Lenalidomide induced miR-181a expression sensitizes leukemia cells to conventional chemotherapy. (A) Untransfected THP-1 cells were cultured for 6 days in the presence of 3μM lenalidomide alone, 1μM ara-C alone, both drugs together (as indicated by “+” or “−” below the graph), or vehicle control (PBS; the left-most bar) and MTS proliferation assay was performed. The bars represent averages from 3 to 4 readings. SEM and relative percentages of proliferation rate are shown. (B) Quantitative real-time RT-PCR analysis for the expression of miR-181a in THP-1 cells transiently transfected with nontargeting control (negative control) or antagomiR-181a. Endogenous expression of miR-181a was found to be lower in those cells transfected with antagomiR-181a before drug treatments (black bars). After the treatment with 3.0μM lenalidomide, miR-181a expression was increased among those cells previously transfected with nontargeting control (red bars). In contrast, the expression of miR-181a was relatively unchanged among those cells transfected with antagomiR-181a followed by 3.0μM lenalidomide treatment (blue bars). Data are shown as an average of measurements and error bars denote SEM. (C) Western blot for those cells described in panel A with the additional treatment with either 3.0μM lenalidomide or vehicle control for 3 days. The expression of C/EBPα (p42 and p30) was found to be higher in those cells treated with lenalidomide, regardless of earlier transfection status described in panel A. Actin served as an internal loading control. (D) THP-1 cells transfected with nontargeting control, or antagomiR-181a were cultured with various concentrations (0-5μM) of cytarabine (Ara-C) in the presence of 3.0μM lenalidomide (lenalid.; solid lines), or vehicle (broken lines) for 72 hours and cellular proliferation was measured by MTS assay. Each datapoint represents an average of 3 measurements.