Figure 6

CDK6 expression is inhibited by miR-124 and miR-137 in glioblastoma multiforme cells. (A) Transfection of 100 nM miR-124 or miR-137 reduces cyclin-dependent kinase 6 mRNA transcript levels by 50% in U251 cells at 48 hours relative to cells transfected with 100 nM control oligonucleotide. Cyclin-dependent kinase 6 expression was determined by TaqMan relative to control genes Gus (black bars), GAPDH (gray bars) and 18S (white bars). Values represent average +/- standard deviation of independent experiments. (B) Cyclin-dependent kinase 6 protein expression is dramatically reduced as determined by western blotting following transfection of miR-124 or miR-137. Levels of phosphorylated RB (pSer 807/811) are also markedly reduced in response to miR-124 or miR-137 transfection. (C) miR-137 sequence in relation to the pMIR-REPORT vector containing the predicted cyclin-dependent kinase 6 miR-137 binding site (wild type). Mutated bases (underlined) were also introduced into the miR-137 seed region (boxed) of the cyclin-dependent kinase 6-3'UTR (mutated). Vertical lines denote Watson-Crick base pairing. (D) Relative luminescence of U251 cells following transfection miR-137 or negative control microRNA in conjunction with wild type or mutated cyclin-dependent kinase 6 reporter constructs. *P < 0.0001.