Either down-regulation of WT1 by siRNA significantly inhibited the proliferation of leukemic cells. Thus, these data suggest that miR-15a/16-1 may function as a tumor suppressor to influence the proliferation of leukemic cells through down-regulating WT1 protein level. However, enforced expression of miR-15a/16-1 can not reduce the activity of a luciferase reporter carrying the 3′-untranslated SIS3 cell line region (3′UTR) of WT1. This result means that miR-15a/16-1 down-regulated the expression of WT1 not through miRNA-mRNA base pairing. Whether miR-15a/16-1 downregulate other genes which interact with WT1 is

not decided. Therefore more study are required to shed light of the new mechanism, which will open new avenues in understanding the mechanisms of miRNA action. Materials and methods cell

lines and primary leukemic cells K562 and HL-60 cell lines were employed for the present study. All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Groud Island, USA) in humidified 37°C incubator with 5% CO2. Primary AML cells were obtained from 20 patients with AML (2 M1 5 M2, 5 M3, 2 M4, 6 M5, the First Affiliated Hospital of Wenzhou Medical College). this website None of these patients had received any treatment. The diagnosis was established according to French-American-British classification. All patients gave informed consent Glutamate dehydrogenase in accordance with the Declaration of Helsinki for cryopreservation and use of the samples for molecular studies. RNA extraction Bone marrow mononuclear cells from normal individuals and patients with AML were aspirated by Ficoll density gradient centrifugation (GE Healthcare, Uppsala, Sweden). Total RNA from cultured cell

lines and bone marrow mononuclear cells were extracted by TRIzol (Invitrogen) Following the manufacture’s protocol. RNA concentrations and quality were determined with Beckman DU6400 spectrophotometer (Beckman, USA) and gel analysis. qPCR for miRNA and mRNA expression Quantitative real-time polymerase chain reaction (qRT-PCR) analysis for miR-15a and miR-16-1 was performed in triplicate with the NCode™ miRNA First-strand cDNA synthesis and SYBR® Green PCR Master Mix (Applied Biosystems, MM-102 Foster City, CA) according to the manufacturer’s instructions. U6 snRNAs was used as the internal control. The fold-change for miR-15a/16-1 expression levels was calculated using ΔCT and 2-ΔΔCT. WT1 transcript was determined by quantitative real-time PCR using specific primer sets[16] and ABL housekeeping gene was used for normalization[17].