干扰素诱导的三角形四肽重复蛋白2抗体基因在胃癌进展中的作用  

Role of anti - interferon - induced protein with tetratricopeptide repeats 2 in gastric cancer cell lines

作　　者：翟文斯 陈陆俊[1] 郑晓[1] 胡文蔚[1] 吴晨[1] 王琦[1] 卢斌峰[1] 蒋敬庭[1] Zhai Wensi;Chen Lujun;Zheng Xiao;Hu Wenwei;Wu Chen;Wang Qi;Lu Binfeng;Jiang Jingting(Department of Tumor Biological Treatment, Jiangsu Engineering Research Center for Tumor Immunotherapy and Institute of Cell Therapy, the Third Affiliated Hospital of Soochow University, Changzhou 213003, China)

机构地区：[1]苏州大学附属第三医院肿瘤生物诊疗中心、江苏省肿瘤免疫治疗工程技术研究中心、苏州大学细胞治疗研究院,常州213003

出　　处：《中华实验外科杂志》2018年第4期638-641,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（31570877、31570908）;国家科技支撑计划资助项目（2015BA112B12）;国家自然科学基金海外及港澳学者合作研究基金（31729001）

摘　　要：目的探讨干扰素诱导的三角形四肽重复蛋白2抗体（IFIT2）在人胃癌进展中的作用及机制。 方法通过使用RNA干扰（RNAi）技术构建IFIT2稳定下调表达的人胃癌细胞株SGC-7901及AGS，聚合酶链反应（PCR）和Western blot分别在RNA水平和蛋白水平验证两株细胞株LV-IFIT2-短发卡RNA（shRNA）组的IFIT2的表达水平，进一步研究IFIT2在人胃癌细胞株中的生物学功能。 结果Real-time PCR检测结果显示，SGC-7901以及AGS细胞系中LV-IFIT2-shRNA组IFIT2 mRNA表达水平均显著低于LV-NC组（SGC-7901：0.426±0.140比1.000±0.157，P＝0.004；AGS：0.232±0.090比1.000±0.194，P＝0.003）；蛋白质印迹检测结果显示，SGC-7901以及AGS细胞系中LV-IFIT2-shRNA组的IFIT2的蛋白表达水平均显著低于LV-NC组（SGC-7901：0.344±0.041比1.000±0.062，P＝0.002；AGS：0.091±0.001比1.000±0.106，P＝0.000）。对稳定转染LV-IFIT2-shRNA及LV-NC的人胃癌细胞系SGC-7901和AGS进行CCK-8增殖实验、划痕实验、Transwell实验和细胞周期测定。在培养的48、72 h shRNA组的吸光度值显著高于LV-NC组，提示下调IFIT2的表达可以显著抑制人胃癌细胞系SGC-7901和AGS的增殖能力（SGC-7901：48 h：0.348±0.031比0.276±0.026，P＝0.013；72 h：0.523±0.042比0.411±0.027，P＝0.004；AGS：48 h：0.454±0.034比0.362±0.038，P＝0.011；72 h：0.696±0.053比0.553±0.030，P＝0.003）；划痕实验显示SGC-7901和AGS细胞IFIT2干扰组的迁移能力明显高于NC组的迁移能力，提示IFIT2表达的降低可明显增强SGC-7901和AGS细胞的迁移能力（SGC-7901：0.640±0.029比0.447±0.056，P＝0.006；AGS：0.444±0.036比0.166±0.034，P＝0.001）；Transwell侵袭实验中，LV-IFIT2-shRNA组的结晶紫染色迁移细胞数明显多于LV-NC组，表明SGC-7901和AGS细胞下调IFIT2表达后的侵袭能力提高（SGC-7901：299.3±45.6比162.3±25.9，P＝0.011；AGS：404.0±44.5比235.0±30.0，P＝0.003）；流式细胞术分析细胞周�ObjectiveTo investigate the role of anti-interferon-induced protein with tetratricopeptide repeats 2 （IFIT2） in human gastric cancer. MethodsIFIT2 was down-regulated by RNA interference （RNAi） method, and the role of IFIT2 in regulation of biological behaviors in human gastric cancer cell lines SGC-7901 and AGS was investigated. Polymerase chain reaction （PCR） and Western blotting were used to examine the mRNA and protein level of IFIT2 expression in the LV-IFIT2-short hairpin RNA （shRNA） and LV-NC groups in both SGC-7901 and AGS cells. ResultsThe Real-time PCR results showed that the mRNA expression level of IFIT2 in SGC-7901 as well as AGS cell lines in LV-IFIT2-shRNA group was significantly lower than that in LV-NC group （SGC-7901： 0.426±0.140 vs. 1.000±0.157, P=0.004; AGS： 0.232±0.090 vs. 1.000±0.194, P=0.003）. Western blotting results indicated that the IFIT2 expression at the protein level was significantly decreased in the LV-IFIT2-shRNA group compared with the LV-NC group in both SGC-7901 and AGS cells （SGC-7901： 0.344±0.041 vs. 1.000±0.062, P=0.002; AGS： 0.091±0.001 vs. 1.000±0.106, P=0.000）. We performed cell counting kit-8 （CCK-8） assay, wound healing assay, transwell assay and cell cycle assay to examine the role of IFIT2 in cell viability, migration and cell cycle, respectively in human gastric cancer cell lines SGC-7901 and AGS. After 48 and 72 hours of cell culture, the absorbance valueof LV-IFIT2-shRNA group was significantly higher than LV-NC group （SGC-7901： 48 h： 0.348±0.031 vs. 0.276±0.026, P=0.013; 72 h： 0.523±0.042 vs. 0.411±0.027, P=0.004; AGS： 48 h： 0.454±0.034 vs. 0.362±0.038, P=0.011; 72 h： 0.696±0.053 vs. 0.553±0.030, P=0.003）, which indicated decreased IFIT2 could markedly inhibit SGC-7901 and AGS cell proliferation. Wound healingassay showed decreased IFIT2 expression in human gastric cancer cell lines SGC-7901 and AGS significantly increasedcell migration （SGC-7901： 0.640±0.029 vs. 0.447±0.056, P=0.006; A

关 键 词：干扰素诱导的三角形四肽重复蛋白2抗体 胃癌 RNA干扰 

分 类 号：R735.2[医药卫生—肿瘤]