Previous studies have demonstrated that proper concentration of 5-aza-2'-deoxycytidine (5-aza-CdR) treatment was advantageous to decrease DNA methylation level, but the relationships between 5-aza-CdR treatment and methylation status of imprinted genes are seldom detected. The aim of this study was to investigate the effect of low concentration 5-aza-CdR treatment on the methylation status of imprinted gene Xist in different genders of buffalo bone marrow mesenchymal stem cells (BMSCs). BMSCs were isolated and the cell gender was identified through polymerase chain reaction (PCR). Then different concentrations of 5-aza-CdR (0, 0.02, 0.1 μM) were applied for the treatment. The results showed cellular morphology, growth, Xist gene expression pattern, and adherent ability were not significantly affected with the treatment of 5-aza-CdR for 24 hours. Meanwhile, immunofluorescence analysis indicated that the expression of 5-methylcytosine (5-mC) was also not influenced after the treatment. However, bisulfite sequence PCR (BS-PCR) analysis revealed that the methylation level of Xist differentially methylated region (DMR) decreased significantly when the concentration of 5-aza-CdR increased to 0.1 μM in the ♀BMSCs group (p < 0.05), while there was no significant difference among the ♂BMSCs-treated groups. Our results implied that low concentrations of 5-aza-CdR treatment had little impacts on cellular morphology, growth Xist gene expression pattern, adherent ability, and global DNA methylation level of BMSCs in both genders, but the treatment could significantly decrease the methylation level of Xist DMR in ♀BMSCs. Thus, we conclude 5-aza-CdR treatment can affect the methylation status of Xist DMR, furthermore, the influence is also related to sex differences.

中文翻译：

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5-氮杂2'-脱氧胞苷对水牛（Bubalus bubalis）骨髓间充质干细胞不同性别Xist基因甲基化状态的作用。

先前的研究表明，适当浓度的5-氮杂-2'-脱氧胞苷（5-氮杂-CdR）处理可降低DNA甲基化水平，但5-氮杂-CdR处理与印迹基因甲基化状态之间的关系很少检测到。这项研究的目的是调查低浓度5-氮杂-CdR处理对不同性别的水牛骨髓间充质干细胞（BMSCs）印迹基因Xist甲基化状态的影响。分离BMSC，并通过聚合酶链反应（PCR）鉴定细胞性别。然后将不同浓度的5-氮杂-CdR（0、0.02、0.1μM）用于治疗。结果显示细胞形态，生长，Xist基因表达模式，5-氮杂-CdR治疗24小时对粘附力和粘附能力没有显着影响。同时，免疫荧光分析表明，处理后5-甲基胞嘧啶（5-mC）的表达也不受影响。然而，亚硫酸氢盐序列PCR（BS-PCR）分析显示，♀BMSCs组中5-氮杂-CdR的浓度增加至0.1μM时，Xist差异甲基化区域（DMR）的甲基化水平显着降低（p <0.05）， ♂BMSCs治疗组之间无显着差异。我们的研究结果表明，低浓度的5-氮杂-CdR处理对男女BMSCs的细胞形态，生长Xist基因表达模式，粘附能力和总体DNA甲基化水平影响很小，但是这种处理可以显着降低♀BMSCs中Xist DMR的甲基化水平。因此，我们得出结论：5-氮杂-CdR处理可以影响Xist DMR的甲基化状态，此外，这种影响还与性别差异有关。