Reversible silencing and function of MIR34B in myeloma cells. (A) Effect of 5-azadC treatment on myeloma cells. M-/U-MSP analysis of MIR34B/C promoter methylation status and stem-loop quantitative RT-PCR analysis of mature MIR34B expression in KMS-12-PE and LP-1 cells on the seventh days after treatments. 5-azadC treatment resulted in progressive demethylation of MIR34B/C promoter and reexpression of mature MIR34B in cell lines harboring homozygous MIR34B/C methylation. SNORD48 was used as internal control for stem-loop quantitative RT-PCR analysis. (B) Effect of MIR34B in myeloma cells. KMS-12-PE myeloma cells, homozygously methylated for MIR34B/C, were transfected with precursor MIR34B oligo compared with nontargeting precursor mimic. (i) Stem-loop quantitative RT-PCR analysis of mature MIR34B expression at 48 hour after transfection. (ii) Cellular proliferation of myeloma cells in response to overexpression of MIR34B was measured by MTT assay, whereas (iii) cell death was measured by trypan blue exclusion assay and (iv) the percentage of sub-G1 cells from propidium iodide staining. Error bar represents SD.
