Fig. 2.
BCL2 is a target of miR-15 and miR-16.(A) Transfection of miR-15a/miR-16-1 cluster in MEG-01 BCL2+ leukemia cells is followed by a significant reduction in protein levels. Data were confirmed in duplicate experiments. The pSR-mir15/16-WT-transfected cells shows cleavage of APAF-1 (a cytochrome c interactor), pro-caspase 9 (intrinsic pathway), and PARP (a final effector of various apoptotic pathways). (B) Transfection with RNA oligos miR-15 and miR-16 separately or combined significantly reduce Bcl2 protein levels. Normalization was performed with β-actin. The Northern blot (NB) showed the miR-16-sense transfection efficiency. The same results were obtained for the other three oligos used (data not showed). The mRNA levels of the BCL2 gene in the same cells are shown and normalized against β-actin mRNA expression. WB, Western blot. (C) The 3′ UTR of BCL2 enables miR-15/16 regulation. Relative repression of firefly luciferase expression was standardized to a transfection control, Renilla luciferase. pGL-3 (Promega) was used as the empty vector. (Left) miR-15-a and miR-16-1 oligoRNAs (sense and antisense) were used for transfections. (Right) pSR-mir15/16-WT was used. Two different types of 3′ UTR mutants were constructed, one without all 9 bp of miRNA::mRNA interaction (3′M1) and the other with a deletion of the first 5 bp in the same complementarity region (3′M2). All of the experiments were performed twice in triplicate (n = 6).