MLN2238-inducedmiRprofiling in MM cells and expression of miR33b in MM and normal cells. (A) MM.1S MM cells was treated with vehicle or MLN2238 (12nM) for 3 hours and cells were harvested. RNAs were isolated for miR profiling using ABI Gene Card A Version 2. The data were analyzed by dCHIP and a minimum difference of ≥ 1.5 ΔΔCt value of a miR between control and test samples is shown. Blue represents down-regulated and red represents up-regulated miRs. (B) MM.1S cells were treated with vehicle or MLN 2238 (12nM) for indicated times and then cells were harvested. RNAs were isolated and subjected to qRT-PCR to examine the expression of pri-miR33b, pre-miR33b, and mature miR33b. (C) MM.1S cells were treated with Act-D (5 μg/mL), MLN2238, or Act-D + MLN 2238 (12nM) for the indicated times. The cells were harvested and RNAs were prepared to examine the expression of miR33b. (D) INA6 and ANBL6 MM cells were treated with vehicle or MLN 2238 (12nM) for the indicated times and then harvested. RNAs were isolated and subjected to qRT-PCR to examine the expression of miR33b. (E) RNAs were isolated from purified patient MM cells, normal CD138⁺ cells derived from BM, and PBMCs from healthy donors, followed by analysis of basal expression level of miR33b using qRT-PCR. (F) MM.1S cells were treated with vehicle, dexamethasone (30nM), lenalidomide (7μM), or SAHA (160nM) for the indicated times and then cells were harvested. RNAs were isolated and subjected to qRT-PCR to examine the expression of miR33b. Results shown are means ± SD (n = 3). ***P ≤ .001.