The BHRF1 miRNA negatively regulates PTEN expression.

<p>(a) The miR-BHRF1-3 target site in the PTEN 3’UTR according to a PAR-CLIP assay is shown (Skalsky et al.). (b) Primary B-cells were infected with B95-8 wild type (wt) or with the Δ123 recombinant and harvested at the indicated time points after infection for immunoblotting with a PTEN-specific antibody. Actin served as a loading control. Exactly the same amounts of Elijah and Oku-BL extracts were loaded on both gels to allow comparison of signal intensity between the two blots. The right panel shows the ImageJ-based quantification of the PTEN protein levels over time, normalized for differences in actin levels and signals obtained with Elijah and Oku-BL. PTEN expression in LCLs transformed by wt EBV, miR-BHRF1-3 (Δ3) single knockout (c), or 3SM (d). Actin served as a loading control. The right panels show the ImageJ-based quantification of PTEN expression for multiple blood samples. All values were normalized for differences in actin levels. (e) Luciferase assays performed with the PTEN 3’UTR or a seed-match mutant thereof by transfection of an expression plasmid that drives miR-BHRF1-3 expression or an empty pcDNA3.1 negative control. The results of three transfections are given relative to the luciferase activity signals recorded with the negative control. (f) 14 days old B-cells from two donors transformed with wild type EBV were treated for 24h with the PI3K inhibitor wortmannin (10nM) or the solvent DMSO. The cell cycle profile was determined by BrdU incorporation and quantified by FACS analysis. The results obtained with one B-cell primary sample are shown. This right panel depicts the ratio of cells present in the G2/M phase and in the S phase determined for two donors.