title: [Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells].
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).
  
  
  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.
authors.affiliation: Department of Dermatology and Venereology, Second Xiangya Hospital, Central South University, Changsha 410011, China. xiaorong@medmail.com.cn
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Yan
authors.initials: Y
authors.lastname: Ding
authors.forename: Qian-jin
authors.initials: QJ
authors.lastname: Lu
authors.forename: Ya-ping
authors.initials: YP
authors.lastname: Li
authors.forename: Yong-jian
authors.initials: YJ
authors.lastname: Li
authors.forename: Xin-jie
authors.initials: XJ
authors.lastname: Yang
authors.forename: Yu-wen
authors.initials: YW
authors.lastname: Su
authors.forename: Yun-sheng
authors.initials: YS
authors.lastname: Liang
authors.forename: Gui-ying
authors.initials: GY
authors.lastname: Zhang
authors.forename: Hai-quan
authors.initials: HQ
authors.lastname: Wen
mesh_terms: 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*
chemical_list: D054353:Perforin; D001374:Azacitidine