The Role of 5-aza-2′-Deoxycytidine on Methylation Status of Xist Gene in Different Genders of Buffalo (Bubalus bubalis) Bone Marrow Mesenchymal Stem Cells
The Role of 5-aza-2′-Deoxycytidine on Methylation Status of Xist Gene in Different Genders of Buffalo (Bubalus bubalis) Bone Marrow Mesenchymal Stem Cells
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5-氮杂-2-脱氧胞苷对不同性别水牛骨髓间充质干细胞Xist基因甲基化状态的影响
DOI：
10.1089/cell.2018.0040
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发表时间：
2019
影响因子：
1.6
通讯作者：
Fenghua Lu
中科院分区：
文献类型：
--
作者：
Xin Zhao;Ziyun Ruan;Xiling Qin;Yun Feng;Qing Yu;Jie Xu;Yanfei Deng;Penglei Shen;Deshun Shi;Fenghua Lu
已有研究表明，适当浓度的5-aza-2′-脱氧胞苷（5-aza-CdR）处理有利于降低DNA甲基化水平，但很少检测到5-aza-CdR处理与印迹基因甲基化状态的关系。本研究旨在探讨低浓度5-aza-CdR处理对不同性别水牛骨髓间充质干细胞（BMSCs）中存在的印迹基因甲基化状态的影响。分离骨髓间充质干细胞，采用聚合酶链反应（PCR）鉴定细胞性别。然后分别用不同浓度（0、0.02、0.1 μM）的5-aza-CdR进行处理。结果显示，5-aza-CdR处理24h后，细胞形态、生长、Xistgene表达模式和贴壁能力均无显著影响。同时，免疫荧光分析显示，治疗后5-甲基胞嘧啶（5-mC）的表达也未受影响。然而，亚硫酸酯序列PCR （BS-PCR）分析显示，当5-aza-CdR浓度增加到0.1 μM时，♀BMSCs组的xistdifferentially methylated region （DMR）甲基化水平显著降低（p< 0.05），而♂BMSCs处理组间差异不显著。结果表明，低浓度5-aza-CdR处理对两性骨髓间充质干细胞的细胞形态、生长、xist基因表达模式、粘附能力和整体DNA甲基化水平影响不大，但可显著降低♀骨髓间充质干细胞中xistdmr的甲基化水平。因此，我们认为5-aza-CdR治疗可以影响xistdmr的甲基化状态，而且这种影响还与性别差异有关。
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 geneXistin 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,Xistgene 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 ofXistdifferentially 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, growthXistgene expression pattern, adherent ability, and global DNA methylation level of BMSCs in both genders, but the treatment could significantly decrease the methylation level ofXistDMR in ♀BMSCs. Thus, we conclude 5-aza-CdR treatment can affect the methylation status ofXistDMR, furthermore, the influence is also related to sex differences.