miR-566 inhibitor blocked EGFR/Akt signaling. (A) qRT-PCR analysis of miR-566 in total RNA from indicated glioma cell lines and normal astrocytes. The data shown are average fold changes (the mean ± s.e. of 3 individual experiments) of individual miR-566 expression in each cell line relative to astrocytes. (B) Western blot (top) and real-time RT-PCR (bottom) were used to detect the expression of EGFR in a panel of glioma cell lines and astrocytes. GAPDH was used to normalize the initial input of total RNA. The expression levels of the transcripts of EGFR were based on the amount of target message relative to GAPDH. Bar graphs show the ratio of the expression level in each cell line to that in astrocytes. (C) qPCR was used to measure the expression of miR-566 in glioma cells that were infected with or without lenti-AS-566. The data are shown as the mean ± SD. **, P < 0.01. (D, E) U87 and LN229 glioma cells were either infected with lenti-NC or lenti-AS-566, and 48 h later, real-time RT-PCR (D) and Western blot (E) were used to detect the expression of EGFR and Akt.