DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.
DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy.
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通过图像分析，免疫荧光和振动微光谱法评估，丙戊酸处理的HELA细胞的DNA甲基化变化。
DOI：
10.1371/journal.pone.0170740
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Vidal BC
中科院分区：
文献类型：
--
作者：
Veronezi GM;Felisbino MB;Gatti MS;Mello ML;Vidal BC
丙戊酸（Valproic acid，VPA）是一种组蛋白去乙酰化酶抑制剂，除诱导组蛋白高乙酰化外，还能影响DNA甲基化状态。在HeLa细胞中，VPA促进组蛋白乙酰化和染色质重塑。然而，在该细胞模型中没有检查DNA去甲基化的持续效应比组蛋白乙酰化提供的持续效应更长，组蛋白乙酰化是一种快速和短暂的现象。证明VPA诱导的DNA去甲基化在HeLa细胞将有助于了解VPA对侵袭性肿瘤细胞系的影响。在目前的工作中，VPA处理的HeLa细胞中的DNA去甲基化进行了评估，通过图像分析的染色质纹理，丰富的5-甲基胞嘧啶（5 mC）的免疫荧光信号和傅立叶变换红外（FT-IR）显微光谱集中在光谱区域的振动相关的-CH 3基团。图像分析表明，在不存在药物的情况下，用1.0 mM VPA处理4 h促进的染色质解包增加持续24 h，表明发生了DNA去甲基化，这通过5 mC免疫荧光信号减少得到证实。FT-IR光谱的DNA样品从1 mM或20 mM VPA处理的细胞进行的光谱窗口的峰值拟合分析-CH 3伸缩振动显示，这些基团的振动和能量减少的功能，减少的丰度5 mC诱导VPA浓度增加。仅20 mM-VPA处理导致在1375 cm-1处评估的-CH 3弯曲振动相对于在1492 cm-1处评估的总体胞嘧啶的面内振动的比率增加。正如FT-IR显微光谱法所检测到的那样，CH 3伸缩振动比-CH 3弯曲振动更敏感，用于旨在将振动光谱与DNA 5 mC丰度变化联系起来的研究。
Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than–CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.