Effects Of Vitamin C On H3K9me3 Modification Of Buffalo Fetal Fibroblasts And Its Mechanism

In somatic cell cloning,the reprogramming factors in the oocyte cytoplasm need to remove the original epigenetic markers of the donor nucleus and reprogram the nucleus to a state conducive to embryo development,so that the reconstructed embryo can develop into a new individual.The incomplete reprogramming of histone H3K9 trimethylation(H3K9me3)is one of the main causes of developmental arrest of somatic cell cloning embryos.Overexpression of histone demethylases 4A(KDM4A)down-regulated the H3K9me3 modification level of donor cells can significantly increase the development rate of cloned embryos.In recent years,several research have found that Vitamin C(Vc)supplementation in donor cell culture medium or cell cloned embryo culture medium can improve the development rate of somatic cloned embryos,but the underlying mechanism still unclearly.Vc is a cofactor of Fe2+/α-ketoglutarate-dependent oxidative oxygenase,and KDM4 belongs to this family of enzymes.It is inferred that Vitamin C may affect the modification level of H3K9me3 by regulating the expression of KDM4A.Therefore,this study aims to explore the effects of Vc on the proliferation,metabolism and epigenetic modification of Buffalo fetal fibroblasts(BFFs),focusing on the effect of Vc on KDM4A expression and the molecular mechanism of its regulation.The results of all the works are summarized in the following:1.Effect of Vc on BFFs proliferation,metabolism and epigenetic modification.BFFs cultured with 20 μg/mL Vc for 48 h could promote cell proliferation and expression of proliferation-related genes(P<0.05)while reduce the apoptosis rate and expression of apoptosis-related genes were decreased(P<0.05).When BFFs were cultured in the medium supplemented with 20 μg/mL Vc,the intracellular ATP production of BFFs began to decrease,and when the supplement of Vc increased to 30 μg/mL,the intracellular ATP production of BFFs was significantly decreased(P<0.05),and the metabolic phenotype of BFFs shifted to glycolysis.With the increasing of Vc supplement,Vc the H3K9me3 modification level of BFFs significantly decreased(P<0.05),whereas the H3K9ac modification level of BFFs significantly increased(P<0.05),and the expression of HP1α were significantly inhibited(P<0.05).Vc can also reduce heterochromatin in the nucleus and increase chromatin accessibility of BFFs.2.Vc affects BFFs apoptosis,H3K9me3 modification and HP1α expression by regulating KDM4A expressionVc can significantly up-regulate the expression of KDM4A,KDM4B,KDM4C and KDM4D.Inhibition of KDM4A expression by 0.25 μM Toxoflavin(KDM4A inhibitor)significantly increased apoptosis rate(P<0.05),significantly increased H3K9me3 modification level and up-regulated HP1αexpression(P<0.05).When the expression of KDM4A was inhibited,and 20μg/mL Vc can relieve the inhibition of KDM4A and significantly decreased apoptosis,H3K9me3 modification level and HP1α expression level(P<0.05).3.Vc mainly affects the expression of KDM4A through PI3K signaling pathwayThe PI3K,MEK/MAPK and mTOR pathways all affect the expression of KDM4A,but the PI3K signaling pathway has the greatest influence on KDM4A.When 10μg/mL LY294002(PI3K inhibitor),0.25 nM Rapamycin(mTOR inhibitor)and 1.25 μM PD98059(MEK inhibitor)were used to inhibit the expression of PI3K,mTOR and MEKrespectively,the expression of KDM4A was significantly decreased(P<0.05).Adding 20 μg/mL Vc could rescue the inhibitory effect of LY294002 on PI3K and significantly increase the expression of KDM4A(P<0.05).However,the addition of 20 μg/mL Vc could not rescue the inhibition of mTOR or MEK,and could not significantly increase the expression of KDM4A(P>0.05).4.Vc affects the expression of KDM4A through PI3K/PDK1/SGK1 signal axisVc regulates the expression of KDM4A in BFFs through PI3K/PDK1/SGK1 signaling pathway,and reduces the modification level of H3K9me3 and the expression of HP lα.20 μg/mL Vc could significantly up-regulate the expression of PI3K,PDK1 and SGK1 genes.Inhibition of PDK1 expression by 250 nM MP7(PDK1 inhibitor)or inhibition of SGK1 expression by 3 μM EMD(SGK1 inhibitor)significantly down-regulated KDM4A expression,increased H3K9me3 modification level and HPla expression level(P<0.05).The down-regulation of KDM4A expression,H3K9me3 modification level and HPla up-regulation could be rescued by 20 μg/mL Vc supplementation(P<0.05).In conclusions,Vc can regulate cell proliferation,metabolism and epigenetic modification of BFFs,suggesting that Vc can shift the of epigenetic modification level of donor cells to a more easily reprogrammed state,which will be further verified infuture.These results provide theoretical and experimental basis for improving the efficiency of somatic reprogramming.