The Effect Of Insulin On Promoter Methylation Of EB Pluripotency And Differentiation Marker Genes

Object:To observe whether different concentration of insulin has effect on promoter methylation of Nanog, Sox2, Gata6, Brachyury and Fgf5genes.Method:In order to simulate the early development of embryo in vitro, we chose embryonic body(EB) which was formed by mouse ES cell line SF1-G, treated with insulin of different concentrations (60pM,6nM), then collected DNA of EB at day0,5,7separately. Promoter region methylation was tested by using MeDIP-chip.NimbleScan software, SignalMap were used to analyze the change of promoter methylation of Nanog, Sox2, Gata6, Brachyury and Fgf5under different conditions.Results:1. In terms of the morphology, the capsule cavity tendency appeared at day5in60pM group and became more obvious at day7; however, in6nM group, this trend was not evident.2. Both60pM and6nM groups displayed obvious CpG islands methylation of Sox2gene promoter region at day5, while the methylation level of60pM group was higher than the6nM one.3. At day5,60pM group displayed a high methylation level of Nanog gene promoter region, but no change happened in the6nM group.4. Compared with EB at day0, both6nM and60pM insulin groups displayed methylated CpG islands of Fgf5gene promoter region at day5, while the methylation level of60pM group was lower than the6nM one.5. Both60pM and6nM insulin groups displayed methylation of Gata6gene promoter region at day7, while the methylation level of60pM group was higher than the6nM one.6. Compared with EB of60pM insulin at day5, the CpG islands methylation level of Brachyury gene promoter region of60pM at day7was higher.Conclusion:1. Insulin can affect methylation level of promoter regions of Sox2, Nanog, Gata6, Fgf5and Brachyury, moreover, the changes are different between different concentrations; the genes’expression may be connected with the methylation level of gene promoter regions.2. The methylation level of promoter region of Sox2, Nanog, Gata6, Fgf5and Brachyury is also different at different culture time, even though cultured with the same insulin concentration.