华蟾素介导miR-497-5p/VEGFA通路调控肺癌细胞的增殖、凋亡及血管生成

目的:探讨华蟾素介导miR-497-5p/VEGFA通路对肺癌细胞增殖、凋亡及血管生成的影响。方法:选取肺癌细胞株A549、H460为研究对象，以不同浓度华蟾素（0、25、50、75、100、125、150 μg/mL）作用细胞株，采用MTT检测细胞活力。随后，设置组别为空白组、华蟾素组（100 μg/mL华蟾素）、华蟾素+inhibitor NC组、华蟾素+miR-497-5p inhibitor组，通过MTT检测细胞活力；克隆形成实验检测克隆形成能力；流式细胞术检测细胞凋亡；Western blot检测细胞中VEGF、VEGFA蛋白表达；RT-qPCR检测细胞中miR-497-5p表达；并采用荧光素酶报告实验确定miR-497-5p靶向VEGFA。结果:随着华蟾素浓度的升高，A549、H460细胞增殖活力明显下降（P＜0.01）；与空白组相比，华蟾素组A549、H460细胞克隆形成能力明显降低、细胞凋亡明显增加、VEGF和VEGFA蛋白表达明显降低、miR-497-5p mRNA表达明显升高（P＜0.01）；荧光素酶报告实验显示miR-497-5p靶向VEGFA；与华蟾素+inhibitor NC组相比，华蟾素+miR-497-5p inhibitor组A549、H460细胞中miR-497-5p mRNA表达明显降低、VEGF和VEGFA蛋白表达明显升高、细胞克隆形成能力明显增加、细胞凋亡明显被抑制、细胞增殖活力明显增加（P＜0.05）。结论:华蟾素可能通过调控miR-497-5p/VEGFA通路从而抑制肺癌细胞增殖和血管生成，并诱导细胞凋亡。

Cinobufotalin-mediated miR-497-5p/VEGFA pathway regulates proliferation,apoptosis and angiogenesis of lung cancer cells

Objective:To investigate the effect of miR-497-5p/VEGFA pathway on proliferation,apoptosis and angiogenesis of lung cancer cells mediated by cinobufotalin.Methods:Lung cancer cell line A549 and H460 were selected for the study,and the cell lines were treated with different concentrations of cinobufotalin (0,25,50,75,100,125,150 μg/mL),and the cell viability was detected by MTT.Subsequently,groups were set up as control group,cinobufotalin group (100 μg/mL cinobufotalin),cinobufotalin+inhibitor NC group,and cinobufotalin+miR-497-5p inhibitor group,and cell viability was detected by MTT.Clone formation assay was performed to detect clone formation ability.Apoptosis was detected by flow cytometry.The protein expression of VEGF,VEGFA was detected by Western blot.miR-497-5p expression was detected by RT-qPCR.And miR-497-5p targeting to VEGFA was determined using luciferase reporter assay.Results:The proliferative viability of A549,H460 cells decreased significantly with the increase of cinobufotalin concentration (P＜0.01).Compared with the control group,the clone formation ability of A549,H460 cells was significantly reduced,apoptosis was significantly increased,VEGF and VEGFA protein expression was significantly reduced,and miR-497-5p mRNA expression was significantly increased in the cinobufotalin group (P＜0.01).Luciferase reporter assay showed that miR-497-5p targeted VEGFA.Compared with the cinobufotalin+inhibitor NC group,miR-497-5p mRNA expression was significantly lower,VEGF and VEGFA protein expression was significantly higher,cell clone formation ability was significantly increased,apoptosis was significantly inhibited,and cell proliferation viability was significantly increased in the A549,H460 cells of cinobufotalin+miR-497-5p inhibitor group (P＜0.05).Conclusion:Cinobufotalin may inhibit proliferation and angiogenesis of lung cancer cells,and induce cells apoptosis by regulating miR-497-5p/VEGFA pathway.