Hereditary Study On Abnormality Of Methylation Regulatory System Of Adult And Offspring Of Developmental Mouse Exposed To 5-aza

Objective: To investagate the heritability of abnormal DNA methylation patterns by studying on the methylation regulation system of adult mice exposed to 5-Aza during development period;In addition,a series of behavioral experiments were performed to study whether the mother with anxiety and depression-like behaviors coluld affect the behavior on the offspring mice,to disscuss the stability of the heritability of abnormal DNA methylation patterns.Method: The 2-weeks-old mice(F0)were randomly divided into 2 groups.The mice in the 5-Aza group were given 5-Aza in the development period,and the mice in control group were given an equal amount of saline.When the mice were 7-8-weeks-old,the behavioral tests carried out.After the behavioral experiment,some of the mice were sacrificed,and the hippocampus were took for next experiments,and the other mice were divided into four groups: control group female mated with control group male(control group),5-Aza group female mated with control group male(Group 2),control group female mated with 5-Aza group male(Group 3),and 5-Aza group male mated with 5-Aza group female(Group 4),obtained the F1 mice.The F1 mice were subjected to behavioral tests when they grew to 7 to 8 weeks of age.After behavioral tests,the mice's hippocampus were took for next experiments.The mRNA level of DNA methyltransferase(Dnmt1),DNA methyltransferases 3a(Dnmt3a),DNA methyltransferases 3b(Dnmt3b),5-hydroxymethylcytosine(5hmC),methylation-CpG-binding domain protein 1(MBD1),methylation-CpG-binding protein 2(MeCP2)were detected by real-time quantitative PCR;the level of 5-methylcytosine(5mC)and 5hmC were measured by ELISA;the protein expression levels of Dnmt1,Dnmt3 a,MBD1 and MeCP2 in hippocampus were assayed by Western Blot.Then arrange control group female mice(F0)to receive the group 2 mice(F1)(Raised by control female group),and arrange 5-Aza female(F0)mice to receive the control group mice(F1)(Raised by 5-Aza female group).When grew to 7 to 8-weeks-old,the mice were subjected to behavioral tests,include the open field test,the elevated cross maze test,the forced swimming test,and the tail suspension test.Results: In this study,the results of Real-time quantitative PCR showed that compared with the control group,the Dnmt1,Dnmt3 a and Dnmt3 b mRNA level of hippocampus in adult mice(F0)exposed to 5-Aza during development was decreased(p<0.05);and the Dnmt1,Dnmt3 a and Dnmt3 b mRNA level in hippocampus of Group 2,3 and 4 of their offspring mice(F1)was lower than that in hippocampus of control group mice(p<0.05).Compared with the control group mice,the 5hmC mRNA level in hippocampus of the F0 was increased(p<0.05);and 5hmC mRNA level in hippocampus of the Group 2,3 and 4 of the F1 was increased(p<0.05).Compared with the control group,the MBD1 and MeCP2 mRNA level in hippocampus of the F0 were decreased(p<0.05);the MBD1 and MeCP2 mRNA level in hippocampus of Group 2,3 and 4 of the F1 was higher than those in the control group(p<0.05).Our results suggested that compared with the control group,the 5mC content in hippocampus of the F0 and Group 2,3 and 4 of the F1 was lower than that in hippocampus of the control group(p<0.05);the 5hmC content in hippocampus of the F0 and Group 2,3 and 4 of the F1 was significantly higher than that in hippocampus of the control group(p<0.05)via ELISA.Our western blot data suggested that the protein expression level of Dnmt1 and Dnmt3 a in hippocampus of the F0 were significantly lower than that in the control group mice(p<0.05);and compared with the control group mice,the protein expression level of Dnmt1 and Dnmt3 a in hippocampus of the Group 2,3 and 4 of the F1 was decreased(p<0.05).The protein expression level of MBD1 and MeCP2 in hippocampus of the F0 was decreased(p<0.05);and the protein expression level of MBD1 and MeCP2 in hippocampus of Group 2,3 and 4 of the F1 was significantly lower than that of the control group(p<0.05).In the open field test,compared with control group,rearing times,shuttle times and the time stay in the central region of the mice raised by control female were decreased(p<0.05).For the mice raised by 5-Aza female,their rearing times,shuttle times and the time stay in the central region showed no difference(p> 0.05).In the elevated plus maze test,the time that the mice raised by control female stay in the open arm was lower than that in the control group(p<0.01).There was no difference in the time of the open arms between the mice raised by 5-Aza female and the control group(p>0.05).And the rearing times showed no difference in the mice raised by control female and the control group,or in the mice raised by 5-Aza female and the control group(p>0.05).In the forced swimming test,the immobility time of the mice raised by control female was higher than that in the control group(p<0.01),while the immobility time of the mice raised by 5-Aza female was not different from that in the control group(p>0.05).In the tail suspension test,there was no difference in the immobility time between the mice raised by control female and the control group,or between the mice raised by 5-Aza female and the control group(p>0.05).Conclusion: In the study,our results shown that exposure to 5-Aza during development caused the abnormalities of the DNA methylation regulatory system of adult mice can be inherited to offspring.Behavioral studies have shown that the offspring of the female mice with anxiety-depressed behavior and normal male mice showed the anxiety-depressed behavior,and were not altered by normal mothers' raise.Further indicating that changes in DNA methylation can be stably inherited.