组蛋白H3乙酰化介导Nanog基因对胶质瘤细胞生物学活性的影响

发布时间：2018-11-21 21:26

【摘要】：目的：研究正常脑组织、不同级别胶质瘤组织以及胶质瘤细胞系U87中H3乙酰化组蛋白的表达，使用去乙酰化酶抑制剂Apicidin干扰U87胶质瘤细胞系，研究组蛋白H3乙酰化程度改变对胶质瘤细胞中Nanog的影响，并探索组蛋白H3低乙酰化对胶质瘤细胞体内及体外生物学活性的影响。为后续进一步研究Nanog的表观遗传学干预治疗奠定基础。 方法：①使用Western-Blot方法检测53例不同级别脑胶质瘤组织及11例取自颅脑损伤行内减压术患者的正常脑组织中H3乙酰化组蛋白的表达情况，RealtimePCR检测高级别和低级别胶质瘤组织内Nanog表达情况。②通过组蛋白去乙酰化酶抑制剂Apicidin干预U87胶质瘤细胞，不同浓度和时间干预过后，，通过RT-PCR和Western-blot实验分别检测U87细胞株中H3乙酰化组蛋白和Nanog的mRNA和蛋白表达水平，ChIP检测U87细胞系中Nanog启动子区域H3组蛋白乙酰化水平。③通过MTT、细胞划痕实验、流式细胞术检测细胞凋亡、细胞周期、透射电镜检测凋亡、裸鼠成瘤实验、免疫组织化学染色等方法检测组蛋白H3低乙酰化水平抑制Nanog后对胶质瘤细胞体内外生物学活性的影响。 结果：①与正常脑组织相比，乙酰化组蛋白H3在不同级别胶质瘤中的表达均明显上调（P＜0.05）。WHO Ⅲ～Ⅳ级胶质瘤组织中乙酰化组蛋白H3表达明显高于WHOⅠ～Ⅱ级（P＜0.05）。与正常脑组织相比，在胶质瘤细胞系U87中的表达亦显著升高（P＜0.05）。②利用组蛋白去乙酰化酶抑制剂Apicidin干预胶质瘤U87细胞系后，同时检测乙酰化组蛋白H3及Nanog表达水平的变化，发现Nanog启动子区域的组蛋白H3乙酰化水平降低，同时Nanog的表达亦明显降低；③与空白对照组相比，U87-Apicidin胶质瘤细胞的增殖水平和迁移能力均减弱，细胞凋亡增加； U87-Apicidin皮下胶质瘤的体积与质量都明显小于空白对照组，提示Apicidin处理组U87细胞裸鼠皮下成瘤能力亦明显下降（P＜0.05）。 结论：①组蛋白H3乙酰化可能与胶质瘤的发生发展关系密切。胶质瘤级别越高，乙酰化组蛋白H3的表达量越高；②Nanog启动子区域的组蛋白H3乙酰化修饰水平可以影响Nanog的表达；③Apicidin可以特异性的降低Nanog启动子区域乙酰化组蛋白H3水平，从而影响Nanog的表达，最终对胶质瘤的生物学活性产生抑制性作用。
[Abstract]:Objective: to study the expression of H3 acetylated histone in normal brain tissue, glioma tissue of different grades and glioma cell line U87, and to use deacetylase inhibitor Apicidin to interfere with U87 glioma cell line. The effect of acetylation of protein H3 on Nanog in glioma cells and the effect of low acetylation of histone H3 on the biological activity of glioma cells in vivo and in vitro were investigated. To lay a foundation for the further study of epigenetic intervention therapy of Nanog. Methods: 1the expression of H3 acetylated histone was detected by Western-Blot in 53 gliomas with different grades and 11 normal brain tissues from patients with craniocerebral injury undergoing internal decompression. RealtimePCR was used to detect the expression of Nanog in high grade and low grade gliomas. (2) U87 glioma cells were treated with histone deacetylase inhibitor (Apicidin). The mRNA and protein expressions of H3 acetylated histone and Nanog in U87 cell line were detected by RT-PCR and Western-blot assay respectively. The H3 histone acetylation level of Nanog promoter region in U87 cell line was detected by ChIP. 3 the H3 histone acetylation level was detected by MTT, cell scratch assay. Flow cytometry was used to detect cell apoptosis, cell cycle, transmission electron microscopy (TEM), and tumorigenesis in nude mice. The effects of low acetylation of histone H3 on the biological activity of glioma cells in vivo and in vitro were detected by immunohistochemical staining. Results: 1Compared with normal brain tissue, The expression of acetylated histone H3 was significantly up-regulated in different grade gliomas (P < 0. 05). The expression of acetylated histone H3 in gliomas of grade 鈪

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