c-Myc通过缺氧诱导因子-1α调控结肠癌细胞HT-29的增殖

摘要

目的 研究癌基因c-Myc对人结肠癌细胞HT-29中缺氧诱导因子-1α(HIF-1α)的影响及其对肿瘤细胞增殖的作用.方法 构建高表达c-Myc的质粒pcDNA3.1-c-Myc；将构建好的pcDNA3.1-c-Myc质粒转染入HT-29细胞中,通过Western blot印迹法检测c-Myc在HT-29细胞中的表达情况.应用Real-time PCR和Western blot印迹法检测常氧和乏氧状态下HT-29细胞系中HIF-1α的mRNA和蛋白水平,同时检测HIF-1α下游靶基因血管内皮生长因子(VEGF)、内皮型一氧化氮合酶(eNOS)的mRNA水平.共转染pcDNA3.1-c-Myc质粒和HIF-1α的siRNA入HT-29细胞中.应用噻唑蓝(MTT)法检测HT-29细胞的增殖情况.结果 成功构建pcDNA3.1-c-Myc质粒；常氧、乏氧状态下转染pcDNA3.1-c-Myc质粒后,HT-29细胞系中HIF-1α的mRNA水平无变化,VEGF和eNOS的mRNA水平明显升高,HIF-1α蛋白水平明显增高.高表达c-Myc的HT-29细胞增殖能力明显增强.共转染pcDNA3.1-c-Myc质粒和HIF-1α的siRNA后,HT-29细胞的增殖能力明显降低.结论 在HT-29细胞系中c-Myc通过HIF-1α调控HT-29细胞的增殖.%Objective To investigate the influence of c-Myc on hypoxia-inducible factor 1, α subunit (HIF-1α) and proliferationof HT-29 cells. Methods The plasmid pcDNA3. 1-c-Myc was constructed and transfected into human colon carcinoma cell line HT-29. Efficiency of transfection was detected by Western blotting. The mRNA and protein levels of HIF-lα in normoxia and hypoxia were measured by real-time PCR and Western blotting. The mRNA levels of vascular endothelial growth factor (VEGF) and eNOS (the target genes of HIF-1α) were measured by real-time PCR. PcDNA3. 1-c-Myc plasmid and HIF-1α siRNA were co-transfected into HT-29 cells. MTT was used to detect the proliferation of the HT-29 cells. Results The plasmid pcDNA3. 1-c-Myc was successfully constructed. After transfection into HT-29 cells in normoxia and hypoxia, real-time PCR showed that the mRNA level of HIF-1α had no change, while Western blotting showed the protein level of HIF-lα was significantly increased. The mRNA levels of VEGF and eNOS were significantly increased. The proliferation of HT-29 cells was stimulated through the over-expression of c-Myc. But co-transfection of pcDNA3. 1-c-Myc plasmid and HIF-1α siRNA decreased cell proliferation in HT-29 cell line. Conclusion c-Myc regulates the proliferation of HT-29 cells through HIF-1α.