shRNA-hIAP2增强CNE-1鼻咽癌细胞对放射敏感性的实验研究

发布时间：2018-04-22 07:40

本文选题：人凋亡抑制蛋白 + 鼻咽癌细胞　；参考：《中国耳鼻咽喉颅底外科杂志》2013年05期

【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(P0.05),以hIAP-2-shRNA2沉默效果最显著(P0.05);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(P0.05);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(P0.05);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(P0.05)。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。
[Abstract]:Objective to construct the shRNAs of human inhibitor of apoptosis protein 2 hIAP-2 and to investigate the radiosensitivity of CNE1 nasopharyngeal carcinoma cell line by targeting hIAP-2 gene in combination with radiotherapy. Methods four hIAP-2-shRNA silencing fragments were constructed and transfected into CNE1 cells by liposome method, and the best silencing shRNA sequences were screened by real-time fluorescence quantitative PCRQ-PCR and Western blot. CNE1 cells were transfected with the best silencing shRNA sequence. The gene and protein expression of hIAP-2 was detected by real-time quantitative PCR and WB. Apoptosis was measured by flow cytometry and cell invasion was measured by transwell. Results the results of Q-PCR and WB detection showed that 4 Gy was the best dose of MTT. The results of Q-PCR and WB detection showed that all four hIAP-2-shRNA could inhibit hIAP-2 mRNA and protein expression effectively. HIAP-2-shRNA2 silencing effect was the most significant. The results of hIAP-2-shRNA2 silencing and WB detection showed that after hIAP-2-shRNA2 transfection, the irradiation group was not transformed. The expression of hIAP-2 gene and protein in CNE1 cells exposed to hIAP-2-shRNA2 was compared. The results of flow cytometry showed that the apoptosis rate of the irradiated group after transfection of hIAP-2-shRNA2 was significantly higher than that of the group without hIAP-2-shRNA2 transfection. The result of transwell test showed that the rate of apoptosis in the irradiated group after hIAP-2-shRNA2 transfection and that in the untransfected hIAP-2-shRNA2 group was higher than that in the non-transfected hIAP-2-shRNA2 group. The difference of invasion ability was statistically significant (P 0.05). Conclusion the silencing of hIAP-2 gene can increase the apoptosis rate and decrease the invasive ability of nasopharyngeal carcinoma cells.
【作者单位】：中南大学湘雅医院肿瘤科;中南大学湘雅医学院附属肿瘤医院淋巴瘤血液放疗科;中南大学湘雅医学院附属肿瘤医院消化内二科;
【分类号】：R739.63

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