Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY, 40202, USA.
Medical University of Warsaw and Center for Preclinical Research and Technology, Warsaw, Poland.
Stem Cell Rev Rep. 2018 Dec;14(6):823-836. doi: 10.1007/s12015-018-9838-5.
The paternally-imprinted genes insulin-like growth factor 2 (IGF2), H19, delta-like homologue 1 (DLK1), and maternally-expressed gene 3 (MEG3) are expressed from the tandem gene loci IGF2-H19 and DLK1-MEG3, which play crucial roles in initiating embryogenesis and development. The erasure of imprinting (EOI) at differentially methylated regions (DMRs) which regulate the expression of these genes maintains the developmental quiescence of primordial germ cells (PGCs) migrating through the embryo proper during embryogenesis and prevents them from forming teratomas. To address the potential involvement of the IGF2-H19 and DLK1-MEG3 loci in the pathogenesis of embryonal carcinoma (EC), we investigated their genomic imprinting at DMRs in the human PGC-derived EC cell line NTera-2 (NT2). We observed EOI at the IGF2-H19 locus and, somewhat to our surprise, a loss of imprinting (LOI) at the DLK1-MEG3 locus. As a result, NT2 cells express imprinted gene ratios from these loci such that there are i) low levels of the proliferation-promoting IGF2 relative to ii) high levels of the proliferation-inhibiting long noncoding RNA (lncRNA) H19 and iii) high levels of proliferation-promoting DLK1 relative to iv) low levels of the proliferation-inhibiting lncRNA MEG3. Consistent with this pattern of expression, the knockdown of DLK1 mRNA by shRNA resulted in decreased in vitro cell proliferation and in vivo tumor growth as well as decreased in vivo organ seeding by NT2 cells. Furthermore, treatment of NT2 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaD) inhibited their proliferation. This inhibition was accompanied by changes in expression of both tandem gene sets: a decrease in the expression of DLK1 and upregulation of the proliferation-inhibiting lncRNA MEG3, and at the same time upregulation of IGF2 and downregulation of the lncRNA H19. These results suggest that the DLK1-MEG3 locus, and not the IGF2-H19 locus, drives the tumorigenicity of NT2 cells. Based on these results, we identified DLK1 as a novel treatment target for EC that could be downregulated by 5-azaD.
父系印迹基因胰岛素样生长因子 2 (IGF2)、H19、Delta-like 同源物 1 (DLK1)和母系表达基因 3 (MEG3) 从串联基因座 IGF2-H19 和 DLK1-MEG3 表达，这些基因在启动胚胎发生和发育中起着至关重要的作用。印迹消除 (EOI) 在调节这些基因表达的差异甲基化区域 (DMRs) 上，维持了在胚胎发生过程中通过胚胎 proper 迁移的原始生殖细胞 (PGCs) 的发育静止，并防止它们形成畸胎瘤。为了研究 IGF2-H19 和 DLK1-MEG3 基因座在胚胎癌细胞 (EC) 发病机制中的潜在作用，我们研究了人类 PGC 衍生的 EC 细胞系 NTera-2 (NT2) 中这些基因座在 DMRs 上的基因组印迹。我们观察到 IGF2-H19 基因座上的 EOI，并且有些出乎我们的意料，DLK1-MEG3 基因座上的印记丢失 (LOI)。结果，NT2 细胞表达这些基因座的印迹基因比例为：i) 增殖促进 IGF2 的低水平，ii) 增殖抑制长非编码 RNA (lncRNA) H19 的高水平，iii) 增殖促进 DLK1 的高水平，以及 iv) 增殖抑制 lncRNA MEG3 的低水平。与这种表达模式一致，通过 shRNA 敲低 DLK1 mRNA 导致 NT2 细胞体外细胞增殖和体内肿瘤生长减少，以及 NT2 细胞体内器官播种减少。此外，用 DNA 甲基转移酶抑制剂 5-氮杂-2'-脱氧胞苷 (5-azaD) 处理 NT2 细胞可抑制其增殖。这种抑制伴随着串联基因簇表达的变化：DLK1 的表达减少和增殖抑制 lncRNA MEG3 的上调，同时 IGF2 的上调和 lncRNA H19 的下调。这些结果表明，DLK1-MEG3 基因座而非 IGF2-H19 基因座驱动 NT2 细胞的致瘤性。基于这些结果，我们确定了 DLK1 作为 EC 的一个新的治疗靶点，5-azaD 可以下调它。
