DNA依赖性蛋白激酶催化亚基在电离辐射诱发HeLa细胞自噬中的作用  

The role of DNA-PKcs in autophagy induced by ionizing radiation

作　　者：韩艳勤 王豫[1] 刘晓丹 周平坤[1] 

机构地区：[1]军事医学科学院放射与辐射医学研究所北京市放射生物学重点实验室,北京100850

出　　处：《军事医学》2016年第7期537-540,共4页Military Medical Sciences

基　　金：国家自然科学基金资助项目(31370843)

摘　　要：目的了解DNA依赖性蛋白激酶催化亚基(DNA-PKcs)在电离辐射诱发细胞自噬中的作用。方法通过慢病毒载体(p SicoR)构建DNA-PKcs短发夹RNA(shRNA)表达载体,并转染HeLa细胞敲低DNA-PKcs表达。CCK-8实验检测细胞增殖活性及细胞放射敏感性,免疫印迹检测自噬相关蛋白表达,免疫荧光实验检测自噬标志物GFP-LC3的表达。结果成功构建shRNA敲低DNA-PKcs表达的HeLa细胞模型,通过该细胞模型观察到抑制DNA-PKcs蛋白表达导致细胞增殖能力降低、放射敏感性增高。P62和LC3蛋白表达变化,以及细胞内绿色荧光蛋白(GFP)-LC3免疫荧光斑检测结果显示,DNA-PKcs基因敲低后,明显增加X射线诱发细胞自噬。抑制DNA-PKcs导致哺乳动物西罗莫司(雷帕霉素)靶蛋白(mTOR)第2481位丝氨酸磷酸化水平降低。结论抑制DNA-PKcs增强受照射宫颈癌HeLa细胞的自噬及放射敏感性,mTOR信号通路可能参与DNA-PKcs对自噬的调节作用。Objective To investigate the effect of DNA-dependent protein kinase catalytic subunit （DNA-PKcs） on autophagy induction by ionizing radiation （IR）. Methods The cell model of knocking-down DNA-PKcs expression was constructed by transfecting HeLa cells with a pSicoR-based lentivirus vector expressing DNA-PKcs specific shRNA. Cellular growth activity and radiosensitivity were detected by cell countingkit （CCK） -8 assay. The expression of autophagy related proteins was detected by Western blotting hybridization. Autophagy was also detected by monitoring the autophagic marker green fluorescere protein （ GFP）-light chain 3 （ LC3 ） puncta per cell under an immunofluorescent microscope. Results A cellular model of knocking-down DNA-PKcs expression was successfully generated by transfecting the specific shRNA against DNA-PKcs. Depression of DNA-PKcs significantly decreased the growth activity of HeLa cells and increased the cellular sensitivity to ionizing radiation. Both the expression changes of P62 and LC3 proteins and immunofluorescent GFP- LC3 puncta observation indicated that knocking-down DNA-PKcs prompted the induction of autophagy by ionizing radiation. Moreover, inactivation of DNA-PKcs led to a decreased phosphorylation of mammalian target of sirolimus （ Rapamycin, RAPA） （mTOR） at S2481 site. Conclusion Depression of DNA-PKcs expression prompts the induction of autophagy by IR and cellular radiosensitivity, mTOR signaling may be involved in the regulation of autophagy processing by DNA-PKcs.

关 键 词：DNA依赖性蛋白激酶催化亚基 电离辐射 H La细胞 细胞增殖 自噬 西罗莫司 哺乳动物西罗莫司靶蛋白(mTOR) 

分 类 号：R818[医药卫生—放射医学]