title: [Functional study of hENT1 on SKM-1 cell resistance to decitabine].
abstract: OBJECTIVE
  To investigate the effect of human equilibrative nucleoside transporters 1 (hENT1) silencing on proliferation, apoptosis and demethylation of human myelodysplastic syndrome (MDS) derived cell line SKM-1 treated with 5-aza-2'-deoxycytidine (decitabine, DAC).
  
  
  METHODS
  hENT1 was silenced in SKM-1 cells mediated by lentivirus transfection. The infection efficiency was detected by flow cytometry, and the mRNA expression level of hENT1 was confirmed by qRT-PCR. The proliferation ratio of SKM-1 cells treated with different concentrations (0.5, 1, 5 mmol/L) of DAC for 24, 48 and 72 h was detected by CCK-8 method after hENT1 silencing. The apoptosis of SKM-1 cells was detected by Western blot for cleaved level of caspase-3 and evaluated by flow cytometry after staining with anti-Annexin V-PE and 7-AAD. The p15(INK4B) DNA methylation status was measured by methylation specific PCR using EZ DNA Methylation-Gold™ Kit.
  
  
  RESULTS
  The expression level of hENT1 silenced group (0.253±0.030) was statistically decreased compared with that in control group (1.000±0.091) (P<0.01). Compared with control, the proliferation inhibition rate of hENT1 silenced group was significantly decreased by different concentrations of DAC (0.5, 1, 5 μmol/L) treatment for 24, 48, 72 h (P<0.05), which was (49.41±4.02)% and (33.03±2.47)%, respectively (P=0.007) at 5 μmol/L DAC treatment for 72 h in hENT1 silenced group and the control group. Western blot showed that cleaved caspase3 of hENT1 silenced group was also significantly inhibited. The percentage of Annexin Ⅴ⁺ cells and demethylation status of p15(INK4B) were significantly decreased.
  
  
  CONCLUSIONS
  Apoptosis of hENT1 silenced SKM-1 cells induced by DAC was decreased, and the susceptibility of these cells to demethylation treatment was also decreased.
authors.affiliation: Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Wenhui
authors.initials: W
authors.lastname: Shi
authors.affiliation: Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xu
authors.affiliation: Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Chunkang
authors.initials: C
authors.lastname: Chang
authors.affiliation: Department of Hematology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
mesh_terms: D017209:Apoptosis; D001374:Azacitidine / Q000031:analogs & derivatives; D053148:Caspase 3; D002460:Cell Line; D019175:DNA Methylation; D000077209:Decitabine; D004351:Drug Resistance; D033721:Equilibrative Nucleoside Transporter 1; D006801:Humans; D016086:Lentivirus; D009190:Myelodysplastic Syndromes
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; C438649:SLC29A1 protein, human; D000077209:Decitabine; C505407:CASP3 protein, human; D053148:Caspase 3; D001374:Azacitidine