槲寄生素对人骨肉瘤U20S细胞的抑制及对PI3K/Akt/mTOR通路的调控作用

目的:探讨槲寄生素对人骨肉瘤U20S细胞的抑制作用,并探讨其对磷脂酰肌醇-3激酶(phosphatidylinositol 3-kinase,PI3K)/Akt/m TOR通路的调控作用。方法:对数期生长U20S细胞分为5组:空白组,阳性组,槲寄生素低剂量组、中剂量组、高剂量组,空白组、槲寄生素各剂量组分别用0、5、10、20 mg·L^-1槲寄生素溶液处理,阳性组用245 mg·L^-15-FU处理,干预24 h后比较各组细胞形态学变化;采用MTT法检测并对比干预24、48、72 h后细胞增殖情况;分别采用Transwell法、流式细胞术检测并对比干预48 h时穿膜细胞数、凋亡率;分别采用RT-qPCR、Western blot法检测并对比干预48 h时细胞中PI3K、Akt、mTOR mRNA和蛋白及磷酸化Akt(pAkt)蛋白表达情况。结果:空白组细胞生长状态良好,不同干预组细胞数量减少、轮廓及遮光性变差,部分细胞变圆,呈漂浮状态,其中槲寄生素高剂量干预组生长状态最差。不同时刻各干预组MTT实验OD值从大至小、穿膜细胞数从多至少依次为:空白组>槲寄生素低剂量组>阳性组>槲寄生素中剂量组>槲寄生素高剂量组,除阳性组与槲寄生素中剂量组比较,差异无统计学意义(P>0.05),其他各组两两比较,差异均有统计学意义(P<0.05);槲寄生素高剂量组48 h、72 h OD值均高于24 h(P<0.05),48 h、72 h OD值比较,差异无统计学意义(P>0.05),其他各组OD值均随时间的延长呈升高趋势(P<0.05)。各干预组凋亡率从低至高依次为:空白组<槲寄生素低剂量组<阳性组<槲寄生素中剂量组<槲寄生素高剂量组,除阳性组与槲寄生素中剂量组比较,差异无统计学意义(P>0.05),其他各组两两比较,差异均有统计学意义(P<0.05)。各干预组PI3K、mTOR mRNA和蛋白相对表达量、p Akt/Akt从高至低依次为:空白组>槲寄生素低剂量组>阳性组>槲寄生素中剂量组>槲寄生素高剂量组,除阳性组与槲寄生素中剂量组比较,差异无统计学意义(P>0.05),其他各组两两比较,差异均有统计学意义(P<0.05)。结论:槲寄生素可有效抑制人骨肉瘤U20S细胞增殖和侵袭,促进凋亡,其中20 mg·L^-1的槲寄生素干预效果最佳,其机制可能与抑制PI3K/AKT/mTOR通路发挥调控作用有关。

Inhibition of Viscumin on Human Osteosarcoma U20S Cells and Regulation on PI3K/Akt/mTOR Pathway

Objective:To investigate the inhibitory effect of viscumin on human osteosarcoma U20S cells,and to explore its regulatory effect on the phosphatidylinositol 3-kinase(PI3K)/Akt/mTOR pathway.Methods:U20S cells in logarithmic phase were divided into 5 groups:blank group,positive group,low dose group,medium dose group,high dose group of quercetin,blank group,and various dose groups of quercetin with 0,5,10 And 20 mg·L^-1quercetin solution,the positive group was treated with 245 mg·L^-15-FU,and the morphological changes of the cells in each group were compared after 24 hours of intervention.Cell proliferation after72 h;Transwell method,flow cytometry were used to detect and compare the number of transmembrane cells and apoptotic rate at48 h after intervention;RT-qPCR and Western blot were used to detect and compare the cells at 48 h Expression of PI3K,Akt,mTOR mRNA and protein and phosphorylated Akt(pAkt)protein.Results:The cells in the blank group grew well.The number of cells in the different intervention groups decreased,their contours and shading became worse,and some of the cells became round and floating.The growth status of the high dose intervention group was the worst.At different times,the OD values of the MTT experiments of each intervention group were from large to small,and the number of transmembrane cells was at least in order:blank group>low dose group of quercetin>positive group,medium dose group of quercetin>high dose group of quercetin Except for the positive group and the middle dose group of quercetin,the difference was not statistically significant(P>0.05),and the other groups were compared in pairs,the difference was statistically significant(P<0.05);The OD values the high dose group of at 48 h and 72 h were higher than that at 24 h(P<0.05).There was no significant difference in OD values at 48 h and 72 h(P>0.05).The OD values of other groups increased with time,with significant difference(P<0.05).From low to high,the apoptosis rate of each intervention group is as follows:blank group0.05),and the other groups were compared pairwise,and the differences were statistically significant(P<0.05).The relative expression levels of PI3K,mTOR mRNA and protein,and p Akt/Akt in each intervention group are as follows:blank group>low dose group of quercetin>positive group>medium dose group of quercetin>high dose group of quercetin Except for the positive group and the middle dose group of quercetin,the difference was not statistically significant(P>0.05),and the other groups were compared in pairs,the differences were statistically significant(P<0.05).Conclusion:Viscumin can effectively inhibit the proliferation and invasion of human osteosarcoma U20S cells and promote apoptosis.Among them,20 mg·L^-1quercetin has the best intervention effect,and its mechanism may related to the regulatory role played in inhibiting the PI3K/AKT/mTOR pathway.