Effects Of Developmental Arsenic Exposure On DNA Methylation And MicroRNA Expression In The Liver Of Mice

Objective:Arsenic is a metal like element widely existing in nature,and ranks first in the list of toxic chemicals of American toxicology and Disease Registry（ATSDR）.Arsenic poisoning in drinking water has accumulated in more than 70 countries and regions in the world,which is an important public health problem.Developmental arsenic exposure will continue to affect human health,leading to adverse birth outcomes and disease effects in adulthood.Epigenetic modification is considered to be one of the main mechanisms of developmental arsenic exposure leading to increased susceptibility to diseases in adulthood,but the specific mechanism remains unclear.DNA methylation and miRNA changes are important components of epigenetic modification,and their disorders are closely related to the occurrence of diseases.Nrf2is a key transcription factor in antioxidant response system and can be activated by inorganic arsenic exposure.The relationship between DNA methylation and diseases has attracted more and more attention.The aim of this study was to investigate the effects of arsenic exposure on DNA methylation and miRNA expression in the liver of weaning mice,It further revealed the mechanism of Nrf2 expression induced by arsenic exposure during development.Methods:1.In this study,male weaned C57BL/6J mice（4 weeks old）were used as the research object to establish a mouse model of arsenic exposure in drinking water during development（spermatogenesis,pregnancy and lactation）.The parental mice were exposed to 0.5 ppm arsenic（NaAsO₂:NA₂HASO₄=1:1）in drinking water from5 weeks before pairing（spermatogenesis）until weaning（4 weeks old）;The mice in the control group drank double distilled water.The mice were fed and drank freely.The liver of weaning mice was collected for study.2.Differential miRNA screening and analysis:high throughput miRNA microarray technology was used to detect the liver of mice.The difference of miRNA expression profile between the two groups was analyzed by limma software package in R/bioconduct.The heat map and fire map of differential miRNAs were constructed by pheatmap and ggplot software packages respectively;Online software DIANA miRPath v.3 was used to analyze the KEGG pathway of different miRNAs,and the combined pathway with statistical significance（P<0.05）was selected for thermography.3.Mi RNA verification:RT-qPCR was used to verify the reliability of miRNA chip,and 9 different miRNAs were selected by random number table method for verification.4.Detection of whole genome DNA methylation:the content of 5-methylcytosine（5mc%）in liver was detected by ELISA;Western blot and RT-qPCR were used to detect the protein and mRNA expression of DNA methyltransferase（DNMT1,DNMT3a/b）and DNA demethylase（Tet1,Tet2,Tet3）.5.mRNA and protein expression of Nrf2:the mRNA and protein expression levels of Nrf2 was detected by Western blot and RT-qPCR.6.Detection of Nrf2promoter DNA methylation:primers were designed according to the enrichment status of Cp G island in Nrf2 promoter region,and bisulfite sequencing PCR（BSP）was used to analyze Nrf2 promoter DNA methylation level.6.Statistical analysis:differential miRNAs KEGG pathway was analyzed by DIANA mirpath v.3.BSP results are processed in R language matrix 2×2.statistical analysis.The results of Western blot,RT-qPCR and ELISA were analyzed by Graphpad prism 6.0,and the data were expressed as the average±Student’s t test was used to compare the two groups,and one-way ANOVA was used to compare the multiple groups.Results:1.Using microarray technology,86 differentially expressed miRNAs were screened in the liver of arsenic exposed male mice at developmental stage,including55 up-regulated miRNAs and 31 down regulated miRNAs.The most significant differences were miR-192-5p,miR-21a-5p,miR-7083-5p and miR-7052-5p.2.DIANA miRPath v.3 was used to identify the KEGG pathways that may involve differential miRNAs,10 of which were significantly different.The top four KEGG pathways were fatty acid biosynthesis pathway,ECM receptor interaction pathway,mucin type O-glycan biosynthesis pathway and fatty acid metabolism pathway.3.There was no significant difference in the content of 5mc%in the liver between arsenic exposed group and control group.4.Compared with the control group,the level of DNMT1protein in the arsenic exposure group was increased,and the difference was statistically significant（P<0.05）,but there was no significant difference in the mRNA level between the two groups;There was no significant difference in the mRNA and protein levels of Dnmt3a and DNMT3b between arsenic exposure group and control group;There was no significant difference in the mRNA levels of Tet1,Tet2 and Tet3 in arsenic exposure group.5.The expression of Nrf2 mRNA and protein increased after developmental arsenic exposure.6.The DNA methylation rate of Nrf2 promoter region was 1.7%in the control group and 0.9%in the 0.5 ppm arsenic exposure group.There was no significant difference in the DNA methylation level between the two groups（P=0.065）.Conclusion:1.Developmental arsenic exposure caused the change of miRNA expression profile in the liver of weaning mice,and produced 10 KEGG pathways with significant differences.2.Developmental arsenic exposure promoted the expression of DNMT1 and increased the mRNA level of Dnmt3a,but did not affect the expression of DNMT3b and Tet1,Tet2,Tet3 and did not change the whole genome DNA methylation level of 0.5 ppm group.3.Developmental arsenic exposure had no effect on the DNA methylation level of Nrf2 promoter region,suggesting that there are other potential mechanisms of Nrf2 activation induced by arsenic exposure at developmental stage.