title: [Effect of methylation inhibitor on demethylation  pattern of the PD-1 gene in promoter region and  PD-1 expression in  human T lymphocyte cell line].
abstract: OBJECTIVE
  To observe the demethylation effect of demethylation inhibitor 5-azacytidine (5-Zac) on programmed death receptor 1 (PD-1) in Molt-4 cells (T lymphocyte cell line) and to investigate the relationship between DNA demethylation and expression of PD-1.
  
  
  METHODS
  Molt-4 cells were cultured in the medium containing different concentrations of 5-Zac(0, 5, 10 μmol/L) for 72 h. According to the concentrations of 5-Zac, the Molt-4 cells were divided into a 0 μmol/L 5-Zac group, a 5 μmol/L 5-Zac group, and a 10 μmol/L 5-Zac group. The expression of PD-1 in Molt-4 cells was detected by flow cytometry and the apoptosis rate was calculated. The mRNA transcription level of PD-1 was detected by real-time polymerase chain reaction; Molt-4 cell DNA in all groups were treated by sodium bisulfite. The PD-1 promoter fragment was amplified by PCR, the amplification fragments were transformed into E. coli., the positive clones were selected for equencing, and the methylation status of the fragments of PD-1 promoter was examined. RESULTS Seventy-two hours after the 5-Zac treatment, the expression rate of PD-1 in the Molt-4 cells in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac group was (1.13 ± 0.01)%, (18.96 ± 1.87)%, and (63.09 ± 6.25)% respectively, in a low concentration-dependent way. The PD-1 mRNA expression level was increased significantly with the 5-Zac treatment. Cells apoptosis showed that:compared with the 0 μmol/L 5-Zac group, the apoptosid rate in the 5 μmol/L 5-Zac group and 10 μmol/L 5-Zac group was signficantly increased, which was (1.9 ± 0.06)%, (8.89 ± 1.36)%, and (24.50 ± 3.68)% in the 0 μmol/L 5-Zac group, the 5 μmol/L 5-Zac group, and the 10 μmol/L 5-Zac mol/L group respectively. The bisulfite genomic sequencing showed that the demethylation probability of CpG points on -601 bp and -553 bp was significantly increased in the 5-Zac treated cells compared with those untreated.
  
  
  CONCLUSIONS
  5-Zac can result in the increase of PD-1 expression in the human lymphoid cell series Molt-4 in vitro, and the apoptosis rate increases, which is related to PD-1 gene promoter demethylation.
authors.affiliation: Department of Infectious Diseases, Second Xiangya Hospital, Central South University, Changsha, China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.forename: Xinqiang
authors.initials: X
authors.lastname: Xiao
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Liang
authors.forename: Minyuan
authors.initials: M
authors.lastname: Peng
authors.forename: Yongfang
authors.initials: Y
authors.lastname: Jiang
authors.forename: Yun
authors.initials: Y
authors.lastname: Xu
authors.forename: Guozhong
authors.initials: G
authors.lastname: Gong
mesh_terms: D017209:Apoptosis / Q000235:genetics; D001374:Azacitidine / Q000494:pharmacology*; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D061026:Programmed Cell Death 1 Receptor / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; C105992:PDCD1 protein, human; D061026:Programmed Cell Death 1 Receptor; D012333:RNA, Messenger; D001374:Azacitidine