The Mechanism Of SLC7A11 And CDKN1A Methylation In The Activation Of Hepatic Stellate Cells Induced By Sodium Arsenite

Objective:To investigate the effects of different concentrations of sodium arsenite（Na As O₂）on the activation of human hepatic stellate cells（LX-2）and the expression of iron death related proteins,and to explore the mechanism of SLC7A11 and CDKN1A methylation in the activation of LX-2 cells induced by sodium arsenite.Methods:1)The relative survival rate of Na As O₂ infected LX-2 cells was detected by MTT colorimetric method,and the IC₅₀ of Na As O₂ infected LX-2 cells was determined according to the specific situation,and the Naas O₂ infected concentration was divided into low,medium and high dose groups;2)Observe cell migration and healing through scratches;3)Screen differential DNA Methylation sites through 850K methylation chips;4)Real-time fluorescence quantitative（RT-q PCR）detection of slc7a11,Cyclin Dependent Kinase Inhibitor 1A（cdkn1a）,Transforming growth factor-β1（transforming growth Factor-β1,tgf-β1）m RNA transcription levels of Anti-Collagen TypeⅢ（collagenⅢ）;Protein expression levels of SLC7A11,CDKN1A,SLC7A4,collagen III and TGF-β1 were detected by Western blot;5)The cells were treated with methylation inhibitor（5-Aza-cd R）and the median lethal dose was determined;6)The mechanism of methylation of promoter regions of SLC7A11,CDKN1A and SLC7A4 genes in the activation of hepatic stellate cells induced by Na As O2 was investigated by mass spectrometry;7)The protein expression levels of SLC7A11,CDKN1A,SLC7A4,collagen III,TGF-β1 were detected by Western blot after the intervention of methylation inhibitor（5-Aza-Cd R）+Na As O₂.Results:1)The IC₅₀ of LX-2 cells infected with Na As O₂ was 27.24μmol/L.The low,middle and high concentrations of Na As O2group were determined to be 5μmol/L,10μmol/L and 15μmol/L,respectively,and the control group was determined to be0μmol/L;2)The scratch healing rate of the Na As O₂ intervention group was much lower than the control group,and the scratch healing rate decreased gradually with the increase of Na As O₂ concentration;3)Using gene chip technology,74,267 differentially methylated loci were detected after screening,among which 29,583 loci were hypermethylated,44,684 loci were hypomethylated,and the number of genes mapped to these loci were hypermethylated,12,083genes were hypermethylated,and 13,734genes were hypomethylated.SLC7A11 was found to be hypermethylated at cg22659014.CDKN1A hypermethylated at promoter cg17964532;4)After exposure to Na As O₂solution,compared with the control group,the m RNA expression of SLC7A11 in low,medium and high dose Na As O_2groups was decreased,while that of SLC7A4 in low and high dose Na As O_2groups was increased（P<0.05）.The m RNA expressions of TGF-β1and collagenⅢwere increased（P<0.05）.Compared with the control group,SLC7A11protein expression level was decreased and CDKN1A protein expression level was increased in Na As O₂ low-dose,medium-dose and high-dose groups,while SLC7A4protein expression level was increased in Na As O₂ medium-dose and high-dose groups（P<0.05）.Compared with the control group,TGF-β1 protein expression level in Na As O₂low,medium and high dose groups was increased,and collagenⅢprotein expression level in Na As O_2high dose group was increased,（P<0.05）;5)Different concentrations of5-Aza-CDR had certain inhibitory effect on the cell growth of LX-2 cells,and the cell activity decreased with the increase of methylation inhibitor concentration,OD value decreased gradually,and the inhibitory rate of LX-2 showed an increasing trend.There was statistical significance between the methylation inhibitor group and the control group at different concentrations（P<0.05）,and the data showed that the inhibition rate was positively correlated with the dose of 5-Aza-Cd R;6)The m RNA expression level of SLC7A11 in Na As O₂group was decreased,the m RNA expression level of SLC7A4 was increased,and the m RNA expression levels of TGF-β1 and CollagenⅢwere increased（P<0.05）.The m RNA expression level of SLC7A4 in Na As O₂+5-Aza-Cd R group was increased（P<0.05）;Compared with Na As O₂group,SLC7A11 m RNA expression level in Na As O₂+5-Aza-Cd R group was increased（P<0.05）,SLC7A4 m RNA expression level was decreased,and CDKN1A m RNA expression level was increased（P>0.05）.The m RNA expression levels of TGF-β1 and CollagenⅢwere decreased（P<0.05）.Compared with low-dose group,SLC7A11 protein expression levels in low-dose+5-Aza-Cd R group were increased,while CDKN1A and TGF-β1 protein expression levels were decreased（P<0.05）.Compared with medium dose group,SLC7A11 protein expression levels in medium dose+5-Aza-Cd R group were increased,while CDKN1A,SLC7A4 and TGF-β1 protein expression levels were decreased（P<0.05）.Compared with high-dose group,SLC7A11 protein expression levels in high-dose+5-Aza-Cd R group were increased,while CDKN1A,SLC7A4,TGF-β1 and CollagenⅢprotein expression levels were decreased（P<0.05）;7)SLC7A4 was found by mass spectrometry Methylation level difference analysis SLC7A4＿FA40＿Cp G＿2,SLC7A4＿FA28＿Cp G＿4,SLC7A4＿FA40＿Cp G＿3,SLC7A4＿FA40＿Cp G＿11,SLC7A4＿FA40＿Cp G＿12,SLC7A4＿FA43＿Cp G＿2,The average methylation degree of Cp G sites in S LC7A4＿FA43＿CPG＿3-4 fragments was statistically significant（P<0.05）.Conclusion:DNA methylation is involved in Na As O₂ induced human hepatic stellate cell activation.Methylation inhibitors may reduce Na As O₂-induced human hepatic stellate cell activation,suggesting that reduced methylation level may inhibit the process of liver fibrosis induced by sodium arsenite and have a certain protective effect on the liver.