Institute of Pathology and Pathophysiology, Shandong University School of Medicine, Jinan 250012, China.
J Hepatol. 2010 Nov;53(5):889-95. doi: 10.1016/j.jhep.2010.05.012. Epub 2010 Jul 14.
BACKGROUND & AIMS: The retinoblastoma-interacting zinc finger gene RIZ1 is inactivated in many cancers, but the underlying mechanisms remain unknown. This study aimed to investigate the epigenetic mechanisms of RIZ1 inactivation by analyzing the relationship between DNA methylation and histone modifications during regulation of RIZ1 expression.
Methylation-specific PCR, RT-PCR, and immunohistochemistry were performed to examine RIZ1 methylation and expression. Dynamic changes in histone H3 lysine 9 (H3K9) modifications and histone deacetylases (HDACs) associated with the promoter were analyzed by chromatin immunoprecipitation (ChIP).
RIZ1 methylation was detected in 66.7% (32/48) HCC tissues, 6.3% (3/48) corresponding non-cancerous tissues, and 66.7% (4/6) HCC cell lines. All 32 HCC tissues with promoter methylation showed complete loss of RIZ1 protein, whereas RIZ1 protein was present in all the corresponding non-cancerous tissues. Neither 5-aza-2-deoxycitidine (5-Aza-dC) nor Trichostatin A (TSA) reversed promoter methylation, but did restore RIZ1 mRNA and resulted in the downregulation of HDAC1 but not HDAC3. However, 5-Aza-dC+TSA induced a partial reversal of promoter methylation and a markedly synergistic reactivation of RIZ1. Moreover, both HDAC1 and HDAC3 were downregulated. The ChIP assays showed 5-Aza-dC and/or TSA also contributed to the dynamic conversion of trimethylated to acetylated H3K9 at the promoter. Furthermore, a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation.
Our results suggest that promoter methylation and H3K9 modifications work together to silence the RIZ1 gene in HCC. 5-Aza-dC can restore the expression of RIZ1, as reflected by its effects on histone modification levels. This finding indicates that cooperative effects between these epigenetic modifications exist.
视网膜母细胞瘤相互作用锌指蛋白基因 RIZ1 在许多癌症中失活，但潜在机制尚不清楚。本研究旨在通过分析 RIZ1 表达调控过程中 DNA 甲基化与组蛋白修饰之间的关系，研究 RIZ1 失活的表观遗传机制。
采用甲基化特异性 PCR、RT-PCR 和免疫组化方法检测 RIZ1 甲基化和表达。通过染色质免疫沉淀（ChIP）分析，研究与启动子相关的组蛋白 H3 赖氨酸 9（H3K9）修饰和组蛋白去乙酰化酶（HDACs）的动态变化。
在 66.7%（32/48）的 HCC 组织、6.3%（3/48）的相应非癌组织和 66.7%（4/6）的 HCC 细胞系中检测到 RIZ1 甲基化。所有 32 例启动子甲基化的 HCC 组织均完全缺失 RIZ1 蛋白，而相应的非癌组织均存在 RIZ1 蛋白。5-氮杂-2-脱氧胞苷（5-Aza-dC）和曲古抑菌素 A（TSA）均不能逆转启动子甲基化，但能恢复 RIZ1 mRNA，并导致 HDAC1 下调，而不是 HDAC3。然而，5-Aza-dC+TSA 诱导启动子甲基化部分逆转，并显著协同地重新激活 RIZ1。此外，HDAC1 和 HDAC3 均下调。ChIP 实验表明，5-Aza-dC 和/或 TSA 也有助于启动子上三甲基化 H3K9 向乙酰化 H3K9 的动态转化。此外，H3K9 三甲基化减少先于 H3K9 乙酰化增加。
我们的结果表明，启动子甲基化和 H3K9 修饰共同作用导致 HCC 中 RIZ1 基因沉默。5-Aza-dC 可通过影响组蛋白修饰水平恢复 RIZ1 的表达。这一发现表明这些表观遗传修饰之间存在协同作用。
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