MiR-24, but not miR-27a regulates 14-3-3β via sites in the 3′UTR and CDS.

<p>(A)–(C) Expression of mature miR-24 and 14-3-3β mRNA were measured via microarray. Correlation plots of 14-3-3β and miR-24 expression in (A) pre-B-ALL (n = 37), (B) T-ALL (n = 20), and (C) AML (n = 36) cell lines and primary samples are shown. Significant inverse correlation was determined via Pearson r (r<−0.03, p<0.05*). (D) The effect of miR-23a cluster members on endogenous 14-3-3β protein expression was measured via Western blot. The left panel is a representative blot from one experiment, and the right panel is a graph of normalized protein expression from 4 independent experiments (±SEM), determined by densitometry analysis (p<0.05*). (E) A schematic showing predicted miR-27a binding sites in the 3′UTR of 14-3-3β (top). HEK293T cells were co-transfected with 14-3-3βL-1537 or 14-3-3βL-1537-del (middle panel), and 14-3-3βL-2061 or 14-3-3βL-2061-del (bottom panel) (500 ng, white bars) alone and with 25 nM of miR-23a (grey bars) or miR-27a (black bars). Normalized mean Luc expression (±SEM, n≥3) is represented on the graph. (F) A schematic shows predicted miR-24 binding sites in the CDS and 3′UTR of 14-3-3β (top). HEK293T cells were co-transfected with 14-3-3βL-3′UTR (or deletion mutants 1 and 2; middle panel) or 14-3-3βL-CDS (or deletion mutants 1 and 2; bottom panel), (500 ng, white bars) alone and with 25 nM of miR-24 (grey bars) or miR-23a (black bars). Normalized mean Luc expression (±SEM, n≥3) is represented.