Atg7-siRNA通过调节精氨酸循环干扰食管癌ECA109细胞放疗敏感性  

Atg7-siRNA interferes with radiosensitivity of esophageal cancer ECA109 cells by regulating arginine circulation

作　　者：张小红 周云 杜秋莹 任慧欣 王超群 ZHANG Xiaohong;ZHOU Yun;DU Qiuying;REN Huixin;WANG Chaoqun(Laboratory of Clinical and Experimental Pathology,Xuzhou Medical University,Xuzhou 221004,Jiangsu,China;Department of Radiotherapy,Xuzhou Clinical School of Xuzhou Medical University,Xuzhou 221004,Jiangsu,China)

机构地区：[1]徐州医科大学临床与实验病理学实验室,江苏徐州221004 [2]徐州医科大学徐州临床学院放疗科,江苏徐州221004

出　　处：《山东大学学报（医学版）》2021年第4期28-34,共7页Journal of Shandong University:Health Sciences

基　　金：徐州市推动科技创新专项资金(KC17111)。

摘　　要：目的探讨siRNA干扰自噬相关基因7(Atg7)后,对ECA109细胞放疗敏感性的影响以及对精氨酸循环相关通路的调控。方法食管癌ECA109细胞分为Ctrl组、siRNA组、Ctrl+R8Gy组(8Gy放射剂量处理的转染Atg7-Ctrl的ECA109细胞)和siRNA+R8Gy组(8Gy放射剂量处理的转染Atg7-siRNA的ECA109细胞);采用Western blotting法分别检测各组细胞Atg7、Beclin-1、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、精氨酸琥珀酸合成酶1(ASS1)和精氨酸琥珀酸裂解酶(ASL)蛋白的表达。CCK8实验检测精氨酸对ECA109细胞的影响,绘制不同浓度(0、8、16、32、64、128 mmol/L)精氨酸处理下的细胞存活曲线,并计算其IC50和IC10,将IC10作为后续实验的作用浓度。采用精氨酸和siRNA分别处理细胞,对细胞进行8Gy照射,24 h后通过计算细胞数量评估细胞的死亡率。结果无论放疗前后,siRNA干扰均可使细胞的Atg7蛋白下调(P<0.01)。与Ctrl组相比,Ctrl+R8Gy组自噬相关蛋白Beclin-1和LC3Ⅱ上调(P<0.01);与Ctrl+R8Gy组相比,siRNA+R8Gy组自噬相关蛋白Beclin-1和LC3Ⅱ下调(P<0.01)。与Ctrl+R8Gy组相比,siRNA+R8Gy组精氨酸循环相关通路蛋白ASS1和ASL上调(P<0.01)。精氨酸对ECA109细胞具有增殖抑制作用,且呈浓度和时间依赖性。放疗后24 h,细胞死亡量分析显示,siRNA干扰可以提高细胞死亡率(F=73.59,P<0.001),添加精氨酸可以提高细胞死亡率(F=158.72,P<0.001),siRNA和精氨酸同时作用时细胞死亡率最高(F=7.88,P=0.02)。结论放疗可以诱导ECA109细胞发生自噬,Atg7敲低可以拮抗放疗诱导的自噬,提高放疗诱导的ECA109细胞死亡率,增加ECA109细胞对放疗的敏感性。Atg7敲低可以调控ECA109细胞放疗后精氨酸循环相关通路蛋白,提高精氨酸浓度增加ECA109细胞的放射敏感性。Objective To investigate the effects of siRNA interference with autophagy related gene 7(Atg7)on the radiosensitivity of ECA109 cells and the regulation of arginine circulation-related pathways.Methods ECA109 cells were divided into four groups:Ctrl group,siRNA group,Ctrl+R8 Gy group(ECA109 cells transfected with Atg7-Ctrl at 8 Gy radiation dose)and siRNA+R8 Gy group(ECA109 cells transfected with Atg7-siRNA at 8 Gy radiation dose).The protein expressions of Atg7,Beclin-1,microtubule associated protein 1 light chain 3Ⅱ(LC3Ⅱ),arginine succinate synthetase1(ASS1)and arginine succinic acid lyase(ASL)in each group were detected with Western blotting.The effects of arginine were detected with CCK8 assay.The cell survival curves were drawn at different concentrations of arginine(0,8,16,32,64,128 mmol/L).IC50 and IC10 were calculated,and IC10 was set as the action concentration in the subsequent experiments.Cells were treated with arginine and siRNA respectively,and irradiated with 8 Gy.After 24 h,the cell mortality was assessed by counting the number of cells.Results siRNA interference down-regulated Atg7 before and after radiotherapy(P<0.01).Compared with the Ctrl group,the Ctrl+R8 Gy group had up-regulated levels of Beclin-1 and LC3Ⅱ(P<0.01).Compared with the Ctrl+R8 Gy group,the siRNA+R8 Gy group had down-regulated levels of Beclin-1 and LC3Ⅱ(P<0.01).Compared with the Ctrl+R8 Gy group,the siRNA+R8 Gy group had up-regulated levels of ASS1 and ASL(P<0.01).Arginine inhibited the proliferation of ECA109 cells in a concentration and time-dependent manner.Cell death analysis showed that siRNA interference(F=73.59,P<0.001)and arginine addition(F=158.72,P<0.001)increased cell mortality.The cell mortality was at the highest level when siRNA and arginine were used in combination(F=7.88,P=0.02).Conclusion Radiotherapy can induce autophagy in ECA109 cells.The Atg7-siRNA interference effectively antagonizes the radiotherapy-induced increase in autophagy,increases the radiotherapy-induced ECA109 cell death,and enhances the

关 键 词：siRNA干扰自噬相关基因7 精氨酸琥珀酸合成酶1 精氨酸琥珀酸裂解酶 食管癌 精氨酸 放疗敏感性 

分 类 号：R310.31[医药卫生—基础医学]