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.
10.3969/j.issn.1672-7347.2011.12.007
- Author:
Min ZHANG
1
;
Xinqiang XIAO
;
Yunsheng LIANG
;
Minyuan PENG
;
Yongfang JIANG
;
Yun XU
;
Guozhong GONG
Author Information
1. Department of Infectious Diseases, Second Xiangya Hospital, Central South University, Changsha, China.
- Publication Type:Journal Article
- MeSH:
Apoptosis;
genetics;
Azacitidine;
pharmacology;
Cell Line;
CpG Islands;
genetics;
DNA Methylation;
drug effects;
genetics;
Enzyme Inhibitors;
pharmacology;
Humans;
Programmed Cell Death 1 Receptor;
genetics;
metabolism;
Promoter Regions, Genetic;
genetics;
RNA, Messenger;
genetics;
metabolism;
T-Lymphocytes;
cytology;
metabolism
- From:
Journal of Central South University(Medical Sciences)
2011;36(12):1163-1169
- CountryChina
- Language:Chinese
-
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.
CONCLUSION: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.
