Silencing of DNA methyltransferase 1 by siRNA influences fetal bovine fibroblast cell proliferation and apoptosis
Silencing of DNA methyltransferase 1 by siRNA influences fetal bovine fibroblast cell proliferation and apoptosis
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siRNA沉默DNA甲基转移酶1影响胎牛成纤维细胞增殖和凋亡
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易建明
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于孟飞;王文璐;易建明
DNA 甲基转移酶 1 (Dnmt1) 在基因组 DNA 复制和早期胚胎发育过程中维持 DNA 甲基化模式方面发挥着至关重要的作用。异常的 Dnmt1 表达会导致表观遗传变化，从而导致体细胞核移植 (SCNT) 功效低下。最近的研究表明，通过 RNA 干扰敲低牛成纤维细胞中的 Dnmt mRNA 有利于提高 SCNT 功效。本研究的目的是确定靶向 Dnmt1 的 siRNA 对胎牛成纤维细胞 (FBFC) 的影响。我们针对 Dnmt1 设计了三种 siRNA，用于转染至 FBFC 中。 Dnmt1-siRNA3在24、48和72 h时mRNA表达水平显着降低（P<0.01）。转染后48小时，siRNA3对Dnmt1的抑制效果达到近80%。 Dnmt1-siRNA3 处理 FBFC 也显着抑制其增殖，降低细胞活力，并在 G0/G1 转变中阻止更多细胞 (P<0.05)。然而，Dnmt1-siRNA3增加了凋亡细胞的数量（P<0.05）。这项研究表明，针对 Dnmt1 的特异性 siRNA 可以有效降低 FBFC 中 Dnmt1 mRNA 的表达。这表明在 siRNA 处理的供体细胞中观察到的 SCNT 功效的改善可能部分归因于细胞周期分布的变化。
DNA methyltransferase 1 (Dnmt1) plays a crucial role in maintaining DNA methylation patterns during genomic DNA replication and early embryonic development. Aberrant Dnmt1 expression leads to epigenetic changes, which result in low somatic cell nuclear transfer (SCNT) efficacy. Recent studies demonstrated that knockdown of the Dnmt mRNA in bovine fibroblast cells via RNA interference was beneficial in improving the SCNT efficacy. The objective of the present study was to determine the effects of siRNAs targeting Dnmt1 on fetal bovine fibroblast cells (FBFCs). We designed three siRNAs against Dnmt1 for transfection into FBFCs. Dnmt1-siRNA3 significantly reduced the mRNA expression level at 24, 48 and 72 h (P<0.01). At 48 h post-transfection, the inhibitory efficacy of siRNA3 against Dnmt1 reached nearly 80%. Dnmt1-siRNA3 treatment of FBFCs also significantly inhibited their proliferation, reduced cell viability and arrested more cells in the G0/G1 transition (P<0.05). However, Dnmt1-siRNA3 increased the number of apoptotic cells (P<0.05). This study demonstrated that specific siRNAs targeting Dnmt1 effectively knocked down Dnmt1 mRNA expression in FBFCs. It suggests that the improved SCNT efficacy observed in the siRNA-treated donor cells might be partially due to changes in the cell cycle distribution.