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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 effect of hypoxia and siRNA on hypoxia inducible factor-1α (HIF-1α) on telomerase catalytic subunit (hTERT), cell cycle and chemotherapeutic resistance in nasopharyngeal carcinoma cells Effect of medicine. Methods The nasopharyngeal carcinoma cells 5-8F and CNE2 were treated with hypoxia (1% O2) in a three-gas incubator. The expressions of HIF-1α and hTERT protein in different hypoxia phases (0 ~ 72h) were detected by Western blotting. The HIF-1α gene-specific siRNA was transfected into nasopharyngeal carcinoma cell lines 5-8F and CNE2 respectively, and the siRNA with the highest silencing efficiency was screened out. The experiment was divided into untreated group (normoxia), untreated group (hypoxia), Negative -siRNA (hypoxia), HIF-1α-siRNA (hypoxia), fluorescence quantitative PCR and Western blotting were used to detect the expression of hTERT and HIF-1α after transient transfection. Flow cytometry (flow cytometry, FCM) analysis of hypoxia or silence HIF-1α on cell cycle. The chemosensitivity of nasopharyngeal carcinoma cells to cisplatin (DDP) and 5-fluorouracil (5-FU) after hypoxia or silencing of HIF-1α was detected by MTT assay. Results The expressions of HIF-1α (F = 37.147, P <0.001) and hTERT (F = 70.069, P <0.001) in nasopharyngeal carcinoma 5-8F cells after hypoxia for 0-72h were significantly up-regulated. HIF-1α-siRNA transiently transfected 5-8F cells> 98%. The expression of hTERT mRNA in HIF-1α-siRNA group was 0.37 ± 0.05, significantly lower than 1.00 ± 0.00 in untreated group (hypoxia) and 0.95 ± 0.01 in Negative-siRNA (hypoxia group), F = 360.339, The expression of hTERT protein was (0.27 ± 0.05), significantly lower than that of untreated group (hypoxia) 0.54 ± 0.00 and Negative-siRNA group (hypoxia) 0.53 ± 0.01, F = 24.010, P <0.001. The proportion of cells in G0 / G1 phase in untreated group (45.63 ± 2.01)% was significantly higher than that in untreated group (26.75 ± 1.28)% in untreated group (P <0.001.5-8F) The IC50 of 5-FU (normoxia) group was (17.30 ± 3.31) μg / mL for untreated group and (32.04 ± 12.75) μg / mL for untreated group and 33.90 ± 0.87 for Negative-siRNA group (13.72 ± 2.36) μg / mL in HIF-1α-siRNA group, F = 3.704, P <0.001.5-8F. The chemosensitivity to DDP in hypoxia group was also decreased. After silencing HIF-1α, The chemosensitivity of -8F cells to DDP was significantly increased. The results of CNE2 and 5-8F were identical except for the cell cycle. Conclusion Hypoxia can induce G1 / S arrest and chemoresistance in nasopharyngeal carcinoma cells, which may be related to up-regulation of hTERT expression and up-regulation of telomerase activity. Silencing of HIF-1α significantly reverses hypoxic-induced tumor resistance and down-regulates telomeres Enzyme activity.