Liu Feng, Yu Zi-jian, Sui Jian-li, Bai Bei, Zhou Ping-kun
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing 100850, China.
Chin Med J (Engl). 2006 May 5;119(9):731-9.
Cockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene.
The vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid P(silencer TM 3.1). The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G(1) population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis.
Two constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to (gamma-ray and UV light. siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G(1) population were induced in control HeLa cells at 18 hours after being exposed to 10 J/m(2) of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells, but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G(1) phase population was observed in UV-irradiated CSB-siRNA transfected HeLa cells. In addition, siRNA-mediated silencing of CSB promoted the elimination of G(2)/M phase cells after UV light radiation.
siRNA-mediated silencing of CSB causes cells to proliferate more slowly, sensitize cells to genotoxicants, and modify UV radiation-induced cell cycle changes. siRNA-mediated inactivation of CSB could be an attractive strategy for ameliorating cancer therapy, which can be fulfilled via the combination of gene therapy and sensitization of radiotherapy or chemotherapy.
科凯恩综合征（CS）是一种罕见的人类遗传性疾病，其特征为紫外线敏感性增加、发育异常和早衰。从CS患者分离出的细胞在转录偶联DNA修复方面存在缺陷。尽管存在修复缺陷，但CS患者发生自发或紫外线诱导癌症的风险并未增加。本研究采用RNA干扰策略，探讨靶向CS组B（CSB）基因的潜在放射增敏和抗癌活性。
通过将编码双链小干扰RNA（siRNA）的寡核苷酸插入质粒P（silencer TM 3.1）构建编码CSB特异性siRNA的载体。用上述载体转染HeLa细胞，构建表达CSB-siRNA的细胞系。采用集落形成能力检测细胞存活率。通过FACScan流式细胞术分析细胞周期。通过检测亚G1期细胞群的积累以及荧光染色试验测定细胞凋亡。采用逆转录聚合酶链反应（RT-PCR）半定量mRNA表达。通过蛋白质印迹分析检测蛋白质水平。
构建了两种编码CSB特异性siRNA的载体，它们均显著抑制HeLa细胞中CSB的表达，并导致细胞对γ射线和紫外线的敏感性增加。siRNA介导的CSB沉默降低了细胞增殖率，增加了自发凋亡以及紫外线或顺铂诱导凋亡的发生率2至3.5倍。在对照HeLa细胞暴露于10 J/m2紫外线后18小时，诱导了显著的S期阻滞和G1期细胞群的显著减少。在紫外线照射的CSB-siRNA转染的HeLa细胞中也观察到S期阻滞，但S期细胞群增加的程度低于紫外线照射的对照细胞。在紫外线照射的CSB-siRNA转染的HeLa细胞中未观察到或观察到G1期细胞群相对较弱的减少。此外，siRNA介导的CSB沉默促进了紫外线照射后G2/M期细胞的清除。
siRNA介导的CSB沉默导致细胞增殖更缓慢，使细胞对基因毒性剂敏感，并改变紫外线辐射诱导的细胞周期变化。siRNA介导的CSB失活可能是改善癌症治疗的一种有吸引力的策略，这可以通过基因治疗与放疗或化疗增敏相结合来实现。
