Primary leukemic cells were isolated by Ficoll density gradient centrifugation (GE Healthcare, Uppsala, Sweden). Pure curcumin (Sigma-Aldrich, St Louis, MO) was dissolved in DMSO as 20 mM stock solution and kept at -20°C. For experiments, leukemic cells and primary AML cells were cultured in serial concentrations of curcumin and control cultures were treated with DMSO only. Table 1 The data of acute myeloid leukemia patients NO Sex Age(y) FAB subtype Chromosome karyotype 1 M 24 M5 46, XY 2 M 36 M3 46, XY PML-RARa+ 3 F 47 M5 46, XX 4 F 53
M4 46, XX MYH11-CBFβ+ 5 M 29 M3 46, XY PML-RARa+ 6 F 48 M2 46, XX AML-ETO+ 7 F 35 Selleck MK-4827 M4 46, XX MYH11-CBFβ+ 8 M 41 M5 46, XY 9 F 58 M2 46, XX AML-ETO+ 10 M 47 M4 46, XY 11 M 41 M2 46, XY 12 F 26 M5 46, XX Plasmids transfection pRETROSUPER vector expressing miR-15a/16-1 (pRS-15/16) was constructed as previously described. The same empty plasmid (pRS-E) was served as negative control. K562 and HL-60 cells were transiently transfected with 1 μg/mL (final concentration) pRS-15/16 or pRS-E vector mediated by Lipofectamine™ LTX and PLUS™ Reagents (Invitrogen) according to the manufacturer’s instructions. RNA extraction Total RNA from curcumin-treated or untreated leukemic cells were extracted by TRIzol (Invitrogen) Following the manufacture’s protocol. RNA
concentration CB-5083 clinical trial and quality were quantified by measuring the absorbance at 260 nm 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 by the aid of the NCode™ miRNA First-strand cDNA synthesis (Invitrogen) and SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. U6 snRNA level was used for normalization.
The fold change for each miRNA in curcumin-treated leukemic cells relative to untreated cells was calculated using the 2-ΔΔCT method [14]. WT1 transcript was determined by quantitative real-time PCR using specific primer. ABL and GAPDH housekeeping genes were used for normalization [15, 16]. The following primers were used respectively, miR-15a: 5′-TAG CAG CAC ATA ATG GTT TGT G-3′, miR-16-1: 5′-TAG CAG CAC GTA AAT ATT GGC G-3′, U6: 5′-CGC AAG GAT GAC ACG CAA ATT C-3′, WT1: sense Thalidomide 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 SB525334 cell line 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′). GAPDH: (sense strand: 5′-CCA GGT GGT CTC CTC TGA CTT C-3′, antisense strand: 5′-GTG GTC GTT GAG GGC AAT G-3′, Taqman probe: 5′- Fam-ACA GCG ACA CCC ACT CCT CCA CCT T-Tamra-3′).