Beclin1调控鼻咽癌细胞放疗抵抗的体外研究

Beclin1 promotes radioresistance in nasopharyngeal carcinoma in vitro

摘要:

目的探讨Beclin1基因对鼻咽癌细胞放疗抵抗的影响。方法Western blotting法检测CNE2、610B及其放疗抵抗细胞CNE2Rs、610BRs中Beclin1蛋白的表达，CNE2细胞经不同条件放射线照射后Beclin1蛋白的表达改变；通过脂质体转染Beclin1 cDNA和慢病毒介导的Beclin1 shRNA，在610B细胞和CNE2Rs细胞中分别过表达及沉默Beclin1基因，平板克隆集落形成实验检测鼻咽癌细胞的放疗抵抗能力的改变；细胞免疫荧光法检测DNA双链损伤相关指标γH2AX表达的改变。结果Beclin1蛋白在放疗抵抗细胞中表达增加，同时随着放疗时间的延长及放疗剂量的增加，Beclin1蛋白表达迅速升高，随后趋于基础水平；Beclin1过表达后610B细胞克隆集落形成能力增强，生存分数增加，γH2AX焦点数量减少；相反，Beclin1沉默后，CNE2Rs细胞克隆集落形成能力下降，生存分数下降，γH2AX焦点数量增加。结论Beclin1能促进鼻咽癌细胞的放疗抵抗性，其与放疗后DNA双链损伤修复调控可能相关。

Abstract:

ObjectiveTo study the function of Beclin1 on the radioresistance in nasopharyngeal carcinoma．MethodsWestern blotting was used to check the Beclin1 protein in CNE2, 610B and their radioresistant cell lines CNE2Rs, 610BRs,as well as the dynamic changes of Beclin1 protein in CNE2 cell exposed to irradiation under different conditions. Plate colony formation assay was applied to examine the alterations of radioresistant capacity in CNE2 and 610B after the expression of Beclin1 was upregulated with Lipofectaminemediated Beclin1 cDNA and inhibited with lentivirusmediated Beclin1 shRNA．Immunofluorescence was adopted to detect the expression of γH2AX，which was a surrogate marker of DNA doublestrand breaks．ResultsThe expression of Beclin1 protein was increased in radioresistant cell lines and after irradiation，which displayed an irradiation dosedependent and timedependent manner．Beclin1 overexpression in 610B cells resulted in enhanced ability to form colony on plate and high survival fraction, increased γH2AX foci formation. On the contrary，inhibition of Beclin1 expression in CNE2Rs cells led to declined ability of plate colony formation and low survival fraction, decreased γH2AX foci formation．ConclusionBeclin1 promotes radioresistance of nasopharyngeal carcinoma, which may be related to modulating the repair process of DNA doublestrand breaks．