MicroRNA and mRNA expression in B-CLL. (A) Results of TaqMan PCR in B-CLL and control CD19⁺ cells (Ctrl). The y-axis represents relative abundance of mature miR-155 (number of B-CLL patients, N(B-CLL) = 118, number of controls, N(Ctrl) = 7) and of miR-150 (N(B-CLL) = 75, N(Ctrl) = 4) relative to RNU44; and of MYB mRNA (N(B-CLL) = 119, N(Ctrl) = 10) and PU.1 mRNA (N(B-CLL) = 119, N(Ctrl) = 5) relative to GAPDH. Data (shown as a fold change [FC]) are baseline normalized to RNA levels obtained in normal controls (control measurements were set equal to 1) and shown in log2 scale. Error bars represent SEM. P values (t test) are shown in parentheses. (B) Immunoblotting analysis of MYB and PU.1 in B-CLL (N = 5) and control (C87, N = 1) B-cell samples. The NB4 cell line was used as a positive control. The optical density (below each lane) of each protein band was measured by Scion Image software for Windows Beta 4.0.2, the values below the Western blots represent a fold change of densities of the specific bands (or their expected positions) in the patient, and control lanes normalized to the levels of the housekeeping protein β-actin. (C) The MYB mRNA levels in primary B-CLL cells positively associate with Rai staging. Three patient subgroups are shown. White bars represent currently investigated patients (OS 0) either treated by first-line therapy or untreated (Th 0–1); gray bars, currently investigated patients (OS 0) who received second or more lines of therapy (Th > 1); and black bars, previously investigated, deceased patients (OS 1) who had received at least one line of therapy (Th ≥ 1). The patient numbers in each expression assay and patient group are indicated in parentheses. Error bars represent SEM. P values (t test) are shown in parentheses on top of the brackets indicating the groups being compared. (D) RNA levels of miR-155 (white bars), PU.1 (light gray bars), MYB (dark gray bars), and miR-150 (black bars) categorized by the B-CLL prognostic parameters. Favorable outcome group = IgVH mutated, CD38 negative, ZAP70 negative. Unfavorable outcome group = IgVH unmutated, CD38⁺, ZAP70⁺. The patient numbers in each expression assay are indicated in parentheses. Error bars represent SEM. P values (t test) are shown in parentheses on top of the lines indicating the groups being compared. (E) Expression levels of miR-155 (white bars) and miR-150 (gray bars) in MYB underexpressors (< 1.5-fold decrease, left) and MYB overexpressors (> 1.5 increase; right) relative to control B cells. Patient numbers in each expression assay are indicated in parentheses. Error bars represent SEM. P values (t test) are shown in parentheses on top of the brackets indicating the groups being compared.