Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, BC, Canada Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada.
Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
Mol Hum Reprod. 2015 May;21(5):452-65. doi: 10.1093/molehr/gav007. Epub 2015 Feb 19.
Previously we showed that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were affected by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450 K array). We identified 444 differentially methylated CpG loci in dNK-treated EVT compared with medium control (P < 0.05). The genes associated with these loci had critical biological roles in cellular development, cellular growth and proliferation, cell signaling, cellular assembly and organization by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltransferase IV) were chosen for follow-up studies because of their biological relevance from research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced compared with control (P < 0.01 for both CLDN4 and FUT4 mRNA expression; P < 0.001 for CLDN4 and P < 0.01 for FUT4 protein expression), and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by small interfering RNA reduced trophoblast invasion, possibly through the altered matrix metalloproteinase (MMP)-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduces protein expression. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility.
先前我们表明，在胶原凝胶外植体模型中，滋养细胞外胚层（EVT）的外突和迁移受到蜕膜自然杀伤细胞（dNK）的影响。本研究旨在探讨这一现象背后的分子原因。我们使用 Illumina Infinium HumanMethylation450 BeadChip 阵列（450 K 阵列）评估了暴露和未暴露的 EVT 的全基因组 DNA 甲基化。与培养基对照相比，dNK 处理的 EVT 中有 444 个差异甲基化 CpG 位点（P < 0.05）。通过 IPA 分析，与这些基因座相关的基因在细胞发育、细胞生长和增殖、细胞信号传导、细胞组装和组织中具有关键的生物学作用。此外，IPA 从 dNK 处理的 EVT 中鉴定出 23 个与迁移相关的基因。在这些基因中，CLDN4（编码紧密连接蛋白 4）和 FUT4（编码岩藻糖基转移酶 IV）被选择用于后续研究，因为它们在肿瘤细胞研究中的生物学相关性。结果表明，与对照组相比，dNK 处理的 EVT 中 CLDN4 和 FUT4 的 mRNA 和蛋白表达均显著降低（CLDN4 和 FUT4 mRNA 表达的 P 值均<0.01；CLDN4 的 P 值<0.001，FUT4 的 P 值<0.01），且与 DNA 甲基化呈负相关。用小干扰 RNA 敲低 CLDN4 和 FUT4 可降低滋养层的侵袭，这可能是通过改变基质金属蛋白酶（MMP）-2 和/或 MMP-9 的表达和活性。综上所述，dNK 通过改变 DNA 甲基化谱至少部分改变 EVT 的迁移能力。CLDN4 和 FUT4 的高甲基化降低了蛋白表达。CLDN4 和 FUT4 是参与调节滋养层迁移的代表性基因。
