Figure 3

MiR-125b suppresses the expression of the luciferase reporter with Nestin 3’-UTR. (A) Schematic representation of the constructs used in the luciferase reporter assay. The Nestin 3’-UTR sequences containing the putative binding sites of miR-125b were inserted into the the pGL3 luciferase reporter vector. Site-directed mutagenesis was performed to mutate base pairs in the predicted seed region targeted by miR-125b in the Nestin 3'-UTR. The mutant binding site is underlined. (B) Luciferase assay was performed 72 hours after transfection using the Dual Luciferase. MiR-125b down-regulated luciferase activities controlled by wild-type Nestin 3’UTR, but did not affect luciferase activity controlled by mutant Nestin 3’UTR. The firefly luciferase activity was standardized to renilla luciferase control. (**P < 0.01 vs. WT; *P < 0.05 vs. WT).The results are means of three independent experiments±SD. (C) The expression level of Nestin was analysed by qRT-PCR and the Western blot in NS/PCs after transfection with miR-125b mimics, inhibitors, NC and INNC. Bars show mean ± SD. All experiments were repeated three times. * P < 0.05 vs. ctr, ** P < 0.01 vs. ctr.