Figure 2

PUMA was a direct target gene of miR-221/222. (A) Schematic representation of the putative binding sites in PUMA mRNAs 3'UTR for miR-221/222 (identical seed sequences AGCUACAU as shown). Sequence alignment of miR-221, miR-222 and the conserved binding sites among the different vertebrate species (Red, G:C pair or A:U pair; Blue, G:U pair). (B) U251 cells were transfected with As-miR-221/222, and PUMA protein level was detected by Western blot assay. β-actin protein was regarded as endogenous normalizer. (C) H4 cells were transfected with pMSCV-miR-221/222, and miR-221/222 and PUMA expression levels were detected by Northern blot and Western blot assay. U6 and β-actin protein were regarded as endogenous normalizer. (D) pGL3-WT-PUMA-3'UTR-Luc and pGL3-MUT-PUMA-3'UTR-Luc reporters were transfected into U251 cells transfected with As-miR-221 and/or As-miR-222. Luciferase activity was determined 48 h after transfection. The ratio of normalized sensor to control luciferase activity is shown. Error bars represent standard deviation and were obtained from three independent experiments. (E) U251 and LN229 cells were transfected with As-miR-221/222, and p53 protein level was detected by Western blot assay. β-actin protein was regarded as endogenous normalizer. * P < 0.05 compared with control group, ** P < 0.01 compared with control group.