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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 investigate the effect of hypoxia-inducible factor-1alpha (HIF-1α) on glycolysis and cell proliferation in human glioma SHG44 cells in hypoxia. Methods Cobalt chloride (CoCl 2) was used to simulate hypoxia. HIF-1α gene expression was silenced by small interfering RNA (siRNA) technique. The cells were divided into 4 groups: normal culture group, hypoxic culture group, negative control group and siRNA interference group. Each group was divided into the normal sugar group (2 g / L) and high glucose group (5 g / L). Real-time PCR and Western blot were used to detect the mRNA and protein expression of HIF-1α and glycolytic enzyme in tumor cells respectively. The levels of ATP in cells were detected by bioluminescence method. Mitochondrial membrane potential was measured by laser scanning confocal microscopy. Cell proliferation Flow cytometry was used to detect the changes of apoptosis, necrosis and intracellular reactive oxygen species. Results ① Under hypoxic conditions, the efficiency of HIF-1α siRNA silencing HIF-1α gene was 71%. ② The mRNA and protein expressions of HIF-1α and glycolytic enzymes in hypoxia group were significantly higher than those in normal group (P <0.05), the levels of ATP in cells were decreased, necrosis and apoptosis were increased, while the cell proliferation ability was significantly enhanced P <0.05), meanwhile mitochondrial membrane potential increased (P <0.05). ③ The expression of HIF-1α and glycolytic enzyme in siRNA interference group was lower than that in hypoxia group and negative control group (P <0.05); the level of ATP in hypoxia group was lower than that in hypoxia group (P <0.05) Necrosis rate was higher than that of hypoxia group (P <0.05), mitochondrial membrane potential recovered. Conclusion Hypoxia induces the expression of HIF-1α in tumor cells and promotes the malignant progression of the tumor. Silencing HIF-1α by siRNA silencing can increase the growth inhibition and necrosis of SHG44 cells under hypoxia, and its mechanism is related to inhibition of glycolysis and restoration of mitochondrial function.