CAJ | 학술논문

目的探讨人食管鳞状细胞癌中缺氧诱导因子-1α(HIF-1α)和己糖激酶-Ⅱ(HK-Ⅱ)的表达变化及对糖酵解的影响.方法缺氧条件(1％氧浓度)下培养TE13及Eca109细胞,设置不同缺氧时间(分别为6、12、24、48 h),并以常氧培养(20％氧浓度)为对照.Western印迹检测HIF-1α及HK-Ⅱ的蛋白表达变化；经RNA干扰技术特异性静默HIF-1α,利用实时定量PCR及Western印迹检测HIF-1α和HK-Ⅱ的表达变化；分光光度法检测常氧和缺氧条件下细胞培养液中细胞的乳酸浓度变化.结果 ①缺氧条件下,TE13及Eca109细胞中HIF-1α表达均随缺氧时间延长而逐渐升高(P＜0.05),在缺氧12 h表达达到高峰,后表达又随时间延长而下降.TE13及Eca109细胞缺氧12 h后HK-Ⅱ表达均较常氧培养明显增强(P＜0.05).②实时定量PCR法检测显示,常氧条件下被干扰的TE13/shRNA和Eca109/shRNA细胞中HIF-1α RNA表达量较未干扰的TE13、Eca109细胞明显减弱(P＜0.05).HK-ⅡRNA表达结果与HIF-1 RNA一致.③常氧及缺氧培养时,HK-Ⅱ蛋白在TE13/shRNA和Eca109/shRNA细胞的表达均较未干扰的TE13、Eca109细胞明显减弱,差异有统计学意义(P＜0.05).④缺氧时TE13和Eca109细胞乳酸分泌量为14.707±3.594和15.062±3.901,较常氧时增多(6.070±1.839和6.891±1.592,P＜0.05).TE13/shRNA、Eca109/shRNA乳酸分泌量较未静默TE13、Eca109细胞显著减少,差异均有统计学意义(P＜0.05).结论食管鳞癌中HIF-1α及HK-Ⅱ在缺氧条件下表达明显增强,HK-Ⅱ表达及乳酸浓度与HIF-1α表达密切相关,HIF-1α可能通过HK-Ⅱ影响细胞的糖酵解水平.
목적 탐토인식관린상세포암중결양유도인자-1α(HIF-1α)화기당격매-Ⅱ(HK-Ⅱ)적표체변화급대당효해적영향.방법 결양조건(1％양농도)하배양TE13급Eca109세포,설치불동결양시간(분별위6、12、24、48 h),병이상양배양(20％양농도)위대조.Western인적검측HIF-1α급HK-Ⅱ적단백표체변화；경RNA간우기술특이성정묵HIF-1α,이용실시정량PCR급Western인적검측HIF-1α화HK-Ⅱ적표체변화；분광광도법검측상양화결양조건하세포배양액중세포적유산농도변화.결과 ①결양조건하,TE13급Eca109세포중HIF-1α표체균수결양시간연장이축점승고(P＜0.05),재결양12 h표체체도고봉,후표체우수시간연장이하강.TE13급Eca109세포결양12 h후HK-Ⅱ표체균교상양배양명현증강(P＜0.05).②실시정량PCR법검측현시,상양조건하피간우적TE13/shRNA화Eca109/shRNA세포중HIF-1α RNA표체량교미간우적TE13、Eca109세포명현감약(P＜0.05).HK-ⅡRNA표체결과여HIF-1 RNA일치.③상양급결양배양시,HK-Ⅱ단백재TE13/shRNA화Eca109/shRNA세포적표체균교미간우적TE13、Eca109세포명현감약,차이유통계학의의(P＜0.05).④결양시TE13화Eca109세포유산분비량위14.707±3.594화15.062±3.901,교상양시증다(6.070±1.839화6.891±1.592,P＜0.05).TE13/shRNA、Eca109/shRNA유산분비량교미정묵TE13、Eca109세포현저감소,차이균유통계학의의(P＜0.05).결론 식관린암중HIF-1α급HK-Ⅱ재결양조건하표체명현증강,HK-Ⅱ표체급유산농도여HIF-1α표체밀절상관,HIF-1α가능통과HK-Ⅱ영향세포적당효해수평.
Objective To investigate the changes of hypoxia-inducible factor(HIF)-1α and hexokinase-Ⅱ(HK-Ⅱ)expression in human esophageal squamous cell carcinoma and its effect in glycolysis.Methods TE13 cells and Eca109 cells were cultured under hypoxic condition(1 ％O2)for different hypoxic time(6,12,24 and 48 hours).Cells cultured under normal oxygen condition(20％O2)were set as control.The changes of HIF-1α and HK-Ⅱ expressions at protein level were detected by Western blot.HIF-1α genes were specifically silenced with RNA interference technology(RNAi),and then the changes of HIF-1α and HK-Ⅱ expression were determined by realtime PCR and Western blot.Under normal oxygen and hypoxic condition,the changes of lactic acid concentration in cell culture medium were detected by spectrophotometric method.Results Under hypoxic condition,the expression of HIF-1α and HK-Ⅱ gradually increased as hypoxic time extended(P＜0.05),reached a peak at 12h and then gradually decreased as time extended.Compared with that under normal oxygen condition,the expression of HK-Ⅱ in TE13 cells and Eca109 cells significantly increased under hypoxic condition(P＜0.05),which was more significant after 12 hours hypoxia.The result of realtime PCR indicated that under normal oxygen condition the expression of HIF-1α at RNA level in TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference(P＜0.05).The expression of HK-Ⅱ at RNA level was consistent with the result of HIF-1α.Under normal and hypoxia condition,the expression of HK-Ⅱ at protein level in TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference,and the difference was statistic significant(P＜0.05).The lactic acid secretion of TE13 cells and Eca109 cells under hypoxia condition(14.707 ± 3.594 and 15.062 ±3.901)was higher than that under normal oxygen condition(6.070±1.839 and 6.891±1.592,P＜0.05).The lactic acid secretion of TE13/shRNA cells and Eca109/shRNA cells significantly decreased compared with TE13 cells and Eca109 cells without interference,and the difference was statistic significant(P＜0.05).Conclusion The expressions of HIF-1α and HK-Ⅱ in human esophageal squamous cell carcinoma significantly increased under hypoxia conditions.The expression of HK-Ⅱ is closely correlated with lactic acid concentration and HIF-1α expression.HIF-1α may affect cell glycolysis through HK-Ⅱ.