The Rb1 gene inhibits the viability of retinoblastoma cells by regulating homologous recombination.
The Rb1 gene inhibits the viability of retinoblastoma cells by regulating homologous recombination.
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DOI：
10.3892/ijmm.2013.1374
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发表时间：
2013-07
影响因子：
5.4
通讯作者：
Ying Yang;Sijia Tian;Bryce Brown;Pei Chen;Huan Hu;Lei Xia;Jing Zhang;Xiaoxiao Cai;Zhao Chen;Xueke Pan;J. Ge;Keming Yu;J. Zhuang
中科院分区：
文献类型：
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作者：
Ying Yang;Sijia Tian;Bryce Brown;Pei Chen;Huan Hu;Lei Xia;Jing Zhang;Xiaoxiao Cai;Zhao Chen;Xueke Pan;J. Ge;Keming Yu;J. Zhuang
视网膜母细胞瘤是由视网膜母细胞瘤基因（Rb1）的两个等位基因失活引起的儿童眼部肿瘤。没有Rb1基因功能，在视网膜母细胞瘤细胞中观察到染色体畸变。基因组的不稳定性与DNA双链断裂（DSBs）的修复密切相关。然而，Rb1在DNA DSB修复中的确切分子机制尚不清楚。因此，在本研究中，我们旨在通过检测DNA DSB修复来研究Rb1基因是否影响DNA稳定性，以及Rb1基因是否调节视网膜母细胞瘤细胞的增殖。Rb1免疫荧光和RT-PCR结果显示，Rb1基因在SO-Rb50视网膜母细胞瘤细胞中沉默，对SO-Rb50细胞的核型分析表明，Rb1功能缺失导致基因组不稳定；在我们的研究中观察到数量和结构染色体畸变。此外，采用γ-H2AX免疫荧光法（一种常用的DNA DSB原位标记物）检测辐照（2.5 Gy和5.0 Gy）后SO-Rb50细胞的DNA DSB修复效率。我们发现DNA修复效率在ir诱导损伤后显著提高（P0.05）。筛选Rb1对DNA DSB修复亚通路、非同源末端连接（non-homologous end joining， NHEJ）和同源重组（homologous recombination， HR）活性的影响结果表明，Rb1对NHEJ活性没有影响，但显著促进了HR通路的活性（HR水平较对照提高了2.46倍）（P<0.01）。此外，我们发现外源性Rb1转染的SO-Rb50细胞的细胞活力明显受到抑制（P<0.01），细胞周期试验表明外源性Rb1诱导S期阻滞（P<0.001），并抑制视网膜母细胞瘤细胞（SO-Rb50）的体外增殖。因此，本研究为Rb1基因调控视网膜母细胞瘤细胞增殖的作用机制提供了新的认识。
Retinoblastoma is a childhood ocular tumor caused by the inactivation of both alleles of the retinoblastoma gene (Rb1). Without Rb1 gene function, chromosomal aberrations are observed in retinoblastoma cells. The instability of the genome is closely associated with the repair of DNA double-strand breaks (DSBs). However, the precise molecular mechanism of action of Rb1 in DNA DSB repair remains unclear. Thus, in this study, we aimed to investigate whether the Rb1 gene affects DNA stability by assaying DNA DSB repair and also whether it regulates the proliferation of retinoblastoma cells. Rb1 immunofluorescence and RT-PCR were performed, demonstrating that the Rb1 gene is silenced in SO-Rb50 retinoblastoma cells, and the karyotype analysis of SO-Rb50 cells indicated that the loss of Rb1 function led to genomic instability; both numerical and structural chromosomal aberrations were observed in our study. In addition, the DNA DSB repair efficiency of the SO-Rb50 cells was measured by γ-H2AX immunofluorescence, a commonly used in situ marker of DNA DSBs, following exposure to ionizing radiation (IR) (2.5 and 5.0 Gy). We found that the DNA repair efficiency was significantly increased following IR-induced damage (P0.05). The assay for the screening of the effect of Rb1 on the sub-pathway of DNA DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR), indicated that Rb1 did not affect NHEJ activity, although it significantly promoted the HR pathway (HR levels increased by 2.46-fold) compared with the control (P<0.01). Furthermore, we found that the cell viability of the SO-Rb50 cells transfected with exogenous Rb1 was significantly inhibited (P<0.01) and cell cycle assay indicated that exogenous Rb1 induced S phase arrest (P<0.001) which also inhibited the proliferation of retinoblastoma cells (SO-Rb50) in vitro. Therefore, this study provides new insight into the mechanisms of action of the Rb1 gene in regulating the proliferation of retinoblastoma cells.