小干扰RNA抑制缺氧诱导因子-1α表达对食管癌细胞糖酵解的影响  

Effects of suppression hypoxia-inducible factor-1α expression by small interfering RNA on glycolysis inesophageal carcinoma

作　　者：唐娜娜[1] 朱宏[1] 何桂钧 郝波[1] 施瑞华[1] 

机构地区：[1]南京医科大学第一附属医院消化科,210029 [2]上海交通大学附属第一人民医院松江分院消化科

出　　处：《中华消化杂志》2013年第2期115-118,共4页Chinese Journal of Digestion

基　　金：国家自然科学基金（30770991）;国家自然科学基金青年基金（30800511）

摘　　要：目的观察人食管鳞状细胞癌细胞中缺氧诱导因子-1α（HIF-1α）对糖酵解的影响并探讨其可能的作用机制。方法TEl3、Ecal09细胞常氧（O2体积分数为0．20）及缺氧（0z体积分数为0．01）培养，缺氧时间为6、12、24、48h，RNA干扰法稳定静默HIF-1α获得TE13/小干扰（si）HIF、Ecal09／siHIF细胞，培养方法和时间同TEl3、Ecal09。Western印迹法检测HIF-1α的表达变化；分光光度法检测培养上清液中乳酸浓度变化；实时定量PCR检测葡萄糖转运蛋白-1（GLUT-1）、乳酸脱氢酶A（LDHA）的mRNA表达变化；Western印迹法检测GLUT-1、LDHA蛋白表达变化。t或t’检验用于分析不同缺氧时间对HIF-1a蛋白表达的影响，组间差异比较采用单因素方差分析。结果TEl3、Ecal09细胞缺氧后HIF-1α表达增强，12h达高峰（t=6．11，8．31，P〈0．05）。常氧条件下，TEl3／siHIF和Ecal09／siHIF细胞乳酸分泌分别为（1．24±0．33）、（1．28±0．37）mmol／L，较TEl3和Ecal09细胞显著下降[（3．25±1．34）、（4．91±1．69）mmol／L，t=2．53、3．59，P均〈0．053；TEl3、Ecal09细胞缺氧后乳酸分泌增多[（6．48±1．73）、（8．02±1．95）mmol／L，t=2．715、2．050，P均〈0．053，TEl3／siHIF、Ecal09／siHIF细胞缺氧后乳酸分泌无明显增多（P〉0．05）。TEl3／siHIF和Ecal09／siHIF细胞中，GLUT-1、LDHA的mRNA表达均明显受抑（常氧：t=6．98、3．92、7．25、3．67，P均〈0．05），GLUT-1蛋白表达明显减弱（常氧：t=4．57、16．56，缺氧：t=6．19、6．09，P均〈0．05），LDHA蛋白表达稍减弱（P〉0．05）。结论抑制人食管鳞状细胞癌中HIF-1a可降低细胞的糖酵解水平，该途径可能涉及GLUT-1、LDHA的表达抑制；除HIF-1α以外，LDHA的蛋白表达可能同时存在其他调控因素。Objective To investigate the effects of hypoxia-inducible factor （HIF）-1α on glycolysis of human esophageal squamous carcinoma cells and the possible mechanism. Methods TEl3 and Ecal09 cells were cultured under normal oxygen （20%O2 ） and hypoxia （1%O2 ） conditions. The hypoxia was duration 6 hours, 12 hours, 24 hours and 48 hours. HIF-1α gene was stable silented by RNA interference method and TE13/small interfering HIF cells and Ecal09/siHIF cells were obtained. The cell culture condition and time was same as TEl3 and Ecal09 cells. The changes of HIF-1α expression were detected by Western-blot. The changes of lactic acid concentration in cellculture supernatant were determined by Spectrophotometry. The changes of glucose transporter-1 （GLUT-l） and lactic dehydrogenase A （LDHA） expression at mRNA level were examined by real- time polymerase chain reaction. The changes of GLUT-1 and LDHA expression at protein level were tested by Western blot. Using t or t＇ test to analyze the effects of hypoxia duration on HIF-1α expression at protein level. One-way ANOVA was applied for the difference analysis between the groups. Results In TEl3 and Ecal09 cells, the HIF-1α expression significantly increased under hypoxia condition and reached the peak at 12 hour （t=6. 11, 8.31; both P〈0.05）. The lactic acid secretion of TEl3/siHIF cells and Ecal09/siHIF cells was （1.24±0.33） and （1.28±0.37） mmol/L, which significantly decreased when compared with TEl3 and Ecal09 cells [（3.25~ 1.34） and （4.91± 1.69） mmol/L, t=2.53, 3.59, both P〈0. 051. The lactic acid secretion of TEl3 and Ecal09 ceils significantly increased after hypoxia [（6.48±1.73） and （8.02± 1.95） retool/L, t= 2. 715, 2. 050, both P〈0.05]. There was no significant lactic acid secretion in TE13/siHIF cells and Ecal09/siHIF cells after hypoxia （P〉 0.05）. The expressions of GLUT-1 and LDHA at mRNA level were significantly suppressed in TEl3/siHIF cells and Ecal09/siHIF cells （normal oxygen., t= 6.98, 3.92

关 键 词：缺氧诱导因子1 Α亚基 糖酵解 RNA 小分子干扰 食管肿瘤 

分 类 号：R735.1[医药卫生—肿瘤]