shRNA-hIAP2增强CNE-1鼻咽癌细胞对放射敏感性的实验研究  

shRNA-hIAP2 enhancing radiation effect on nasopharyngeal carcinoma cell

作　　者：袁媛[1,2] 蒋文娟[3] 姜武忠[1] 

机构地区：[1]中南大学湘雅医院肿瘤科,湖南长沙410008 [2]中南大学湘雅医学院附属肿瘤医院淋巴瘤血液放疗科,湖南长沙410013 [3]中南大学湘雅医学院附属肿瘤医院,消化内二科,湖南长沙410013

出　　处：《中国耳鼻咽喉颅底外科杂志》2013年第5期393-398,共6页Chinese Journal of Otorhinolaryngology-skull Base Surgery

摘　　要：目的构建人凋亡抑制蛋白(human inhibitor of apoptosis protein2,hIAP-2)的shRNA,靶向抑制hIAP-2基因联合放疗,探讨其对CNE1鼻咽癌细胞株放射敏感性的影响。方法首先采用MTT法确定最佳放射剂量;再构建4条hIAP-2-shRNA沉默片段并用脂质体法转染CNE1细胞,最后采用实时荧光定量PCR(Q-PCR)和免疫印迹法(Western blot,WB)筛选出最佳沉默shRNA序列;用最佳沉默shRNA序列转染CNE1细胞,分别设置转染放射线照射组与未转染放射线照射组,采用实时定量PCR和WB检测hIAP-2的基因及其蛋白表达;流式细胞术测细胞凋亡,transwell测细胞侵袭能力。结果 MTT法明确4 Gy为最佳放射剂量;Q-PCR和WB检测结果显示4条hIAP-2-shRNA均能有效抑制hIAP-2 mRNA及蛋白表达(P<0.05),以hIAP-2-shRNA2沉默效果最显著(P<0.05);Q-PCR和WB检测结果显示转染hIAP-2-shRNA2后照射组与未转染hIAP-2-shRNA2照射组的CNE1细胞中hIAP-2基因与蛋白表达比较,差异具有统计学意义(P<0.05);流式细胞术检测结果显示转染hIAP-2-shRNA2后照射组比未转染hIAP-2-shRNA2照射组凋亡率差异具有统计学意义(P<0.05);transwell检测结果显示转染hIAP-2-shRNA2后照射组与未转染hIAP-2-shRNA2照射组侵袭能力差异具有统计学意义(P<0.05)。结论转染hIAP-2基因沉默后放射可增加鼻咽癌细胞凋亡率,降低其侵袭能力,能够起到放疗增敏作用。Objective To construct the inhibitor of the shRNA of apoptosis protein （human inhibitor of apoptosis protein2, hIAP-2 ） for combination of inhibition hIAP-2 gene with radiotherapy, and to investigate its effect on the radiosensitivity of CNE 1 nasopharyngeal carcinoma cell line. Methods MTF assay was used for determing the optimal dose of radiation. Four hIAP-2-shRNA silencing fragments were built and CNE1 cells were transfected by liposome. The best silencing shRNA sequences were screened with real-time quantitative PCR （Q-PCR） and Western blot （WB）. CNE1 cells were transfected with the best silencing shRNA sequences and divided into transfected irradiated group and non-transfected irradiated group. In both groups, expression of hIAP-2 gene and protein was detected by real-time Q-PCR and WB, apoptosis by flow cytometry, and cell invasion by transwell. Results MTT assay demonstrated 4 Gy as the optimal radiation dose. The results of Q-PCR and WB test showed that four hIAP-2- shRNA could effectively inhibit the expression of hIAP-2 mRNA and protein （ P 〈 0.05 ） , and hIAP-2-shRNA2 had the most significant silencing effect （ P 〈 0.05 ） . The results also showed statistically significant differences of hIAP-2 gene and protein expression between the transfected group and the untransfected group （ P 〈0. 05 ）. Flow cytometry showed statistically significant difference of the apoptosis rate between the transfected group and untransfected group （ P 〈 0.05 ） . Meanwhile, transwe11 test showed statistically significant difference of the invasion ability between the two groups （ P 〈 0.05 ）. Conclusions Transfection of silenced hIAP-2 gene can increase nasopharyngeal carcinoma cell apoptosis rate and reduce its invasion, which may play a role in radiosensitization.

关 键 词：人凋亡抑制蛋白 鼻咽癌细胞 RNA沉默 放射敏感性 

分 类 号：R730.5[医药卫生—肿瘤] R739.91[医药卫生—临床医学]