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.
出处
《中华肿瘤防治杂志》
CAS
北大核心
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)
关键词
鼻咽肿瘤
缺氧诱导因子
端粒酶
RNA干扰
逆转耐药
nasopharyngeal neoplasms
hypoxia inducible factor
telomerase
RNA interference reverse drug resistance