微小RNA-203在胶质母细胞瘤干细胞中的表达及其对细胞增殖的影响

摘要

目的 培养胶质母细胞瘤干细胞,检测微小RNA-203(miR-203)在胶质母细胞瘤干细胞中的表达,并观察其对肿瘤干细胞增殖的影响.方法 对胶质母细胞瘤组织行体外原代培养,以CD133为标志物,免疫磁珠法分选胶质母细胞瘤干细胞;免疫荧光染色检测分选所得细胞的CD133、巢蛋白(nestin)、神经胶质纤维酸性蛋白(GFAP)、微管相关蛋白2(MAP2)的表达,从而鉴定肿瘤干细胞.反转录-聚合酶链反应(RT-PCR)检测分选后CD133+细胞、CD133-细胞miR-203的表达;将miR-203模拟体和无意义寡核苷酸链(NC)分别转染胶质母细胞瘤干细胞作为miR-203、NC组,细胞计数试剂盒(CCK-8)法检测转染后24、48、72、96、120 h细胞生存率;流式细胞仪细胞检测转染后3d细胞凋亡率.结果 培养的胶质母细胞瘤干细胞成球样生长,表达干细胞标志物CD133、Nestin,分化后细胞表达星形胶质细胞的标志物GFAP、神经元的标志物MAP2;分选后CD133+细胞中miR-203的表达低于CD133-细胞;转染miR-203后24、48、72、96、120 h组的细胞生存率均降低,差异有统计学意义(P＜0.05);流式细胞仪检测显示miR-203组的细胞凋亡率为(9.74±2.81)％,高于NC组的(3.95±0.91)％,差异有统计学意义(P＜0.05).结论 上调miR-203可能成为针对胶质母细胞瘤干细胞的基因治疗策略.%Objective To isolate glioblastoma multiforme stem cells (GBM-SCs) from glioblastoma multiforme (GBM) specimens and to investigate the expression of microRNA (miR)-203 in GBM stem cells and its impact on cell proliferation.Methods CD133 + cells were separated using magnetic cell sorting technique (MACS) after primary culture.Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze miR-203 expression in CD133 + cells.Lipofectamine RNAiMAX was used to transfect miR-203 mimic and scrambled control oligonucleotides into GBM-SCs.The proliferation ability of miR-203 treated GBM-SCs was determined by CCK-8 and the apoptosis ratio was detected by cell flow cytometry.Results GBM-SCs isolated from GBM specimens formed GBM spheres,expressed markers associated with neural stem cells,and possessed the capacity for self-renewal and multilineage differentiation.MiR-203 expression was down-regulated in CD133+ cells relative to CD133-cells.MiR-203 can repress GBM-SCs growth and induce apoptosis.Conclusion Reactivation of miR-203 expression suggests novel therapeutic strategies for GBM-SCs.