上调microRNA-383对人髓母细胞瘤D341细胞系中PRDX3表达的影响

目的探讨microRNA-383(miR-383)对人髓母细胞瘤D341细胞系中PRDX3基因的表达及细胞增殖活性与凋亡等细胞功能学的影响。方法应用实时荧光定量PCR法检测人髓母细胞瘤D341细胞系、正常脑组织中miR-383及PRDX3 mRNA表达水平;采用Western blot法检测人髓母细胞瘤D341细胞系、正常脑组织中PRDX3基因、蛋白表达水平;人工合成miR-383模拟体(mimics)转染MBD341细胞系,采用cck-8法、流式细胞术等实验方法检测转染人髓母细胞瘤D341细胞系后各组D341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。结果人髓母细胞瘤BD341细胞系中miR-383表达水平为正常脑组织的0.524倍,明显下调。而PRDX3基因mRNA表达水平为正常脑组织的5.214倍,蛋白表达水平较正常脑组织明显上调。miR-383 mimics转染D341细胞系24、48 h,实验组miR-383表达水平均明显高于空白对照组,且48 h作用更加明显;而PRDX3基因mRNA、蛋白表达水平48 h较空白对照组降低。cck-8法检测提示实验组中细胞增殖率降低(P<0.05)。An-nexinV-FITC法检测转染后24、48 h细胞凋亡结果显示miR-383 mimics实验组细胞早期凋亡率明显增高,且24 h作用较明显(P=0.000)。细胞内活性氧水平和线粒体膜电位水平检测结果分别显示,转染后24、48 h实验组较对照组细胞内活性氧水平明显升高,而线粒体膜电位水平较对照组明显降低。结论与正常脑组织相比,miR-383在人髓母细胞瘤D341细胞系中表达降低,PRDX3基因表达升高。上调miR-383可敲低D341细胞系中PRDX3基因表达,并可抑制D341细胞增殖活性,促进D341细胞凋亡,出现D341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。

Upregulation of microRNA-383 influences the PRDX3 expression on human medulloblastoma cell line D341

Purpose To investigate the effects of microRNA-383(miR-383) on PRDX3 expression,cell proliferation and apoptosis and other cell functions of human medulloblastma tumor and cell lines D341.Methods Real-time quantitative RT-PCR was used to detect D341 cells,normal brain tissue in the miR-383 and PRDX3 mRNA expression levels.Western blot was used to detect the PRDX3 protein expression of human D341 cells,and normal brain tissue.Synthetic miR-383 mimics were transfected into D341 cell line by lipofectamine.Using cck-8 method,flow cytometry experiments were conducted to detect the transfected cells in each group,including cell proliferation and apoptosis,cells reactive oxygen species,mitochondrial membrane potential changes.Results The miR-383 expression level was 0.524 times lower than normal brain tissue,and the PRDX3 mRNA expression level was 5.214 times higher than normal brain tissue with high expression.The protein expression levels of PRDX3 in human medulloblatoma D341 cell line were higher than that in nomal brain tissue.After transfected miR-383 mimics 24 h and 48 h,the expression of miR-383 in the experimental group were significantly higher than the control group,and 48 h was more significant,and PRDX3 gene mRNA,protein expression levels in 48 h were significantly reduced,compared with the control group.The detection of cck-8 assay showed that the cell proliferation rate in the experimental group was significantly lower than that in the control group(P<0.05).24 h and 48 h after transfection,annexin V-FITC assay showed that early cell apoptosis rate was significantly higher.The detection of intracellular ROS levels after transfection at 24 and 48 h showed that in the experimental group were significantly increased than that in the control group.The detection of the mitochondrial membrane potential level was significantly lower than the control group.Conclusions Compared with the normal brain tissue,the expression of miR-383 in human medulloblastoma D341 cell lines is reduced,but the expression of PRDX3 is elevated.Up-regulated expression of miR-383 can not only knockdown the expression of PRDX3 in D341 cell line,but also inhibit the D341 cell proliferation and promote apoptosis,elevate the levels of intracellular reactive oxygen species,and reduce the level of mitochondrial membrane potential.