UHRF1 depletion and HDAC inhibition reactivate epigenetically silenced genes in colorectal cancer cells
UHRF1 depletion and HDAC inhibition reactivate epigenetically silenced genes in colorectal cancer cells
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DOI：
10.1186/s13148-019-0668-3
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发表时间：
2019-05-07
影响因子：
5.7
通讯作者：
Suzuki, Hiromu
中科院分区：
文献类型：
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作者：
Niinuma, Takeshi;Kitajima, Hiroshi;Suzuki, Hiromu
背景含有 PHD 和环指结构域 1 (UHRF1) 的泛素样蛋白是表观遗传机制的主要调节因子，并且在各种人类恶性肿瘤中过度表达。在本研究中，我们检查了 UHRF1 在结直肠癌 (CRC) 中异常 DNA 甲基化和基因沉默中的参与情况。结果 用靶向 UHRF1 的 siRNA 瞬时转染 CRC 细胞系，然后使用斑点印迹、亚硫酸氢盐焦磷酸测序和 Infinium HumanMmethylation450 BeadChip 检测分析 DNA 甲基化。使用RT-PCR和基因表达微阵列分析基因表达。 UHRF1 的缺失迅速诱导 CRC 细胞中全基因组 DNA 去甲基化。 Infinium BeadChip 检测和亚硫酸氢盐焦磷酸测序揭示了整个基因组区域的显着去甲基化，包括 CpG 岛、基因体、基因间区域和重复元件。然而，尽管存在大量去甲基化，但 UHRF1 敲除仅最小程度地逆转了 CpG 岛高甲基化相关基因沉默。相比之下，UHRF1 缺失和组蛋白脱乙酰酶 (HDAC) 抑制相结合，重新激活了沉默的基因，并强烈抑制了 CRC 细胞增殖。 UHRF1 去除和 HDAC 抑制相结合也引起基因表达谱的显着变化，从而使细胞周期相关基因显着下调。结论我们的结果表明，（i）CRC 细胞中 DNA 甲基化的维持高度依赖于 UHRF1； (ii) UHRF1 耗竭迅速诱导 DNA 去甲基化，但不足以完全重新激活沉默的基因； (iii)UHRF1和HDAC的双重靶向可能是一种有效的新治疗策略。
BackgroundUbiquitin-like protein containing PHD and RING finger domains 1 (UHRF1) is a major regulator of epigenetic mechanisms and is overexpressed in various human malignancies. In this study, we examined the involvement of UHRF1 in aberrant DNA methylation and gene silencing in colorectal cancer (CRC).ResultsCRC cell lines were transiently transfected with siRNAs targeting UHRF1, after which DNA methylation was analyzed using dot blots, bisulfite pyrosequencing, and Infinium HumanMethylation450 BeadChip assays. Gene expression was analyzed using RT-PCR and gene expression microarrays. Depletion of UHRF1 rapidly induced genome-wide DNA demethylation in CRC cells. Infinium BeadChip assays and bisulfite pyrosequencing revealed significant demethylation across entire genomic regions, including CpG islands, gene bodies, intergenic regions, and repetitive elements. Despite the substantial demethylation, however, UHRF1 depletion only minimally reversed CpG island hypermethylation-associated gene silencing. By contrast, the combination of UHRF1 depletion and histone deacetylase (HDAC) inhibition reactivated the silenced genes and strongly suppressed CRC cell proliferation. The combination of UHRF1 depletion and HDAC inhibition also induced marked changes in the gene expression profiles such that cell cycle-related genes were strikingly downregulated.ConclusionsOur results suggest that (i) maintenance of DNA methylation in CRC cells is highly dependent on UHRF1; (ii) UHRF1 depletion rapidly induces DNA demethylation, though it is insufficient to fully reactivate the silenced genes; and (iii) dual targeting of UHRF1 and HDAC may be an effective new therapeutic strategy.