Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs).
Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs).
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DOI：
10.1042/cbr20110003
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Isik S
中科院分区：
文献类型：
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作者：
Kamaci N;Emnacar T;Karakas N;Arikan G;Tsutsui K;Isik S
人骨髓间充质干细胞(HMSCs)表达拓扑异构酶II(Topo II)的两种亚型。虽然这两种异构体具有相同的催化活性，但它们在细胞中具有不同的功能：Topo IIα对有丝分裂细胞中的染色体分离是必不可少的，而Topo IIβ参与更具体的细胞功能。有许多抑制剂可以抑制这两种Topo II亚型的催化活性。然而，为了研究这两种酶的异构体特异性抑制，有必要使用其他技术，如siRNA(小干扰RNA)干扰来选择性地分别沉默其中一种异构体。根据细胞膜的脂电荷密度和蛋白质种类的不同，以往的研究表明siRNAs对hMSCs的转染率很低。在本文报道的研究中，我们证明了使用脂质体RNAiMAX作为一种有效的转染剂将siRNA导入人骨髓间充质干细胞，具有显著的高效率选择性地沉默Topo IIβ。通过使用未标记的Topo IIβ特异性siRNA寡核苷酸，获得了高水平的转染效率(80%)。具体地说，人们反复证实绿色标记的siRNAs干扰siRNAs的导入。该试剂诱导的细胞毒性最小(3.5-4.5%)，与未处理的细胞相比，转基因hMSCs的细胞活力降低了20-30%，这取决于试剂的浓度。
hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5–4.5%), and cell viability of the transfected hMSCs decreased 20–30% compared with untreated cells, depending on the concentration of the reagent.