[Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells].
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).


CONCLUSIONS
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.
Xiao
Rong
R
Department of Dermatology and Venereology, Second Xiangya Hospital, Central South University, Changsha 410011, China. xiaorong@medmail.com.cn
Ding
Yan
Y
Lu
Qian-jin
QJ
Li
Ya-ping
YP
Li
Yong-jian
YJ
Yang
Xin-jie
XJ
Su
Yu-wen
YW
Liang
Yun-sheng
YS
Zhang
Gui-ying
GY
Wen
Hai-quan
HQ
D000328:Adult; D001374:Azacitidine / Q000494:pharmacology*; D002478:Cells, Cultured; D019175:DNA Methylation / Q000187:drug effects*; D006801:Humans; D054353:Perforin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016176:T-Lymphocyte Subsets / Q000378:metabolism*
D054353:Perforin; D001374:Azacitidine