Abstract:
Objective To investigate the stimulatory effects of miR-218 on the differentiation of glioblastoma cells.
Methods The SNB-19 glioblastoma subcell line overexpressing miR-218 (SNB19-miR218) and the control subcell line overexpressing a nonsense scrambled sequence (SNB19-scr) were established via plasmid transfection and G418 screening. The expression levels of miR-218, CD133, and nestin were detected via quantitative reverse transcription polymerase chain reaction and western blot analysis, whereas the differentiation status was assessed via glial fibrillary acidic protein (GFAP) immunocytochemistry. Using low-copy plasmid transfected cells, the distribution of CD133- and nestin-positive cells was detected via immunofluorescence against an enhanced green fluorescent protein (EGFP) reporter.
Results The expression levels of miR-218, CD133, and nestin in the cells transfected with SNB19-miR218 were 26.23-fold, 17.9%, and 54.2% of those of SNB19-scr. The GFAP labeling index of SNB19-miR218 (96.0% ± 3.81%) was significantly higher than that of SNB19-scr (49.6% ± 5.13%, t = 16.24, P < 0.0001). Immunofluorescence showed that the constituent ratios of the CD133- and nestin-positive cells in the EGFP (+) and (-) subpopulations were significantly uneven (χ 2 = 586.38 and 658.53, respectively, P < 0.0001), confirming that the CD133 and nestin were downregulated specifically in the tumor cells successfully transfected with the miR-218 expression plasmid.
Conclusion miR-218 promotes glioma stem cell differentiation, and therefore downregulates CD133 and nestin expression. This effect may be an important mechanism for inhibiting the proliferation of glioma cells.
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