microRNA383调节人髓母细胞瘤Daoy细胞系中PRDX3的表达

摘要

目的 探讨microRNA383( miR-383)对人髓母细胞瘤Daoy细胞系中PRDX3基因的表达及细胞增殖活性与凋亡等的影响.方法 应用即时荧光定量逆转录聚合酶链反应(qRT-PCR)法检测人髓母细胞瘤组织(15例)、Daoy细胞系、对照脑组织(5例)中miR-383及PRDX3mRNA表达水平；采用Western blot法检测人髓母细胞瘤组织、Daoy细胞系、对照脑组织中PRDX3基因蛋白表达水平；人工合成miR-383模拟体(mimics)转染人髓母细胞瘤Daoy细胞系,采用Cell Counting Kit-8(CCK-8)法、流式细胞术等实验方法检测转染后各组Daoy细胞增殖与凋亡、细胞内活性氧水平、线粒体膜电位水平的变化.结果 15例人髓母细胞瘤组织中13例(13/15) miR-383表达水平较对照脑组织明显下调,14例( 14/15) PRDX3 mRNA表达水平较对照脑组织显著增高.miR-383、PRDX3mRNA在人髓母细胞瘤Daoy细胞系中表达水平分别为对照脑组织的0.353、1.315倍.PRDX3蛋白表达水平在人髓母细胞瘤组织、Daoy细胞系中较对照脑组织增高.miR-383mimics转染后24、48 h,实验组miR-383平均显著高于空白对照组,且48 h更加显著；而PRDX3mRNA、蛋白表达水平48 h较空白对照组显著降低.CCK-8法检测提示实验组中细胞增殖率较对照组明显降低(P＜0.05).Annexin VFITC法检测转染后48 h细胞凋亡结果显示miR-383 mimics实验组细胞早期凋亡率(11.60±0.30)％明显高于空白对照组(2.30±0.20)％(P=0.000).细胞内活性氧水平检测结果显示转染后24、48 h实验组较空白对照组细胞内活性氧水平明显升高,且48 h更加显著.线粒体膜电位水平检测结果显示转染后24h实验组线粒体膜电位水平较空白对照组明显降低.结论 与对照脑组织相比,miR-383在人髓母细胞瘤组织、Daoy细胞系中表达降低,PRDX3基因表达升高.上调miR-383可降低Daoy细胞系中PRDX3基因表达,并有效抑制Daoy细胞增殖活性,促进Daoy细胞凋亡,出现Daoy细胞内活性氧水平升高,线粒体膜电位水平降低的细胞功能学变化.%PRDX3 were higher in human medulloblatoma tumors and Daoy cells than that of normal brain tissue.Transfected miR-383 mimics increased the expression level of miR-383 after 24 h and 48 h was significantly higher than that of the control. In contrast, PRDX3 gene mRNA and protein expression levels were significantly decreased at 48 h compared with the control group.Using CCK-8 assay,the cell proliferation rate in the experimental group was significantly lower than that of the control group ( P ＜ 0.05 ).Annexin VFITC assay demonstrated that early apoptosis rate of the experimental group ( 11.60 ± 0.30 ) ％ was significantly higher than those of the control group (2.3 ± 0.20)％ and negative control group (10.37 ±0.25 ) ％ ( P =0.000) after 48 h of transfeetion.The intracellular ROS levels after transfection at 24 and 48 h significantly increased than those of the control group.Mitochondrial membrane potential level at 24 h after transfection significantly decreased,comparing with the blank control group and the negative control group.Conclusions Compared with normal brain tissue,decreased expression of miR-383 but elevated expression of PRDX3 are medulloblastoma tumour and Daoy cell lines. Up-regulation of miR-383 knockdowns the expression of PRDX3,inhibits proliferation and promotes apoptosis of Daoy cells,leading to increased intracelllar ROS and decreased levels of mitochondrial membrane potential.