Effect of microRNA-153 on biological characteristics of glioblastoma stem cells / 中华神经医学杂志

ABSTRACT

Objective To isolate the glioblastoma multiforme stem cells (GBM-SCs) from GBM specimens and to investigate the biological role ofmiR-153 in GBM-SCs so as to explore the application of gene therapy of GBMs.Methods CD133+ cells were separated using magnetic cell sorting technique (MACS) after primary culture.Immunofluorescence staining was employed to detect the CD133,nestin,glial fibrillary acidic protein (GFAP),microtubule-associated protein 2 (MAP2) expressions; real time-PCR was used to analyze miR-153 mRNA expression in CD 133-cells and CD 133+ cells.Lipofectamine RNAiMAX was used to transfect MiR-153 mimic (miR-153 group) and scrambled control oligonucleotides (NC group) into GBM-SCs; 7 d after that,sphere formation assay was performed to determine the self-renewal ability of GBM-SCs.Real time-PCR and immunofluorescence were carried out to examine the CD133,nestin,GFAP,MAP2 mRNA and protein expressions.At last,the proliferation ability of miR-153 treated GBM-SCs and NC cells was determined by CCK-8 at 24,48,72,96 and 120 h after the transfection and the apoptosis ratio was detected by flow cytometry 3 d after the transfection.Results GBM-SCs isolated from GBM specimens could express stem cell markers CD133 and nestin; after differentiation,the cells could express astrocyte marker GFAP and neuron marker MAP2; miR-153 expression in CD133+ cells was signficantly down-regulated as compared with that in CD133-cells (P＜0.05).Seven d after transfection,the number of spheres in the NC group was significantly larger than that in the miR-153 group (P＜0.05); real time-PCR indicated that the mRNA expressions of CD 133 and nestin in the miR-153 group were significantly decreased,and the GFAP and MAP2 mRNA expressions were statistically increased as compared with those in the NC group (P＜0.05).The results detected by immunofluorescence were in accordance with those by real time-PCR; 48,72,96h after the transfection,cell viability in cells from miR-153 group was statistically significant lower than that in the NC group (P＜0.05); flow cytometry showed that the apoptosis rate of cells from the miR-153 group (9.4 1％±1.98％) was significantlyhigher than that in the NC group (4.28％±0.31％,P＜0.05).Conclusion Reactivation of miR-153 expression suggests novel therapeutic strategy for GBM-SCs.