecting the cadmium-induced cell proliferation and apoptosis.