The Molecular Mechanism Of IP₃Râ…¢ DNA Methylation Regulate The Damage Of Human Embryonic Lung Fibroblasts By Beryllium Sulfate

Objective Through in vitro experiments, observe beryllium sulfate (BeSO4) induced IP3RⅢ DNA methylation changes in human embryonic lung fibroblast (MRC-5), DNAmethylation-related genes DNMT1, DNMT3a, DNMT3b, MBD2, MeCP2’s expression levels,calcium concentration changes in intracellular and inositol triphosphate receptor Ⅲ (IP3RⅢ) expression, investigate whether beryllium sulfate through the DNA methylationmechanisms affects the MRC-5cell damage. Use the DNA methylation inhibitor5-azacytidine (AZC) intervene the MRC-5confirmed beryllium sulfate-induced humanembryonic lung fibroblast cell damage is caused by DNA methylation, while observing theright amount of acetylation of histone deacetylase inhibitor (TSA) synergy exists in the DNAmethylation process.Methods Divid into the vitro human embryo lung fibroblasts in four groups the controlgroup, BeSO4groups (low、medium、high BeSO4group are1、10、100μmol/L), theintervention groups (DNA methylation inhibitor with beryllium sulfate group, acetylationinhibitors and beryllium sulfate groups are10μmol/L AZC+10μmol/L BeSO4,0.5μmol/LTSA+10μ mol/L BeSO4), positive control groups (DNA methylation inhibitor anddeacetylase inhibitor groups are10μ mol/L AZC,0.5μ mol/L TSA). Cells grow tologarithmic phase, according to the experimental needs, reaching the cell density, adding thedrug to the culture cells, collect after24h. Respectively, the following experiments:1. Usingthe nested landed methylation specific PCR(nM-PCR) assay detect to the IP3RⅢ DNAmethylation changes;2. The qPCR determines the DNMT1, DNMT3a, DNMT3b, MBD2, MeCP2mRNA expression;3. IP3RⅢexpression by immunohistochemistry (SP) assay;4. Theconfocal method detects to [Ca2+] levels;5. Nested landed methylation specific PCR(nM-PCR) assay detects to the IP3RⅢ DNA methylation intervention and the qPCR assaydetects to the DNA methylation-related genes expression by AZC, observe the TSA synergy inthis process.Results (1) DNA methylation results showed: IP3RⅢ DNA methylation in the BeSO4medium and high dose groups compared with the control group was statistically significant(P<0.01), and with the increase of drug concentration the IP3RⅢDNA methylation wasreduce.(2) qPCR results showed that: the DNMT1, DNMT3bmRNA expression in the BeSO4high-dose group compared with control group was significantly lower (P<0.01); the DNMT3a,MBD2and MeCP2mRNA expression in the BeSO4low, medium and high dose groups weredecreased compare with the control group ratio (P<0.05or P<0.01).(3)Immunohistochemistry results showed that: IP3RⅢ expression in BeSO4medium and highdose groups compared with control group, the IP3RⅢ expression levels increased (P<0.05orP<0.01).(4) Confocal results showed: the [Ca2+] concentration was high in BeSO4high-dosegroup, the difference was statistically significant (P<0.01).(5) AZC intervention effect on theDNA methylation results showed that:, IP3RⅢ DNA methylation in AZC positive controlgroup and AZC intervention group compared with the control group, the differences werestatistically significant (P<0.01); IP3RⅢ DNA methylation degree reduced significantly inAZC intervention group compared to the AZC positive control group and medium BeSO4group. DNMT1、DNMT3a、DNMT3b、MBD2and MeCP2mRNA expression in AZC positivecontrol group and AZC intervention group compared with the control group, the differenceswere statistically significant(P<0.01), and DNMT1, DNMT3a, DNMT3b, MBD2andMeCP2mRNA expression in AZC intervention group compared to the AZC positive controlgroup and medium BeSO4group was significantly reduced.(6) Effects of TSA on theacetylation experimental study: IP3RⅢdegree of acetylation in TSA positive control group and TSA intervention group compared with the control group, the differences werestatistically significant (P<0.01); IP3RⅢdegree of acetylation in TSA intervention groupcompared to the TSA positive control group and medium BeSO4group was significantlyreduced. DNMT1, DNMT3a, DNMT3b, MBD2and MeCP2mRNA expression in TSApositive control group and TSA intervention group compared with control group, thedifferences were statistically significant (P <0.01) and DNMT1, DNMT3a, DNMT3b, MBD2and MeCP2mRNA relative amount in TSA intervention group compared to the TSA positivecontrol group and medium BeSO4group was significantly reducedConclusions (1) BeSO4causes IP3R DNA methylation in the MRC-5cell.(2) IP3RⅢDNA methylation mechanisms possibly can lead to cell damage by causing BeSO4.(3) BeSO4can be caused calcium concentration increased in the MRC-5cell.(4) Through the AZC,DNA methylation plays a key role in the MRC-5cell damage caused by BeSO4in the processfurther validates.(5) Histone acetylation effect and synergistic in the process of DNAmethylation.