DNA甲基化转移酶1基因沉默干扰胎牛成纤维细胞增殖和凋亡
Silencing of DNA methyltransferase 1 by siRNA influences fetal bovine fibroblast cell proliferation and apoptosis
投稿时间:2017-01-07  修订日期:2017-04-10
DOI:
中文关键词: 凋亡  细胞周期  供体细胞  增殖  RNA干扰
英文关键词: apoptosis  cell cycle  donor cell  proliferation  RNA interfernce  
基金项目:国家自然科学基金青年基金资助项目(81400252);中央高校基本科研业务费专项资金资助项目(2013PY130)
作者单位E-mail
于孟飞 中南民族大学 生命科学学院 alabaster1001@163.com 
王文璐 华中农业大学 动物科技学院,农业动物遗传育种与繁殖教育部重点实验室 phlyysgf@yahoo.com.cn 
易建明 华中农业大学 动物科技学院,农业动物遗传育种与繁殖教育部重点实验室  
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中文摘要:
      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DNA甲基化模式的维持发挥重要作用中起关键作用.Dnmt1表达异常会引起表观遗传改变,并导致体细胞核移植(SCNT)效率低下.近期研究表明,通过RNA干扰在牛成纤维细胞中敲低Dnmt1 mRNA有利于提高SCNT的效率.本研究的目标是阐明Dnmt1-siRNA对胎牛成纤维细胞(FBFCs)的影响.在本研究中,我们设计了三种针对Dnmt1的siRNA来转染FBFCs.研究结果显示:Dnmt1-siRNA3转染FBFCs 24,48和72 h后,Dnmt1 mRNA表达水平显著降低(P <0.01).在转染后48小时,siRNA3对Dnmt1的抑制效率达到近80%.同时,Dnmt1-siRNA3处理的FBFCs的增殖也受到显著,并且FBFC细胞活力下降、处于G0/G1过渡期细胞数量增多(P <0.05).然而,Dnmt1-siRNA3组FBFC细胞的凋亡率也显著增加(P <0.05).这些结果表明通过Dnmt1特异性siRNA能够有效地敲低FBFC中的Dnmt1 mRNA表达量.siRNA处理后FBFC作为核供体可以提高SCNT的效率,这可能归因于细胞周期分布情况的改变.
英文摘要:
      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. The improved SCNT efficacy observed in the siRNA-treated donor cells might be partially due to changes in the cell cycle distribution.
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