BACKGROUND Epigenetic regulations play pivotal roles in tumorigenesis and cancer development. Disruptor of telomeric silencing-1-like (DOT1L), also known as KMT4, is the only identified histone methyltransferase that catalyzes the mono-, di-, and tri-methylation of lysine 79 histone 3 (H3K79). However, little is known about the effect of H3K79 methylation on the modulation of colorectal cancer (CRC) development. METHODS DOT1L expression profiles in different subgroups of CRC tissues and its clinical significances were analyzed from some online datasheets. DOT1L in CRC cell lines was silenced by either lentivirus-mediated knockdown or inhibited by its specific inhibitor, EPZ004777. Then cell proliferation was detected by MTT assay, BrdU assay, and soft agar assay; cell cycle was detected by cytometry; and tumorigenicity was detected by using nude mice xenograft models. Clinical co-expression was analyzed between DOT1L and c-Myc. Chromatin immunoprecipitation (ChIP) assay was used to determine whether the translation of c-Myc was epigenetically regulated by H3K79me2 induced by DOT1L. c-Myc overexpression was used to rescue the cell cycle arrest and tumor growth induced by DOT1L silencing or inhibition in CRC. RESULTS We found that DOT1L was highly expressed in colorectal cancer and was negatively related to the prognosis of patients with CRC. Silencing or inhibition of DOT1L blocked cell proliferation, BrdU incorporation, self-renewal capability in vitro, and tumorigenicity in vivo. Besides, inhibition or silencing of DOT1L also induced cell cycle arrest at S phase, as well as decreased the expression of CDK2 and Cyclin A2. Furthermore, in the clinical databases of CRC, we found that the expression of DOT1L was positively correlated with that of c-Myc, a major regulator in the upstream of cell cycle-related factors. Besides, c-Myc expression was downregulated after DOT1L knockdown and c-Myc restoration rescued decrease of cell proliferation, BrdU corporation, self-renewal capability, cell cycle progression in vitro and tumorigenicity in vivo induced by DOT1L silencing. Then we found that H3K79 methylation was decreased after DOT1L knockdown. ChIP assay showed that H3K79me2 was enriched on the - 682~+ 284 region of c-Myc promoter, and the enrichment was decreased after DOT1L inhibition. CONCLUSIONS Our results show that DOT1L epigenetically promotes the transcription of c-Myc via H3K79me2. DOT1L silencing or inhibition induces cell cycle arrest at S phase. DOT1L is a potential marker for colorectal cancer and EPZ004777 may be a potential drug for the treatment of colorectal cancer.

中文翻译：

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H3K79 甲基转移酶 DOT1L 的沉默或抑制通过表观遗传调节结直肠癌中的 c-Myc 表达来诱导细胞周期停滞。

背景表观遗传调控在肿瘤发生和癌症发展中起关键作用。端粒沉默 1 样 (DOT1L) 的干扰物，也称为 KMT4，是唯一已鉴定的组蛋白甲基转移酶，可催化赖氨酸 79 组蛋白 3 (H3K79) 的单甲基化、二甲基化和三甲基化。然而，关于 H3K79 甲基化对调节结直肠癌 (CRC) 发展的影响知之甚少。方法从一些在线数据表中分析了CRC组织不同亚组中的DOT1L表达谱及其临床意义。CRC 细胞系中的 DOT1L 被慢病毒介导的敲低或被其特异性抑制剂 EPZ004777 抑制。然后用MTT法、BrdU法、软琼脂法检测细胞增殖情况；细胞周期检测；采用裸鼠异种移植模型检测致瘤性。分析了 DOT1L 和 c-Myc 之间的临床共表达。染色质免疫沉淀 (ChIP) 测定用于确定 c-Myc 的翻译是否受到 DOT1L 诱导的 H3K79me2 的表观遗传调控。c-Myc 过表达用于挽救由 DOT1L 沉默或抑制在 CRC 中诱导的细胞周期停滞和肿瘤生长。结果我们发现DOT1L在结直肠癌中高表达，与结直肠癌患者的预后呈负相关。DOT1L 的沉默或抑制可阻断细胞增殖、BrdU 掺入、体外自我更新能力和体内致瘤性。此外，DOT1L的抑制或沉默也诱导细胞周期停滞在S期，并降低CDK2和Cyclin A2的表达。此外，在CRC的临床数据库中，我们发现DOT1L的表达与细胞周期相关因子上游的主要调节因子c-Myc的表达呈正相关。此外，DOT1L 敲低后 c-Myc 表达下调，c-Myc 恢复挽救了 DOT1L 沉默诱导的细胞增殖、BrdU 公司、自我更新能力、体外细胞周期进展和体内致瘤性的降低。然后我们发现 DOT1L 敲低后 H3K79 甲基化降低。ChIP检测显示H3K79me2富集在c-Myc启动子的-682~+284区域，DOT1L抑制后富集减少。结论 我们的结果表明，DOT1L 通过 H3K79me2 表观遗传促进 c-Myc 的转录。DOT1L 沉默或抑制在 S 期诱导细胞周期停滞。