Man CH, Fung TK, Wan H, et al. remarkably decreased than that of K562 after DAC treatment. In K562/DAC, a total of 108 genes were upregulated and 118 genes were downregulated by RNA\Seq. In addition, we also observed aberrant expression of axis (increased and decreased in parental K562 cells rendered cells resistant to the DAC. Taken together, we successfully established DAC\resistant K562 cell line which can serve as a good model for investigating DAC resistance mechanisms, and inhibitors decitabine (5\aza\2\deoxycytidine, DAC) and 5\azacytidine (AZA), which have been recommended as one of the primary treatments for older acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients.6, 7, 8 The DAC is transported into the cell and then phosphorylated by deoxycytidine kinase (was upregulated in hypomethylating agent\resistance cell lines.13 Also, high cytidine deaminase (CDA)/DCK ratio could be a mechanism of primary resistance to DAC in some patients.14 Nevertheless, the detailed mechanisms leading to DAC resistance still remains obscure. In this study, we induced K562 cell line for long periods of time using DAC to obtain the DAC\resistant K562 cell line and investigated the potential mechanisms of DAC resistance. 2.?MATERIALS AND METHODS 2.1. DAC\resistant cell selection and cell culture DAC\resistant K562 cell line (K562/DAC) was established from its parental K562 cell line. The parental K562 cells were exposed continuously to gradually increasing concentrations of DAC. Original inducing DAC concentration was 2.5?mol/L and then increased exponentially in each step till 320?mol/L. The cells acquired resistance to DAC by a series of stepwise selections at last. Selected cells were cultured in DAC\free medium prior to the experiment for at least 2?weeks. K562 and K562/DAC cells were incubated in Iscove’s Modified Dulbecco’s Medium (Wisent, Canada) containing 10% fetal bovine serum (FBS; ExCell Bio, Shanghai, China) and antibiotics at 37C in a humidified, 5% CO2 atmosphere. 2.2. Morphology and measurement of drug sensitivity An inverted light microscope (Nikon) and Wright\Giemsa’s compound stain were used to observe K562 and K562/DAC cells during the exponential phase. The nuclear to cytoplasm ratio of the cells was measured, which was the ratio of the diameter of the nucleus to the thickness of the cytoplasm on both sides. K562 and K562/DAC cells were collected and placed in 6\well plates at a density of 1 1??105/mL with 2?mL medium. Fresh medium containing DAC at final concentration ranging from 0 to 2?mol/L was added immediately, then fresh DAC was supplemented every 24?hours. After 96?hours, the surviving cells were calculated by trypan blue exclusion. The BZS concentration of DAC required for 50% growth inhibition was scored as half maximal (50%) inhibitory concentration (IC50) value. The degree of resistance was evaluated by IC50 value. Each experiment L-Threonine derivative-1 was repeated three times. IC50 value of DAC was analyzed by the method of probit analysis in SPSS21.0 L-Threonine derivative-1 (SPSS Inc, USA). 2.3. Cell survival and proliferation assays Cell viability of the K562 and K562/DAC cells were assessed. Briefly, cells were seeded in 6\well plates at a density of 1 1??105 cells/well with growth medium containing 0% FBS (cell survival assay) or 10% FBS (cell proliferation assay). DAC was added with the final concentration of 1 1?mol/L for 96?hours. The results were presented from three independent experiments. 2.4. Cell apoptosis To study cell apoptosis, cells were treated in 25?cm2 tissue L-Threonine derivative-1 culture flasks without FBS. Then cell apoptosis was evaluated with Annexin\V\FITC and propidium iodide (PI) double staining using an Annexin V apoptosis detection Kit (556547, Annexin V\FITC Apoptosis Detection Kit I; BD, San Jose, CA, USA) according to the manufacturer’s instructions, followed by flow cytometry analysis. 2.5. RNA\Seq analysis Total RNA was extracted from the cell samples by Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. RNA was subjected to RNA\Seq analysis by Beijing BerryGenomics Institute, China. 2.6. RQ\PCR cDNA was reverse transcribed from the RNA. Real\time quantitative PCR (RQ\PCR) was conducted to evaluate the mRNA and miRNA expression levels in the DAC resistant cells as previously described using the primer sets (Table S1).15, 16, 17, 18, 19 2.7. DNA isolation, chemical modification, RQ\MSP and BSP Genomic DNA isolation, chemical modification, real\time quantitative methylation\specific PCR (RQ\MSP) and bisulfite sequencing PCR (BSP) were performed as our previous study.15, 18 2.8. stabled transfected K562 cell line A lenti\virus vector containing cDNA sequence was used to generate stable mRNA and protein were detected by real\time quantitative PCR and western blot, respectively.20 2.9. Statistical analysis All.