缺氧调控端粒酶活性及抑制鼻咽癌细胞增殖实验研究  

Up-regulated telomerase and inhibited proliferation induced by hypoxia in nasopharyngeal carcinoma cells

作　　者：史欣[1] 刘艳慧[2] 申聪香[1] 文忠[1] 李冠雪[1] 付新洒 

机构地区：[1]南方医科大学珠江医院耳鼻咽喉头颈外科,广东广州510282 [2]新疆医科大学第二附属医院耳鼻咽喉科,新疆乌鲁木齐830028

出　　处：《中华肿瘤防治杂志》2015年第7期507-513,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省科技计划(2010B031200009)

摘　　要：目的探讨缺氧及siRNA沉默缺氧诱导因子-1α（hypoxia inducible factor-1α,HIF-1α）后对鼻咽癌细胞中端粒酶催化亚单位（telomerase catalytic subunit,hTERT）、细胞周期和化疗耐药的影响。方法采用三气培养箱对鼻咽癌细胞5-8F和CNE2进行缺氧处理（1%O2）,蛋白质印迹法检测不同乏氧时相（0~72h）HIF-1α和hTERT蛋白的表达。将HIF-1α基因特异性siRNA分别转染鼻咽癌细胞株5-8F和CNE2,筛选出沉默效率最高的siRNA,实验分为未处理组（常氧）、未处理组（缺氧）、Negative-siRNA（缺氧）和HIF-1α-siRNA（缺氧）,荧光定量PCR及蛋白质印迹检测瞬时转染后hTERT及HIF-1α的表达。流式细胞术（flow cytometry,FCM）分析缺氧或沉默HIF-1α后对细胞周期的影响。MTT法检测缺氧或沉默HIF-1α后,鼻咽癌细胞对顺铂（DDP）和5-氟尿嘧啶（5-FU）的化疗敏感性。结果缺氧处理0~72h后鼻咽癌5-8F细胞HIF-1α（F=37.147,P〈0.001）和hTERT（F=70.069,P〈0.001）蛋白的表达上调,差异有统计学意义。HIF-1α-siRNA对5-8F细胞瞬时转染率〉98%。HIF-1α-siRNA组hTERT mRNA表达量为0.37±0.05,显著低于未处理组（缺氧）的1.00±0.00和Negative-siRNA（缺氧）的0.95±0.01,F=360.339,P〈0.001;hTERT蛋白表达量为（0.27±0.05）,显著低于未处理组（缺氧）0.54±0.00和Negative-siRNA组（缺氧）0.53±0.01,F=24.010,P〈0.001。未处理组（缺氧）G0/G1期细胞比例明显增加（45.63±2.01）%,显著高于未处理组（常氧）的（26.75±1.28）%,P〈0.001。5-8F细胞未处理组（常氧）对5-FU的IC50分别为（17.30±3.31）μg/mL,未处理组（缺氧）为（32.04±12.75）μg/mL,Negative-siRNA组为（33.90±0.87）μg/mL,HIF-1α-siRNA组为（13.72±2.36）μg/mL,F=3.704,P〈0.001。5-8F细胞缺氧组对DDP的化疗敏感性也降低,沉默HIF-1α后,5-8F细胞对DDP的化疗敏感性明显提高。除细胞周期外,CNE2与5-8F的结果均一致。结论缺氧促使鼻咽癌细胞发生G1/S阻滞及OBJECTIVE To investigate the influence of hTERT,the cell cycle and chemotherapy resistance in naso- pharyngeal carcinoma cell lines induced by hypoxia and siRNA silencing HIF-1α. METHODS Hypoxia preconditioning was performed as cells cultured in a tri-gas incubator with oxygen concentration in 1%. The expression of HIF-Iα and hTERT at different hypoxia phase （0,4,8,16,24,48 and 72 h） were detected by Western blotting. HIF-1α gene specific siRNA was transferred into nasopharyngeal carcinoma cell lines 5 8F and CNE2, respectively. The most efficient siRNA silencing was screened out. The experiment were divided into untreat group（normoxic） and untreat group（hypoxia）, Neg- ative-siRNA （hypoxia） and HIF la siRNA（hypoxia）. The expressions of HIF 1a and hTERT at different hypoxia phases were detected by Western blotting. The expressions of HIF-lα and hTERT after transient transfection were detected by Fluorescence quantitative PCR and Western blotting. The changes of cell cycle induced by hypoxia or silencing HIF-Iα were analyzed by Flow cytometry（FCM）. The chemosensitivity of nasopharyngeal carcinoma cell to cisplatin（DDP） and 5 fluorouracil（5-FU） on the hypoxia condition or silencing the HIF-1α was detected by MTT assay. RESULTS After hy poxic treatment 0--72 h, the expression of HIF-1a and hTERT protein in 5-8F were upregulated, the differences were statically significant （ HIF-1 a ： F= 37. 147, P〈0.001 ; hTERT ： F= 70. 069, P〈0. 001）. The transfection of HIF-1α-siRNA on 5-8F was more than 98%,the silence rate of HIF-lα-siRNA was as more than 70.3%. The expression of hTERT mR- NA （0.37±0.05） and protein （0.27±0.05） in HIF-lα-siRNA group were significantly lower than those in untreat group （hypoxia） （1.00!0.00,0.54±0.00） and Negative-siRNA group（hypoxia） （0.95±0.01,0.53±0.01）,the differences was statically significant（F= 360. 339, P〈0. 001;F=24. 010, P〈0. 001）. The expressions of HIF-lα and hTERT protein in 5 8F nasopharyngeal c

关 键 词：鼻咽肿瘤 缺氧诱导因子 端粒酶 RNA干扰 逆转耐药 

分 类 号：R739.63[医药卫生—肿瘤]