二氢青蒿素调控miR-219a/MDM2信号通路介导胶质瘤细胞的增殖和凋亡

Dihydroartemisinin Mediates Glioma Cell Proliferation and Apoptosis by Regulating miR-219a/MDM2 Signaling Pathway

目的:探讨二氢青蒿素对胶质瘤细胞增殖和凋亡的影响,及对微小RNA-219a(miR-219a)/鼠双微体基因2(murine doubleminute2,MDM2)信号轴的调节作用。方法:qRT-PCR法检测人正常星形胶质细胞系HA1800和不同胶质瘤细胞系U251、U87和SHG44中miR-219a相对表达;不同浓度二氢青蒿素处理U251细胞,MTT法检测细胞存活率。U251细胞分为空白对照组、miR-219a阴性对照组(转染miR-219a抑制物阴性对照质粒)、miR-219a抑制剂组(转染miR-219a抑制物)、二氢青蒿素+miR-219a抑制剂组(转染miR-219a抑制物24 h后,80μmol/L二氢青蒿素处理),qRT-PCR检测miR-219a和Mdm2 mRNA相对表达;MTT法检测细胞存活率;流式细胞仪检测细胞凋亡率;蛋白印迹法检测MDM2、P53、B淋巴细胞瘤-2基因(BCL-2)和Bcl-2相关X蛋白(BAX)相对表达。结果:U251、U87和SHG44细胞中miR-219a相对表达低于HA1800细胞(P<0.05);U251细胞存活率随二氢青蒿素浓度的升高明显降低(P<0.05);与空白对照组和miR-219a阴性对照组比较,miR-219a抑制剂组miR-219a相对表达、细胞凋亡率以及BAX和P53蛋白相对表达降低,Mdm2 mRNA相对表达以及MDM2和BCL-2蛋白相对表达上调(P<0.05);与miR-219a抑制剂组比较,二氢青蒿素+miR-219a抑制剂组miR-219a相对表达、细胞凋亡率以及BAX和P53蛋白相对表达升高,细胞存活率明显降低、Mdm2 mRNA相对表达以及MDM2和BCL-2蛋白相对表达明显下调(P<0.05)。结论:二氢青蒿素可抑制胶质瘤U251细胞增殖,诱导凋亡,其可能是通过调节miR-219a靶向抑制MDM2表达发挥作用。

Objective:To investigate the effects of dihydroartemisinin on the proliferation and apoptosis of glioma cells and the regulation of miR-219a/murine double minute 2(MDM2)signaling axis.Methods:The relative expression levels of miR-219a in human normal astrocytes cell line HA1800 and different glioma cell lines U251,U87,and SHG44 were detected by real-time quantitative polymerase chain reaction(qRT-PCR).U251 cells were treated with different concentrations of dihydroartemisinin,and the cell survival rate was detected by methyl thiazolyl tetrazolium(MTT)assay.U251 cells were divided into a control group,a miR-219a negative control group(transfected with miR-219a inhibitor negative control plasmid),a miR-219a inhibitor group(transfected with miR-219a inhibitor),and a dihydroartemisinin+miR-219a inhibitor group(80μmol/L dihydroartemisinin treatment 24 h after transfection with inhibitor).The mRNA expression levels of miR-219a and MDM2 were detected by qRT-PCR.MTT assay was used to detect the cell survival rate,the flow cytometry was used to detect the cell apoptosis rate,and Western blot was used to detect the relative protein expression levels of MDM2,B-lymphocytoma-2 gene(Bcl-2),and Bcl-2 associated X protein(Bax).Results:The relative expression levels of miR-219a in U251,U87,and SHG44 were lower than those in HA1800 cells(P<0.05).The survival rate of U251 cells was decreased with the increase in dihydroartemisinin concentration(P<0.05).As compared with the control group and the miR-219a negative control group,the relative expression level of miR-219a,apoptosis rate,and the relative protein expression levels of Bax and p53 in the miR-219a inhibitor group were decreased,while the relative mRNA expression level of Mdm2 and the relative protein expression levels of MDM2 and Bcl-2 were up-regulated(P<0.05).As compared with the miR-219a inhibitor group,the relative expression level of miR-219a,cell apoptosis rate,and the relative protein expression levels of Bax and p53 in the dihydroartemisinin+miR-219a group were increased,while the cell survival rate,the relative mRNA expression level of Mdm2 and the relative protein expression levels of MDM2 and Bcl-2 were decreased(P<0.05).Conclusion:Dihydroartemisinin can inhibit the proliferation of glioma U251 cells and induce apoptosis,which may play a regulatory role by regulating miR-219a to target and inhibit MDM2 expression.