Journal: British Journal of Cancer
Article Title: PA28gamma emerges as a novel functional target of tumour suppressor microRNA-7 in non-small-cell lung cancer
Figure Lengend Snippet: miR-7 post-transcriptionally downregulates PA28gamma expression by directly targeting its 3′-UTR. ( A ) Putative target genes were predicted by PicTar, Target Scan, and miRanda. Each circle represents one prediction algorithm with the number of predicted genes. The number listed in overlapping areas of the circles was simultaneously predicted by different algorithms. ( B ) The potential binding sequences within the 3′-UTR of PA28gamma for miR-7 is conserved in human (Hsa), chimpanzee (Ptr), rhesus (Mml), mouse (Mmu), rat, hedgehog (Eeu), shrew (Sar), horse (Eca), cow (Eta), and elephant (Dno). Seed sequences are highlighted. ( C ) The PA28gamma 3′-UTR and corresponding fragments were inserted into the region immediately downstream of the luciferase gene in the pGL3-con vector. The sequences of the predicted miR-7 binding sites within the PA28gamma 3′-UTR including the wild-type UTR or mutant UTR segments (dotted lines) are shown. ( D ) After co-transfection of miR-7 mimic or miR-NC with the above reporter plasmids and pRL-TK vector in HEK 293T cells, relative luciferase activity was analysed by Dual-Luciferase Reporter Assay. ( E ) A549 and H1299 cells were transfected with miR-7, miR-NC, PA28gamma -siRNA, and miR-7 inhibitor for 48 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). ( F ) H1299 cells were transfected with PA28gamma -siRNA, and/or miR-7 inhibitor for 72 h. PA28gamma protein levels were examined by western blot using GAPDH as a loading control (one of three similar blots is shown). These experiments were performed in triplicate and results are shown as the mean±s.e.m. (*** P
Article Snippet: Vector constructs To construct the wild-type pGL3-con-PA28gamma -UTR-WT plasmid, a wild-type 3′-UTR fragment of the human PA28gamma mRNA (Genbank accession no. NM_005789) containing the putative miR-7 binding sequence (bases 1413–1419) was amplified by PCR and cloned into the XbaI/FseI site of the pGL3-control vector (Promega, Madison, WI, USA), which is downstream of the luciferase reporter gene.
Techniques: Expressing, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Cotransfection, Activity Assay, Reporter Assay, Transfection, Western Blot