Department of Neurosurgery, Henry Ford Health System, 2799 W Grand Blvd, Detroit, MI 48202, USA.
Int J Oncol. 2012 Feb;40(2):494-500. doi: 10.3892/ijo.2011.1229. Epub 2011 Oct 13.
In this study we investigated epigenetic modifications such as DNA methylation, histone acetylation and histone methylation in the regulation of heparanase expression in glioblastoma. We found that heparanase promoters are differentially methylated among three glioblastoma cell lines; however, all these cells expressed baseline levels of heparanase. 5-Aza-2'-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, revoked heparanase expression in all the examined cells. Trichostatin A (TSA), a histone deacetylase inhibitor, activated heparanase expression in promoter unmethylated LN229 and T98G cells but not in promoter methylated U251n cells. To identify the mechanisms of heparanase induction by 5-Aza-dC, heparanase expression-related transcription factors were examined. No detected transcription factors (EGR1, Ets1, GABPα and Sp1) were found to be induced either by 5-Aza-dC or TSA. Furthermore, we found that 5-Aza-dC increased acetylation of histone H3 and di-methylation of histone H3 lysine K4 (H3K4me2) in LN229 and T98G cells. The increased histone acetylation and H3K4me2 were also observed in heparanase-expressing tumor tissues by immunohistochemistry staining. Additionally, we found that nuclear factor κB (NFκB) p65 but not NFκB p50 was correlated with heparanase expression, which could be expressed both by neoplastic cells and angiogenesis-related neovessel cells. However, we did not observe any regulatory mechanism between heparanase and NFκB p65 via transient transfection of their cDNA in T98G and U251n cells. We concluded that heparanase expression is associated with histone modifications and promoter DNA methylation plays a role in the control of gene silencing. Overexpression of both heparanase and NFκB p65 may be the result of excessive histone modifications.
在这项研究中，我们研究了表观遗传修饰，如 DNA 甲基化、组蛋白乙酰化和组蛋白甲基化，以调节神经胶质瘤中肝素酶的表达。我们发现，三种神经胶质瘤细胞系中肝素酶启动子的甲基化程度不同；然而，所有这些细胞都表达了基础水平的肝素酶。DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷（5-Aza-dC）在所有检测的细胞中逆转了肝素酶的表达。组蛋白去乙酰化酶抑制剂曲古抑菌素 A（TSA）在启动子非甲基化的 LN229 和 T98G 细胞中激活了肝素酶的表达，但在启动子甲基化的 U251n 细胞中没有激活。为了确定 5-Aza-dC 诱导肝素酶表达的机制，我们检测了与肝素酶表达相关的转录因子。未发现转录因子（EGR1、Ets1、GABPα 和 Sp1）被 5-Aza-dC 或 TSA 诱导。此外，我们发现 5-Aza-dC 增加了 LN229 和 T98G 细胞中组蛋白 H3 的乙酰化和组蛋白 H3 赖氨酸 K4 的二甲基化（H3K4me2）。免疫组织化学染色也显示，肝素酶表达的肿瘤组织中存在增加的组蛋白乙酰化和 H3K4me2。此外，我们发现核因子 κB（NFκB）p65 而不是 NFκB p50 与肝素酶表达相关，它可以由肿瘤细胞和血管生成相关的新生血管细胞表达。然而，我们在 T98G 和 U251n 细胞中转染其 cDNA 时，并没有观察到肝素酶和 NFκB p65 之间的任何调节机制。我们得出结论，肝素酶的表达与组蛋白修饰有关，启动子 DNA 甲基化在基因沉默的调控中起作用。肝素酶和 NFκB p65 的过度表达可能是组蛋白修饰过度的结果。
