Effect of Recombinant Protein KDM4D on the Growth and Histone Methylation Modification of Buffalo Fibroblasts
This study aimed to investigate the effect of recombinant protein lysine K-specific demethylase 4D(KDM4D)on the growth and histone methylation modification of buffalo fibroblasts(BFFs),providing a theoretical basis for improving the reprogramming effi-ciency of buffalo somatic cells.Firstly,BFFs were treated with different concentrations of recombinant protein KDM4D to optimize the concentrations and time of treatments.Subsequently,the proliferation and apoptosis status of BFFs were detected by using real time quantitative PCR and EdU cell proliferation kit.Finally,the protein expression levels of histone 3 lysine 9 trimethylation(H3K9me3)and heterochromatin protein 1α(HP1α)were assayed by using cellular immunofluorescence and Western blot.The results showed that the treatment with an appropriate concentration of recombinant protein KDM4D(0.10 μg/mL)for 36 h had no significant effect on the morphology of BFFs and could significantly improve cell viability(P＜0.05).Real time quantitative PCR results revealed that recombi-nant protein KDM4D significantly increased the relative mRNA expression levels of cyclin dependent kinase 4(CDK4)and B-cell lym-phoma 2(BCL2),and significantly decreased the relative mRNA expression level of BCL2 associated X(Bax),compared with the con-trol group(P＜0.05).The results of EdU assay showed that the proliferation rate of BFFs after recombinant protein KDM4D treatment was significantly higher than that in the control group(P＜0.05).The results of cellular immunofluorescence and Western blot showed that the protein expression level of H3K9me3 and HP1α in BFFs were significantly reduced after the addition of recombinant protein KDM4D(P＜0.05).In a word,the results indicated that the treatment with an appropriate concentration of recombinant protein KDM4D(0.10 μg/mL)for 36 h could promote the proliferation of BFFs and reduce the expression levels of histone H3K9me3 modifi-cation and HP1α protein in BFFs.This study provides a theoretical basis for further exploring the mechanism of recombinant protein KDM4D on reprogramming of buffalo somatic cells.
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