Upregulation of miR-326 regulated the ability of self-renewal and stemness and prompted differentiation in glioma stem cells. (A) miR-326 upregulation significantly decreased the percentage of neurosphere formation with or without SMO overexpression. (B) and (C) The upregulation of miR-326 decreased Bmi-1, Oct-4, Sox-2, Nanog, and Shh expression, as demonstrated by qRT–PCR and Western blot assays. (D) and (E) The overexpression of miR-326 partly decreased nestin expression and increased the GFAP expression in U251-TSC cells, as determined by immunofluorescence (scale bars, 50 µm). (F) Inhibition of the Hh pathway by antagonists that bind to SMO (cyclopamine,10uM) elevated the miR-326 expression in glioma cells. The data represent mean ± SE of 3 replicates (*P < .05).