纳布啡调节缺氧诱导因子-1α/血管内皮生长因子信号通路对胶质瘤细胞增殖、迁移和侵袭的影响

Effect of nabrphine on proliferation,migration,and invasion of glioma cells by regulating the hypoxia inducible factor-1α/vascularendothelial growth factor signaling pathway

目的探究纳布啡(NAL)对胶质瘤细胞增殖、迁移和侵袭的影响,以及对缺氧诱导因子-1α(HIF-1α)/血管内皮生长因子(VEGF)信号通路的调控机制。方法培养人胶质瘤细胞U251并将其随机分为U251组、NAL低浓度(NAL-L)组、NAL中浓度(NAL-M)组、NAL高浓度(NAL-H)组和NAL-H+HIF-1α激活剂(NAL-H+DMOG)组。采用CCK-8法检测各组U251细胞存活率;采用克隆平板实验检测各组U251细胞的克隆形成能力;采用Transwell实验检测各组U251细胞的迁移及侵袭细胞数;采用蛋白质印迹法(Western blot)检测各组细胞中HIF-1α/VEGF信号通路以及上皮-间充质转化相关蛋白上皮E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)和N-钙黏蛋白(N-cadherin)的相对表达水平。采用GraphPad Prism 9.0软件岁数据进行统计、分析,采用单因素方差分析多组间指标差异,采用Tukey’s事后检验进行两两组间的多重比较分析。结果与U251组比较,NAL-L组、NAL-M组及NAL-H组细胞存活率[(62.33±4.51)%、(41.06±3.37)%、(31.32±2.09)%]、克隆形成数量[(162.38±4.97)、(146.03±3.66)、(127.59±2.96)]、迁移及侵袭细胞数[(106.33±2.57,69.60±2.44)、(92.37±2.09,54.70±2.03)、(80.91±1.76,41.66±1.62)]以及细胞中HIF-1α(0.78±0.07、0.61±0.05、0.48±0.04)、VEGF(0.72±0.06、0.60±0.05、0.43±0.03)、Vimentin(0.80±0.07、0.68±0.05、0.47±0.03)、N-cadherin(0.71±0.06、0.56±0.04、0.40±0.03)的相对表达水平均低于U251组(P<0.05),而E-cadherin表达水平(1.21±0.09、1.47±0.13、1.67±0.17)高于U251组(P<0.05)。在高浓度NAL处理U251细胞的基础上加入HIF-1α激活剂DMOG则逆转了以上指标的变化趋势(P<0.05)。结论NAL能够通过抑制HIF-1α/VEGF信号通路来抑制胶质瘤细胞的增殖、迁移及侵袭过程。

Objective To investigate the effects of nalbuphine(NAL)on the proliferation,migration,and invasion of glioma cells,and its regulatory mechanism on hypoxia inducible factor-1α(HIF-1α)/vascularendothelial growth factor(VEGF)signaling pathway.Methods Human glioma cells U251 were cultured and randomly separated into U251 group,NAL low concentration(NAL-L)group,NAL medium concentration(NAL-M)group,NAL high concentration(NAL-H)group,and NAL-H+HIF-1αactivator(NAL-H+DMOG)group.The CCK-8 method was applied to detect the survival rate of U251 cells in each group.Cloning plate experiment was applied to detect the cloning ability of U251 cells in each group.Transwell experiment was applied to detect the number of migrating and invasive U251 cells in each group.Western blotting was applied to detect the HIF-1α/VEGF signaling pathway and the relative expression levels of epithelial mesenchymal transition related proteins(E-cadherin,Vimentin,and N-cadherin)in each group.All data were analyzed using GraphPad Prism 9.0 software,and one-way ANOVA was used to compare the differences in multiple indicators between groups.Tukey’s post hoc test was used for multiple comparison analysis between two groups.Results The cell survival rates in NAL-L group,NAL-M group and NAL-H group[(62.33±4.51)%,(41.06±3.37)%,(31.32±2.09)%],number of clones formed[(162.38±4.97),(146.03±3.66),(127.59±2.96)],number of migrating and invading cells[(106.33±2.57,69.60±2.44),(92.37±2.09,54.70±2.03),(80.91±1.76,41.66±1.62)],and HIF-1α(0.78±0.07,0.61±1.62,0.48±0.04),VEGF(0.72±0.06,0.60±0.05,0.43±0.03),Vimentin(0.80±0.07,0.68±0.05,0.47±0.03),the relative expression levels of N-cadherin(0.71±0.06,0.56±0.04,0.40±0.03)were lower than those in the U251 group(P<0.05),while the expression levels of E-cadherin(1.21±0.09,1.47±0.13,1.67±0.17)were higher than those in the U251 group(P<0.05).The addition of HIF-1αactivator DMOG to U251 cells treated with high concentration of NAL reversed the trend of changes in the above indicators(P<0.05).Conclusion NAL can inhibit HIF-1α/VEGF signaling pathway to inhibit the proliferation,migration,and invasion of glioma cells.