摘要
背景与目的:Ku80为细胞受辐射致DNA双链断裂(DNA double-strand break,DSB)后的主要修复蛋白之一,Ku80的高表达预示着宫颈癌组织对放射线的抵抗,本研究运用RNAi技术抑制Ku80表达,观察宫颈癌Si-Ha细胞对X线敏感性的变化。方法:构建1个靶向抑制Ku80的siRNA表达载体和1个阴性对照质粒,稳定转染入SiHa细胞后Western blot和RT-PCR检测Ku80表达变化;6 MV X线照射10 Gy后20 h、28 h和48 h收集细胞,流式细胞术检测细胞凋亡率;克隆形成实验检测细胞D0、SF2和α等值。结果:构建表达质粒转染SiHa细胞经筛选后获得2个稳定转染细胞株,Western blot和RT-PCR分析表明转染pKu80-siRNA的阳性克隆Ku80在mRNA和蛋白质水平均受到明显抑制;X线照射28 h及48 h后,Ku80表达受抑细胞其凋亡率均显著高于转染阴性质粒组和空白对照组(P<0.05);Ku80受抑细胞的D0和SF2值分别为1.330和0.425,低于转染阴性质粒细胞(1.748和0.580)和空白对照细胞(1.835和0.597),α值为0.295,高于转染阴性质粒细胞(0.176)和空白对照细胞(0.188)。结论:运用RNAi技术可以有效抑制Ku80的表达从而促进SiHa细胞对X线的敏感性,Ku80可能是一个较理想的肿瘤分子治疗靶点。
Background and purpose： Ku80 protein is one of the players in the repair of DNA double strand break （DSB） after irradiation, the high expression of Ku80 in cervical carcinoma has been found to have an association with its radioresistance. In this study, the radiosensitivity of SiHa cells could perhaps be enhanced through the inhibition of Ku80 expression by RNAi. Methods： pKu80-siRNA and pNeg-siRNA were constructed and were transfected into SiHa cells by lipofectamine. Western blot and RT-PCR were used to measure the expression of Ku80; After 10 Gy irradiation in a single fraction, cells were collected and apoptosis was estimated by flow cytometry at 20 hr, 28 hr and 48 hr; The D0, SF2 and ？ values of three cell lines were calculated by clone formation array. Results： Two stable transfected cell lines-SiHa/Neg-siRNA and SiHa/Ku80-siRNA were screened from the transfected SiHa cells. Western blot analysis and RT-PCR indicated that the expression of Ku80 was suppressed by the Ku80-siRNA; the apoptosis rates of SiHa/Ku80-siRNA were higher than those of control cells at 28 hr and 48 hr after irradiation （P 〈0.05） . The D0 and SF2 values of SiHa/Ku80-siRNA were 1. 330 and 0. 425, respectively, lower than those of SiHa/Neg-siRNA （ 1. 748 and 0. 580, respectively） or control cells （ 1. 835 and 0. 597, respectively）, and the α value was 0. 295, higher than that of both SiHa/Neg-siRNA （0. 176） and control cells（0.188）. Conclusions： Inhibition of Ku80 could enbance the radiosensitivity of SiHa, so Ku80 may be a good candidate for target therapy.
出处
《中国癌症杂志》
CAS
CSCD
2007年第5期385-389,共5页
China Oncology
