Fig 2: miR-200b expression has a critical role in the stemness properties and division of the CD133+ glioma cells. (A) TC1 cells were sorted and CD133+ and CD133- populations were analyzed by RT-qPCR for CD133 and miR-200b levels. **P<0.01 vs. CD133+. (B) TC1 cells were sorted for CD133. The CD133+ population was transfected with scrambled control, miR-200b mimics or miR-200b inhibitor. Overexpression and knockdown confirmed by RT-qPCR. Effects on CD133, Ki67 and GFAP mRNA levels. (C) RT-qPCR of different markers (Nestin, GAP43) from the CD133+ population transfected with scrambled control, miR-200b mimics or miR-200b inhibitor. *P<0.05, **P<0.01 vs. scramble control; #P<0.05, ##P<0.01 vs. inhibitor control. (D) Clonal assay was performed on control, miR-200b mimics and miR-200b inhibitor transfected CD133+ TC1 cells. (E) Neurosphere formation was detected in CD133+ TC1 cells transfected with control, miR-200b mimics and inhibitor. (F) The activity of AKT signaling (CD133, p-AKT, total AKT, Notch) in CD133+ cells with CD133 knockdown or miR-101 overexpression tested by western blot. GAPDH was used as an internal control. **P<0.01 vs. si-control; ##P<0.01 vs. scramble. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; miR, microRNA; GFAP, glial fibrillary acidic protein; GAP43, growth-associated protein 43; si, small interfering RNA; p-, phosphorylated; AKT, AKT serine/threonine kinase 1.

Fig 3: miR-200b suppresses the expression of CD133 in glioma cells. (A) The expression of miR-200b and CD133 in 20 normal brain tissues and 80 glioma samples. (B) Correlation between the expression of miR-200b and CD133 in glioma tissues. miR, microRNA.

Fig 4: miR-200b levels are essential for proliferation and stem-like properties of human glioma cells. (A) Left and middle: Number of TC1 and TC2 cells infected with scrambled control, miR-200b mimics or miR-200b inhibitor; Right: The miR-200b expression was detected by RT-qPCR. (B) Imaging of TC1 and TC2 cells cells positive for BrdU staining. (C) Quantification of TC1 and TC2 cells positive for BrdU staining. (D) RT-qPCR detected Ki67, GAP43, GFAP and CD133 levels in TC1 cells infected with scrambled control, miR-200b mimics or miR-200b inhibitor. *P<0.05, **P<0.01 vs. scramble; #P<0.05, ##P<0.01 vs. inhibitor control. miR, microRNA; GAP43, growth-associated protein 43; GFAP, glial fibrillary acidic protein.

Fig 5: PROM1 is a direct target of miR-200b. (A) Schematic representation of putative miR-200b-binding sites in the 3'UTR of PROM1. (B) U251 cells were cotransfected with wt or mut PROM1 3'-UTR, pGL-3 control and either miR-200b or negative control. Relative firefly luciferase activity was normalized to Renilla luciferase activity. (C) Expression of PROM1 mRNA was detected by reverse transcription-quantitative polymerase chain reaction in TC1 and TC2 cells transfected with miR-200b mimics or control mimic and in these two cells transfected miR-200b inhibitor or control inhibitor. (D) Protein level of CD133 was detected by western blot analysis in TC1 and TC2 cells transfected with miR-200b mimics or control mimic and in these cells transfected with miR-200b inhibitor or control inhibitor. GAPDH was used as an internal control. *P<0.05, **P<0.01 vs. scramble control. 3'UTR, 3' untranslated region; miR, microRNA; PROM1, prominin 1.