title: [Effect of DNA hypermethylation on NOR1 promoter activity and expression].
abstract: OBJECTIVE
  To analyze the effect of DNA hypermethylation on NOR1 promoter activity and expression.
  
  
  METHODS
  NOR1 promoter plasmids were treated with SssI methyltransferase. The plasmids were modified by sodium bisulfite and purified. Sodium bisulfite-modified plasmids were subjected to PCR with primers designed to analyze the methylation status of 26 CpG sites in a 311-bp region of the NOR1 promoter. Cells were transfected by methylated or mock-methylated promoter plasmids. The promoter activities were assessed by the luciferase levels of cell lysates or by directly observing GFP expression under fluorescence microscope. HL60 cells were treated with different concentrations of 5-aza-dC. Total RNA was isolated from harvested cells. Real-time RT-PCR was used to measure the expression level of NOR1 mRNA.
  
  
  RESULTS
  Bisulfite sequencing confirmed that SssI methyltransferase treatment successfully resulted in intensive hypermethylation of the NOR1 promoter plasmids. The promoter activity of NOR1 promoter plasmids was totally blocked by SssI methyltransferase treatment. NOR1 expression levels in HL60 cells were restored by 5-aza-dC treatment.
  
  
  CONCLUSIONS
  NOR1 promoter plasmids are intensively hypermethylated by SssI methyltransferase treatment. The promoter activity of NOR1 promoter plasmids are totally blocked by SssI methyltransferase treatment. The 5-aza-dC treatment may restore the endogenous NOR1 mRNA level in HL60 cells.
authors.affiliation: Cancer Research Institute, Central South University, Changsha 410078,China.
authors.forename: Bo
authors.initials: B
authors.lastname: Xiang
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Li
authors.forename: Mei
authors.initials: M
authors.lastname: Yi
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Li
authors.forename: Guiyuan
authors.initials: G
authors.lastname: Li
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D045744:Cell Line, Tumor; D018899:CpG Islands; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors; D015257:DNA-Cytosine Methylases / Q000494:pharmacology; D000077209:Decitabine; D044127:Epigenesis, Genetic; D020868:Gene Silencing; D018922:HL-60 Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009303:Nasopharyngeal Neoplasms / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; C479458:OSCP1 protein, human; D012333:RNA, Messenger; D000077209:Decitabine; D015254:DNA Modification Methylases; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases; D001374:Azacitidine