| Objective:The mechanism of liver injury induced by inorganic arsenic is complex and has not yet been elucidated.This paper discusses the following three aspects:（1）Preliminary exploration of differentially methylated genes in the activation of human hepatic stellate cells induced by sodium arsenite,and screening of differentially methylated genes related to fibrosis and autophagy;（2）Explore the effect of changes in methylation level on NOTCH1/HES1/PTEN autophagy regulation pathway and sodium arsenite-induced liver fibrosis;（3）Explore the effect of PTEN gene over-expression on NOTCH1/HES1/PTEN autophagy regulation pathway and the effect of sodium arsenate on the activation of hepatic stellate cells,combined with whole transcriptome sequencing to find PTEN-related non-coding RNAs.Methods:（1）In vitro experiment:LX-2 cells were poisoned with different concentrations of sodium arsenite,and divided into control group,low-dose group（5.00μmol/L）,medium-dose group（10.00μmol/L）,high-dose group（15.00μmol/L）,cell autophagy was observed by transmission electron microscope;the model of hepatic stellate cell activation induced by sodium arsenite was verified by Western Blot;850K methylation chip was used to analyze the differentially methylated sites and genes,GO and KEGG signaling pathway enrichment analysis were performed on different methylated genes,and differentially methylated candidate genes related to fibrosis and autophagy were screened out.（2）Establish a mouse liver fibrosis model induced by sodium arsenite,observe the expression of PTEN at the transcriptional and translational levels in the model;intervene the cells with a methylation inhibitor（5-Aza-cd R）,explore the effect of 5-Aza-cd R on NOTCH1/HES1/PTEN autophagy pathway and fibrosis by cell scratch,q RT-PCR,Western Blot and other methods.（3）In vitro experiment:LX-2 cells were transfected with PTEN over-expression plasmid,and the cells were divided into control group,PTEN blank plasmid group,high-dose sodium arsenite+blank plasmid group,high-dose sodium arsenite+PTEN group over-expression group,PTEN over-expression group,observe the effect of PTEN over-expression on NOTCH1/HES1/PTEN autophagy pathway and fibrosis,and search for PTEN-related factors in the activation of LX-2 cells induced by sodium arsenite through whole transcriptome sequencing non-coding RNAs.Results:The study found that sodium arsenite-induced hepatic stellate cell activation related indicatorsα-SMA and CollagenⅠprotein expression levels were up-regulated in a dose-dependent manner,as confirmed by Western Blot analysis,the difference was statistically significant（P<0.05）.Additionally,autophagy was observed by transmission electron microscopy,and the number of corpuscles increased with the concentration of sodium arsenite.Methylation microarray technology identified 25,817 differentially methylated genes,including 12,083 highly methylated genes and 13,734 low methylated genes.There are 1,910 differentially methylated genes were found to be involved in biological processes,and 4 of them were related to autophagy.Moreover,262 cellular component were enriched,18 of which were related to autophagy.Differentially methylated genes are mainly involved in metabolic pathways,cancer pathways,PI3K-AKT signaling pathways,endocytosis,MAPK signaling pathways,etc.Thirty-three differentially methylated candidate genes related to fibrosis and 316 differentially methylated genes related to autophagy regulation were screened out,and PTEN was selected as a candidate gene related to autophagy and fibrosis.（2）It was observed in the animal model:Compared with the control group,the expression level of PTEN m RNA in the low-concentration sodium arsenite group was significantly increased（P<0.05）;compared with the low-concentration sodium arsenite group,the expression level of PTEN m RNA in the high-concentration sodium arsenite group was significantly down-regulated（P<0.05）.Compared with the control group,the expression levels of LC3,BECN,α-SMA,and HES1 m RNA in the low concentration sodium arsenite group were significantly increased（P<0.05）;compared with the control group,the expression level of PTEN protein in the low and high concentration sodium arsenite group were significantly down-regulated,and the difference was statistically significant（P<0.05）;In terms of cell morphology,as the concentrations of sodium arsenite and methylation inhibitors increased,the number of cells and the growth density decreased.In the cell model,compared with the control group,the expression levels of PTEN m RNA in the low,medium and high dose groups was decreased;compared with the high dose group,the expression levels of PTEN m RNA in the high dose sodium arsenite+methylation inhibitor group increased,the difference both were statistically significant（P<0.05）;compared with the low-dose sodium arsenite group,the protein expression level of PTEN was up-regulated in the low-dose sodium arsenite+methylation inhibitor group,and the protein expression levels of HES1 and LC3B were down-regulated;Compared with the medium/high dose sodium arsenite group,the protein expression level of PTEN in the medium/high dose sodium arsenite+methylation inhibitor group was up-regulated;the protein expression level of HES1,NOTCH1,LC3B,BECN1,α-SMA,CollagenⅠwas down-regulated,the difference was statistically significant（P<0.05）;（3）After the PTEN over-expression plasmid transfected the cell model:compared with the high concentration sodium arsenite+blank plasmid group,the expression level of NOTCH1 protein in the PTEN over-expression group was significantly down-regulated（P<0.05）.Compared with the control group,the protein expression levels of HES1,BECN1,and LC3B in the high-concentration sodium arsenite+blank plasmid group were significantly increased（P<0.05）;compared with the high-concentration sodium arsenite+blank plasmid group,the protein expression levels of HES1,BECN1,and LC3B in PTEN overexpression group were significantly down-regulated（P<0.05）;compared with the high-concentration sodium arsenite+blank plasmid group,the protein expression levels ofα-SMA and CollageⅠwere significantly down-regulated in the PTEN overexpression group（P<0.05）.Whole-transcriptome sequencing results found that there were 19,499 differentially expressed m RNAs in the control group and the high-dose sodium arsenite group,of which4,674 were up-regulated m RNAs and 14,825 were down-regulated m RNAs;a total of17,272 differentially expressed lnc RNAs were found,of which 11,571 were up-regulated lnc RNAs,and 5,701 were down-regulated lnc RNAs;a total of 34 differentially expressed circ RNAs,of which 19 were up-regulated and 15 were down-regulated.The top 10pathways enriched by differentially expressed m RNAs were:metabolic pathway,MAPK signaling pathway,herpes simplex virus type 1 infection,cancer pathway,glycerophospholipid metabolism,etc.Two mi RNAs and one circle RNAs related to PTEN were screened out.Conclusion:（1）There are many differentially methylated genes in the Na As O2-induced LX-2 cell activation model,which are involved in the occurrence of digestive system tumors and autophagy;differentially methylated genes are mainly related to metabolism,cancer,PI3K-AKT signaling,MAPK signaling and endocytosis.PTEN is involved in the formation of cell vesicles and membrane-bound vesicles,and it can be used as a candidate gene for studying Na As O2-induced activation and autophagy in LX-2cells.（2）In vivo and in vitro experiments proved that PTEN was lowly expressed in sodium arsenite-induced liver fibrosis.After 5-Aza-Cd R intervention,PTEN transcription and protein levels increased,NOTCH1 and HES1 expressions decreased,and hepatic stellate cells activation and autophagy levels were reduced,NOTCH1/HES1/PTEN pathway may be involved in the regulation of sodium arsenite-induced liver fibrosis and autophagy.（3）The cell level proved that the over-expression of PTEN can reduce the expression of NOTCH1 and HES1,reduce the degree of autophagy and activation in the model of sodium arsenite-induced LX-2 cell activation.Two mi RNAs and one circle RNAs related to PTEN were screened by whole transcriptome sequencing. |
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