Eukemic cells through down-regulating WT1 protein level. However, enforced expression of miR-15a/16-1 can not reduce

Eukemic 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 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.RNA extractionBone 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 expressionQuantitative RG7800 mechanism of action real-time polymerase chain reaction (qRTPCR) analysis for miR-15a and miR-16-1 was performed in triplicate with the NCodeTM miRNA First-strand cDNA synthesis and SYBR?Green PCR Master Mix (Applied Biosystems, 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 CT and 2-CT. WT1 transcript was determined by quantitative real-time PCR using specific primer sets [16] and ABL housekeeping gene was used for normalization [17]. The following primers were used respectively, WT1: (sense strand: 5′-CAG GCT GCA ATA AGA GAT ATT TTA AG CT-3′, antisense strand: 5′-GAA GTC ACA CTG GTA TGG TTT CTC A-3′, Taqman probe: 5′-Fam-CTT ACA GAT GCA CAG CAG GAA GCA CAC TGA-Tamra-3′), ABL: (sense strand: 5’GAT GTA GTT GCT TGG GAC CCA-3′, antisense strand: 5′-TGG AGA TAA CAC TCT AAG CAT AAC TAA AGG T-3′, Taqman probe: 5′-Fam-CCA TTT TTG GTT TGG GCT TCA CAC CAT T-Tamra-3′).Plasmids TransfectionMaterials and methodscell lines and primary leukemic cellspRETROSUPER vector expressing miR-15a/16-1 (pRS15/16) was constructed as previously described [10,18]. The same empty plasmid (pRS-E) was served as control. Leukemic cells were transiently transfected with 1 g/mL (final concentration) pRS-E or pRS-15/16 vector using LipofectamineTM LTX and PLUSTM Reagents (Invitrogen) according to the manufacturer’s instructions.Cell counting kit-8 (CCK-8) assay and trypan-blue exclusion assayK562 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 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). None of these patients PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26437915 had received any treatment. The diagnosis was established according to French-American-British classification. All patients gave informed consent in accordance with the Declaration of Helsinki for cryopreservation and use of the samples for molecular studies.The mock or transfected K562, HL-60 and U937 cells were seeded into 96-well plates (6.0 ?103 cells/well). Cell viability was assessed by CCK-8 assay (Dojin Laboratories, Kumamoto, Japan). The absorbance at 450 nm (A450) of each well was read on a spectrophotometer. Three independent experiments were performed in quadruplica.