RNA干扰HIF-1α对食管癌细胞糖酵解相关基因表达的影响

Effect of RNA interference-based silencing of HIF-1α gene on the expression of glycolysis associated genes in esophageal carcinoma cells

目的:探讨常氧及缺氧培养对RNA干扰的缺氧诱导因子-1α(HIF-1α)及糖酵解相关基因表达的影响。方法:选择3组细胞分别为食管鳞癌Eca-109细胞稳定转染HIF-1α siRNA及空质粒的Eca-109细胞,予以常氧和缺氧培养,缺氧培养时间分别为6、12、24、48h,采用Western blot检测HIF-1α蛋白表达,Western blot和RT-PCR检测3组细胞常氧和缺氧条件下糖酵解相关基因(Glut-1、LDH-A、HK-Ⅱ)的表达,分光光度法分别测定3组细胞常氧和缺氧培养下胞液中乳酸脱氢酶(LDH)、己糖激酶(HK)酶活性。结果:Eca-109细胞缺氧条件下培养,HIF-1α蛋白表达较常氧时增高,干扰细胞常氧下HIF-1α几乎无表达,缺氧后亦无增强。常氧和缺氧条件下,干扰细胞Glut-1、LDH-A、HK-Ⅱ mRNA和蛋白表达较未干扰细胞明显减弱(P<0.05),干扰细胞胞液LDH、HK活性较未干扰细胞明显减少(P<0.05)。结论:RNA干扰能抑制Eca-109细胞的HIF-1α基因表达,进而不同程度减少Glut-1、LDH-A、HK-Ⅱ糖酵解相关基因的表达。

Objective：To investigate the effect of RNA interference-based silencing of HIF-1α gene on the expression of glycolysis associated genes in esophageal squamous carcinoma cells（Eca-109） cultured in either a normoxia or a hypoxia environment.Methods：Eca-109 cells were divided into three groups,namely,untransfected Eca-109 cells,Eca-109 cells stably transfected with HIF-1α siRNA and empty expression vector.After the cells were cultured in a normoxia environment or in a hypoxia environment for 6,12,24,48 h,respectively,the expression of HIF-1α protein was detected by Western blot,and the expression of glycolysis associated genes（Glut-1,LDH-A,HK-Ⅱ） was determined by RT-PCR and Western blot.Spectrophotometry was employed to determine the activities of the glycolysis enzymes（LDH,HK）.Results：After Eca-109 cells were cultured in a hypoxia environment,the expression of HIF-1α protein was upregulated,and expression of HIF-1α was hardly detected in HIF-1α silenced cells,and could not be upregulated in a hypoxia environment.The expression of Glut-1,LDH-A,HK-Ⅱ mRNA and proteins was significantly down-regulated in HIF-1α silenced cells,and the LDH and HK activities were obviously decreased in HIF-1α silenced cells.Conclusion：RNA interference targeting the HIF-1α gene is able to efficiently silence the expression of HIF-1α gene in human esophageal squamous carcinoma cells（Eca-109）and thereby inhibit the expression of glycolysis associated genes（Glut-1,LDH-A,HK-Ⅱ）to different extents.