沉默缺氧诱导因子对人胶质瘤SHG44细胞糖酵解及细胞增殖的影响

Reversal of glycolytic phenotype by HIF-1α siRNA inhibits SHG44 cell growth in vitro

目的探讨沉默缺氧诱导因子(hypoxia-inducible factor-1alpha,HIF-1α)对缺氧状态人胶质瘤SHG44细胞糖酵解及细胞增殖的影响及机制。方法用氯化钴(CoCl2)模拟缺氧,并应用小分子RNA干扰(siRNA)技术沉默HIF-1α基因表达。将细胞分成4组:正常培养组、缺氧培养组、阴性对照组和siRNA干扰组。各组又分为常糖组(2 g/L)和高糖组(5 g/L)。Real-time PCR、Western blot法分别检测肿瘤细胞HIF-1α和糖酵解酶的mRNA及蛋白表达;生物发光法检测细胞内ATP水平;激光共聚焦显微镜检测细胞线粒体膜电位;MTT法检测细胞增殖;流式细胞技术检测细胞凋亡、坏死及细胞内活性氧的变化。结果①缺氧条件下,HIF-1αsiRNA干扰沉默HIF-1α基因的效率达到71%。②缺氧培养组HIF-1α和糖酵解酶的mRNA和蛋白表达高于正常培养组(P<0.05),细胞内ATP水平减少、细胞坏死和凋亡均增加,而细胞增殖能力明显增强(P<0.05),同时线粒体膜电位升高(P<0.05)。③siRNA干扰组HIF-1α和糖酵解酶的表达量低于缺氧培养组、阴性对照组(P<0.05);ATP水平低于缺氧培养组,细胞增殖率下降(P<0.05),细胞坏死率高于缺氧培养组(P<0.05),线粒体膜电位恢复。结论缺氧诱使肿瘤细胞表达HIF-1α,促进肿瘤恶性进展;siRNA干扰沉默HIF-1α能加重缺氧状态下SHG44细胞的生长抑制和坏死,其机制与抑制糖酵解、恢复线粒体功能有关。

Objective To determine the role of hypoxia-inducible factor-1alpha（HIF-1α） on aerobic glycolysis and cell growth in SHG44 cells using siRNA silencing HIF-1α.Methods CoCl2 was used to mimic hypoxia;siRNA technique was used to silence HIF-1α in SHG44 cells.SHG44 cells were divided into 4 groups,including normal culture group,hypoxia culture group,negative control group and HIF-1α siRNA group.Each group was further divided into 2 subgroups,normal glucose intensity group（2 g/L） and high glucose intensity group（5 g/L）.The expression of HIF-1α and glycolysis enzymes was measured by real-time PCR and Western blot analysis.Intracellular ATP levels were measured by bioluminescence assay.Cell viability was measured by MTT assay.Apoptotic and death cells and intracellular reactive oxygen species（ROS） were detected by flow cytometry.The alteration of transmembrane potential of mitochondria was determined by confocal laser scanning microscopy.Results Under hypoxia,the efficiency of siRNA silencing HIF-1α gene reached to 71%.The mRNA and protein levels of HIF-1α,PKM2,PDK1 and LDHA in the hypoxia culture group were significantly higher than those of normal culture group.Tumor cells showed higher proliferation rate and increased necrosis and apoptosis rate than the cells under normal culture.Moreover,tumor cells had lower intracellular ATP concentrations and elevated mitochondrial membrane potential（all P0.05）.The growth inhibition ratio of cells in HIF-1α siRNA group was significantly higher than that of other groups including hypoxia culture group and negative control group（all P0.05）.The levels of glycolytic enzymes PKM2,PDK1 and LDHA in HIF-1α siRNA group were significantly lower than those of hypoxia culture group and negative control group（P0.05）.Treatment with HIF-1α siRNA decreased the elevated mitochondrial membrane potential in cancer cells,which was also associated with a sharp decrease in intracellular ATP level.Conclusion Aerobic glycolysis of cancer cells may contribute directly to carcinogenic progression and malignant transformation by promoting the expression of HIF-1α-regulated genes under hypoxic conditions.HIF-1α gene silencing aggravates growth inhibition and necrosis of SHG44 cells under hypoxia.