The chemical such as Decitabine, RPMI 1640, Fetal bovine serum (FBS), Penicillin and streptomycin, Thiazolyl blue Tetrazolium Bromide (MTT), Eva green, DNA safe stain, RNA extraction kit, cDNA Synthesis Kits were purchase from Sigma Otsuka, America Pharmaceutical Inc., (Thermo scientific, MA, USA), (Gibco, Life technologies, Thermo fisher, USA), ( Bio-idea, Iran), (Solis Biodyne, Estonia), (Biofact, Korea), (RNX-plus solution for total RNA isolation- sinaclon, Iran), (Thermo Fisher scientific, cDNA Inc. USA) respectively.
Human cancer cell lines including NALM-6, HL-60 and normal cell (the Pasteur Institute, Tehran, Iran) were cultured in RPMI-1640 media enriched by 10% heat-inactivated fetal bovine serum (FBS), 50U/ml penicillin and 50μg/ml streptomycin at 37°C and 5% CO2. We have obtained treatment does by the MTT assay and therefor NALM-6 and HL-60 cell lines were treated with 1µM Decitabine for 24 h (21).
Cell viability assay (MTT test):
Cells were cultured at a concentration of 1x105 cells/well in 96-well micro plate and treated with AZad at different concentrations 0.1µM, 1µM and 5µM for 24, 48 and 72 h intervals. After treatment with AZad, 20μl of 5mg/ml MTT solution was added to each well and incubated for 4 h at 37°C. Next, 50µl of 20% acidified SDS added to the cells. Finally, absorbance of each well was measured at 570 nm by EPOCH Microplate Spectrophotometer (synergy HTX, BioTek, USA). Cell viability were expressed as a percentage in comparison to control. All tests were done 3 times (21, 22).
Quantitative polymerase chain reaction (qPCR):
Total RNA was isolated using an RNA extraction kit instruction protocol (RNX-plus solution for total RNA isolation, sinaclon, Iran). Similarly, cDNA was created using the transcriptor first strand cDNA synthesis kit (Thermo fisher scientific, cDNA Inc. USA). The expression level was calculated using the 2‑ΔΔCq method (23). All experiments were repeated at least 3 times. The following primers for qPCR were used: HDAC3, forward 5'‑ CCA AGA CCG TGG CCT ATTT ‑3' and reverse 5'‑ AATGCAGGACCAGGCTATG ‑3'; HDAC7, forward 5'‑ GGACACCATGCAGATCATTCT ‑3' and reverse 5'‑ TGCACGTCC CAGTCTACAAT ‑3'; GAPDH, forward 5'‑ GAGCCACATCGCTCAGACAC ‑3' and reverse 5'‑ CATGTAGTTGAGGTCAATGAAGG ‑3'. Finally, all values were normalized to GAPDH expression levels.
Statistical analysis was performed using two-tailed T-test. P≤0.05 was regarded as statistically significant. All statistical analyses were carried out by Graph Pad Prism 7.00 for windows (Graph Pad Software, San Diego, California, USA). Data were expressed as the mean ± (SD) of at least three independent experiments.