摘要
目的观察细胞内钙变化对氧调节性亚单位HIF-1α表达及其HIF-1转录激活的影响。方法常氧时,采用通透性细胞内钙的螯合剂BAPTA-AM或钙的离子载体Ionomycin降低或升高细胞内钙,用Western-blot检测其对SGC7901细胞中HIF-1α蛋白表达的影响,然后采用间接免疫荧光法、双荧光素酶报告系统及ELISA法检测改变细胞内钙对HIF-1α转位、HIF-1转录活性及其靶基因血管内皮生长因子(Vascular endothelial growth factor,VEGF)分泌的影响。结果用BAPTA-AM降低细胞内钙,能诱导HIF-1α的表达,促进HIF-1α的转位,增强HIF-1的转录活性,增加HIF-1调节基因VEGF的分泌。用Ionomycin升高细胞内钙,虽然也有微弱的促HIF-1α稳定及转位作用,但对HIF-1转录活性及VEGF的分泌并无明显影响。结论螯合细胞内钙能诱导胃癌细胞中HIF-1α的表达及HIF-1的转录激活,提示细胞内钙变化可能在胃癌细胞的缺氧信号转导过程中发挥了重要作用。
Objective To study the possible roles of changed intracellular calcium level in SGC7901 cells on HIF-1a expression and HIF-1 transcriptional activity. Methods Under normoxic conditions, the prorein expression of HIF-1a in SGC7901 cells after treatment with BAPTA-AM （a cell-permeant Ca^2＋ chelator）, or ionomycin （an calcium ionophore） was observed by Western blot. The effect of BAPTA or ionomycin on nuclear location of HIF-1a, HIF-1 luciferase activity as well as secretion of vascular endothelial growth factor （VEGF）, a typical HIF-1 target gene, was determined by indirect immunofluorescence assay, dual luciferase reporter system and enzyme-linked immunosorbent assay （ELISA） respectively. Results Decreasing the intracellular calcium level by BAPTA induced HIF-1a nuclear accumulation, stimulated HIF-1-dependent transcription and enhanced expression of VEGF in SGC7901 cells under normoxic conditions. However, the induction of HIF-1 activity and the secretion of VEGF were not caused by ionomycin application, though it can mediated a weak and transient HIF-1a nuclear accumulation. Conclusion Chelation of cellular calcium modulates HIF-1a expression and HIF-1 transcriptional activity in human gastric cancer cells. Changes in intracellular free calcium concentration appeared to have a critical role in hypoxic signal transduction in gastric cancer cells.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2007年第9期659-662,737,共5页
Cancer Research on Prevention and Treatment
基金
国家自然科学基金资助项目(30100079)
关键词
胃癌细胞
细胞内钙
缺氧诱导因子-1
蛋白表达
转录活性
Gastric cancer cells
Intracellular calcium
HIF-1
Protein expression
Transcriptional activity
