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

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阻滞及化疗耐药,可能与上调hTERT表达,进而上调端粒酶活性有关;沉默HIF-1α显著逆转缺氧诱导的肿瘤耐药并下调端粒酶活性。

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 carcinoma cell were upregulated after hypoxia;after transfection siRNA,the expessions of HIF-la and hTERT mRNA as well as proteins were significantly downregulated. The proportion of Go/GI phase in hypoxic with untreated group was significantly increased （45.63±2.01）, which was significantly higher than that in normoxia group （26.75±1.28;P〈0. 001）. The proportion of S phase had no significant difference the cell cycle was redistributed after si- lencing the HIF-lα The IC50 of 5 8F cell to 5-FU in normoxia group, hypoxia with untreated group, Negative siRNA group and HIF-1α-siRNA group were（17.30±3.31） ,（32.04±12.75）,（33.90±0.87） and （13.72±2.36）μg/mL. The IC50 of hypoxia with untreated group was significantly higher than that of normoxia group （P〈0. 001）, while the IC50 of HIF-la-siRNA group was significantly lower than that of the hypoxia with untreated group （P〈0. 001）. The chemosensi tivity of 5-8F cell in hypoxia group to cisplatin （DDP）was reduced too and after silencing the HIF-lα,the chemosensitivity of cell to the DDP improved significantly. The results of CNE2 were consistented with 5-8F cell excepted for cell cycle. CONCLUSIONS G1/S block and chemotherapy resistance in the nasopharyngeal carcinoma cells induced by hypoxia may be related with upregulate the expressions of hTERT and telomerase activity. Silencing HIF-la significantly reversed re- sistance induced by hypoxia and downregulated telomerase activity.