Yang Yan-Ming, Fang Fang, Li Xin, Yu Lei, Wang Zhi-Cheng
Department of Radiotherapy, Second Hospital of Jilin University, Changchun, Jilin 130041, P.R. China.
Key Laboratory of Radiobiology, Ministry of Health, School of Public Health, Jilin University, Changchun, Jilin 130021, P.R. China.
Oncol Rep. 2017 Jan;37(1):533-539. doi: 10.3892/or.2016.5271. Epub 2016 Nov 23.
Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response 1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0 value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0 value of pc-H/E‑hsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.
电离辐射可上调肿瘤坏死因子相关凋亡诱导配体（TRAIL）的表达水平并增强肿瘤细胞凋亡。早期生长反应1（Egr1）基因启动子具有辐射诱导特性，由Egr1启动子控制的外源基因表达可因电离辐射而增强，但其效率受组织缺氧限制。缺氧反应元件（HREs）是重要的缺氧反应调节序列和敏感性增强子。因此，我们选择TRAIL作为基因放疗药物，观察其是否受Egr1和HRE调控及其对A549细胞的影响和机制。将含有Egr1-hsTRAIL和HRE/Egr1-hsTRAIL的pcDNA3.1-Egr1-TRAIL（pc-E-hsT）和pcDNA3.1-HRE/Egr1-TRAIL（pc-H/E-hsT）质粒转染至A549细胞，对细胞进行缺氧和辐射处理。分别通过逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）检测细胞中的TRAIL信使核糖核酸（mRNA）和培养上清液中的蛋白浓度。用集落形成试验评估平均致死剂量D0值。通过流式细胞术（FCM）和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）分析细胞凋亡率。通过蛋白质印迹法分析死亡受体4（DR4）、死亡受体5（DR5）和裂解的半胱天冬酶-3蛋白的表达。结果显示，在缺氧和/或辐射条件下，TRAIL mRNA表达和TRAIL浓度均显著增加。缺氧条件下pc-H/E-hsT转染细胞的D0值最低，表明其放射敏感性更高。缺氧不会导致pc-E-hsT转染细胞凋亡率增加，但对pc-H/E-hsT转染细胞有促进作用。在常氧和缺氧条件下，pc-E-hsT和pc-H/E-hsT转染细胞中的DR4无明显变化，而在缺氧条件下，pc-H/E-hsT转染细胞中的DR5和裂解的半胱天冬酶-3增加最多。TRAIL的过表达受Egr1和HRE共同调控。TRAIL可能通过依赖DR5至半胱天冬酶-3途径促进缺氧A549细胞的放射敏感性并诱导凋亡。
