芒柄花素调节HIF-1α/VEGF信号通路对胃癌细胞增殖、凋亡和肿瘤血管生成拟态的影响

Effects of formononetin on proliferation and apoptosis of gastric cancer cells and tumor vasculogenic mimicry by regulating HIF-1α/VEGF signaling pathway

目的探讨芒柄花素(FMN)调节低氧诱导因子-1α/血管内皮生长因子(HIF-1α/VEGF)信号通路对胃癌细胞增殖、凋亡和肿瘤血管生成拟态(VM)的影响。方法采用实时荧光定量PCR法检测人胃癌细胞株BGC-823、MGC-803、MKN28、SGC-790和人胃黏膜上皮细胞株GES-1中HIF-1α、VEGF的mRNA表达水平。将生长良好的MGC-803细胞分别设为对照组、30μmol/L FMN组、50μmol/L FMN组、80μmol/L FMN组、80μmol/L FMN+HIF-1α过表达质粒(pc-HIF-1α)组、80μmol/L FMN+阴性对照(pc-vector)组并进行相应处理。MGC-803细胞经上述药物处理及质粒转染后,采用MTT法计算MGC-803细胞增殖率,实时荧光定量PCR法检测各组MGC-803细胞中HIF-1α、VEGF的mRNA表达水平,流式细胞仪检测MGC-803细胞凋亡率,成管实验检测VM形成情况,蛋白质印迹法检测MGC-803细胞中VEGF、HIF-1α的蛋白表达水平。结果各种人胃癌细胞株中HIF-1α、VEGF的mRNA表达水平均较人胃黏膜上皮细胞株GES-1显著升高(P均<0.05);与对照组相比,30μmol/L FMN组、50μmol/LFMN组和80μmol/L FMN组的管样结构形成被不同程度损坏,管样结构数目、HIF-1α和VEGF的mRNA表达水平、VEGF和HIF-1α的蛋白表达水平,以及细胞增殖率均显著降低,细胞凋亡率均显著升高,并均呈剂量依赖性(P均<0.05)。与80μmol/L FMN+pc-vector组相比,80μmol/L FMN+pc-HIF-1α组的管样结构形成被改善,管样结构数目、HIF-1α和VEGF的mRNA表达水平、VEGF和HIF-1α的蛋白表达水平,以及细胞增殖率均显著升高,细胞凋亡率显著降低(P均<0.05)。结论FMN可抑制MGC-803细胞增殖及VM形成,并可诱导MGC-803细胞凋亡,这可能与抑制HIF-1α/VEGF信号通路有关。

Objective This paper aims to investigate the effects of fibronectin(FMN)on the proliferation and apoptosis of gastric cancer cells and tumor vasculogenic mimicry(VM)by regulating hypoxia inducible factor-1α/vascular endothelial growth factor(HIF-1α/VEGF)signaling pathway.Methods Real time fluorescence quantitative PCR(qRT-PCR)was used to detect the mRNA expression levels of HIF-1αand VEGF in human gastric cancer cell lines BGC-823,MGC-803,MKN28,SGC-790,and human gastric epithelial cell line GES-1.MGC-803 cells grown well were assigned into the control group,the 30μmol/L FMN group,the 50μmol/L FMN group,the 80μmol/L FMN group,the 80μmol/L FMN+HIF-1αoverexpression plasmid(pc-HIF-1α)group,and the 80μmol/L FMN+negative control(pc-vector)group,respectively.After MGC-803 cells were transfected with the above drugs and plasmids,MTT was used to calculate the proliferation rate of MGC-803 cells.qRT-PCR was used to detect the mRNA expression levels of HIF-1αand VEGF in MGC-803 cells in each group.Flow cytometry was used to detect the apoptosis rate of MGC-803 cells.A tube forming experiment was conducted to detect VM formation,and Western blotting was used to detect the protein expression levels of VEGF and HIF-1αin MGC-803 cells.Results The mRNA expression levels of HIF-1αand VEGF in human gastric cancer cell lines are obviously higher than those in human gastric epithelial cell line GES-1(P<0.05).Compared with the control group,the formation of tubular structure in the 30μmol/L FMN group,the 50μmol/L FMN group,and the 80μmol/L FMN group are destroyed to varying degrees.The number of tubes,the mRNA expression levels of HIF-1αand VEGF,the protein expression levels of VEGF and HIF-1α,and the cell proliferation rate are significantly reduced,while the cell apoptosis rate is significantly increased in a dose dependent manner(P<0.05).Compared with the 80μmol/L FMN+pc-vector group,the formation of tubular structure in the 80μmol/L FMN+pc-HIF-1αgroup is improved,the number of tubes,the mRNA expression levels of HIF-1αand VEGF,the protein expression levels of VEGF and HIF-1α,and the cell proliferation rate are significantly induced,while the cell apoptosis rate is significantly decreased(P<0.05).Conclusion FMN can inhibit the proliferation and VM formation of MGC-803 cells,and induce their apoptosis,which may be related to the inhibition of HIF-1α/VEGF signaling pathway.