Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells.
- Author:
Rong XIAO
1
;
Yan DING
;
Qian-jin LU
;
Ya-ping LI
;
Yong-jian LI
;
Xin-jie YANG
;
Yu-wen SU
;
Yun-sheng LIANG
;
Gui-ying ZHANG
;
Hai-quan WEN
Author Information
1. Department of Dermatology and Venereology, Second Xiangya Hospital, Central South University, Changsha 410011, China. xiaorong@medmail.com.cn
- Publication Type:Journal Article
- MeSH:
Adult;
Azacitidine;
pharmacology;
Cells, Cultured;
DNA Methylation;
drug effects;
Humans;
Perforin;
genetics;
Promoter Regions, Genetic;
genetics;
T-Lymphocyte Subsets;
metabolism
- From:
Journal of Central South University(Medical Sciences)
2006;31(6):843-847
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells.
METHODS:T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4(+) and CD8(+) subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 alpha and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Western blot.
RESULTS:The perforin mRNA and protein in the CD4(+) and CD8(+) subsets treated with 5-azaC were significantly higher than those in the untreated subsets (P<0.05). The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was significantly reduced in the treated cells compared with the untreated cells (P<0.05).
CONCLUSION:The mRNA and protein expression of perforin significantly increases in the CD4(+) and CD8(+) T cells treated with 5-azaC,which is associated with DNA hypomethylation of perforin promoter in T cells.
