下调UHRF1表达对食管鳞癌Eca-109细胞放射敏感性的影响  

Knock-down of UHRF1 expression enhances the radiosensitivity of Eca-109 cells

作　　者：惠蓓娜[1] 车少敏[1] 孙宇晨 龚拓拓 张晓智[1] HUI Beina;CHE Shaomin;SUN Yuchen;GONG Tuotuo;ZHANG Xiaozhi(Department of Oncology Radiotherapy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China)

机构地区：[1]西安交通大学第一附属医院肿瘤放疗科,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2020年第4期525-529,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：陕西省重点项目-社会发展领域(No.2018ZDXM-SF-043)。

摘　　要：目的研究下调UHRF1对食管鳞癌Eca-109细胞放射敏感性的影响,并对其作用机制进行探讨。方法以人UHRF1基因为靶标设计shRNA序列,慢病毒介导将UHRF1-shRNA转染至Eca-109细胞,qRT-PCR及Western blotting测定转染前后UHRF1 mRNA及蛋白的表达;克隆形成实验评估UHRF1沉默后Eca-109细胞放射敏感性的改变;流式细胞术检测沉默UHRF1对X线照射前后Eca-109细胞凋亡影响;Western blotting检测下调UHRF1、X线照射前后Eca-109细胞中凋亡相关蛋白Bcl-2、caspase-3及AKT/mTOR信号通路相关蛋白表达的影响。结果成功构建了UHRF1表达稳定下调的Eca-109 shUHRF1细胞系;克隆形成实验显示,UHRF1表达下调后Eca-109细胞的放射敏感性提高;流式细胞术分析显示,shUHRF1组凋亡细胞的比例较NC组增加,而照射后这种趋势更加明显;Western blotting检测显示,与NC组相比,shUHRF1组凋亡抑制蛋白Bcl-2表达量下降,凋亡蛋白Active caspase-3表达量升高,AKT/mTOR信号通路关键分子p-AKT、p-mTOR蛋白表达量下降,而6 Gy X线照射后,Bcl-2、Active caspase-3、p-AKT、p-mTOR蛋白变化较照射前更为明显。结论沉默UHRF1可以增加食管癌Eca-109细胞的放射敏感性,其机制与减弱AKT/mTOR信号通路传导活性及诱导凋亡有关。Objective To investigate the effect of ubiquitin-like with PHD and ring finger domains 1(UHRF1)on the radiosensitivity of esophageal squamous carcinoma cells and explore its mechanism.Methods The shRNA sequence was designed with human UHRF1 as the target,and the UHRF1-shRNA was transfected into Eca-109 cells mediated by lentivirus.The expressions of UHRF1 mRNA and protein before and after transfection were determined by fluorescence quantitative PCR and Western blotting.The radiosensitivity of Eca-109 cells after down-regulation of UHRF1 gene was assessed by colony formation assay.The effect of silencing UHRF1 on the apoptosis of Eca-109 cells before and after X-ray irradiation was detected by flow cytometry.Western blotting was used to detect the effects of UHRF1 silencing and X-ray irradiation on the expressions of apoptosis-related proteins Bcl-2,caspase-3 and proteins related to AKT/mTOR signaling pathway in Eca-109 cells.Results We successfully constructed an Eca-109 shUHRF1 cell line with UHRF1 stablely down-regulated.Colony formation assays showed that the radiosensitivity of Eca-109 cells was increased after down-regulation of UHRF1 expression compared with the NC group.The proportion of apoptotic cells in Eca-109 cells was increased,while this change was more obvious after irradiation.Compared with NC group,down-regulation of UHRF1 expression increased the level of active caspase-3 protein and decreased the level of the apoptosis inhibitory protein Bcl-2;the expression levels of p-AKT and p-mTOR protein were decreased.The changes in these four proteins in Eca-109 cells with down-regulation of UHRF1 combined with 6 Gy X-ray irradiation were more obvious compared with the NC group.Conclusion UHRF1 knock-down increased the radiosensitivity of ESCC via inhibiting the Akt/mTOR signaling pathway activity and inducing the apoptosis of the cells.

关 键 词：食管癌 UHRF1 SHRNA 放射敏感性 

分 类 号：R735.1[医药卫生—肿瘤]