title: Epigenetic inactivation of mir-34b/c in addition to mir-34a and DAPK1 in chronic lymphocytic leukemia.
abstract: BACKGROUND
  TP53 mutation/deletion is uncommon in chronic lymphocytic leukemia (CLL). We postulated that components of TP53-centered tumor suppressor network, miR-34b/c, in addition to DAPK1 and miR-34a might be inactivated by DNA hypermethylation. Moreover, we tested if miR-34b/c methylation might correlate with miR-203 or miR-124-1 methylation in CLL.
  
  
  METHODS
  miR-34b/c, miR-34a and DAPK1 methylation was studied in 11 normal controls, 7 CLL cell lines, and 78 diagnostic CLL samples by methylation-specific polymerase chain reaction. MEC-1 cells were treated with 5-Aza-2'-deoxycytidine for reversal of methylation-associated miRNA silencing. Tumor suppressor properties of miR-34b were demonstrated by over-expression of precursor miR-34b in MEC-1 cells.
  
  
  RESULTS
  miR-34b/c promoter was unmethylated in normal controls, but completely methylated in 4 CLL cell lines. miR-34b/c expression was inversely correlated with miR-34b/c methylation. Different MSP statuses of miR-34b/c, including complete methylation and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. 5-Aza-2'-deoxycytidine treatment resulted in promoter demethylation and miR-34b re-expression in MEC1 cells. Moreover, over-expression of miR-34b resulted in inhibition of cellular proliferation and increased cell death. In primary CLL samples, miR-34a, miR-34b/c and DAPK1 methylation was detected in 2.6%, 17.9% and 34.6% of patients at diagnosis respectively. Furthermore, 39.7%, 3.8% and 2.6% patients had methylation of one, two or all three genes respectively. Overall, 46.2% patients had methylation of at least one of these three genes. Besides, miR-34b/c methylation was associated with methylation of miR-34a (P = 0.03) and miR-203 (P = 0.012) in CLL.
  
  
  CONCLUSIONS
  Taken together, miR-34b/c is a tumor suppressor miRNA frequently methylated, and hence silenced in CLL. Together with DAPK1 methylation, miR-34b/c methylation is implicated in the disruption of the TP53-centered tumor suppressor network. Moreover, the association of miRNA methylation warrants further study.
authors.forename: Lu Qian
authors.initials: LQ
authors.lastname: Wang
authors.forename: Yok Lam
authors.initials: YL
authors.lastname: Kwong
authors.forename: Kit Fai
authors.initials: KF
authors.lastname: Wong
authors.forename: Chi Shan Bonnie
authors.initials: CS
authors.lastname: Kho
authors.forename: Dong Yan
authors.initials: DY
authors.lastname: Jin
authors.forename: Eric
authors.initials: E
authors.lastname: Tse
authors.forename: Anders
authors.initials: A
authors.lastname: Rosèn
authors.affiliation: Department of Medicine, Queen Mary Hospital, The University of Hong Kong, Hong Kong, China. jcschim@hku.hk.
authors.forename: Chor Sang
authors.initials: CS
authors.lastname: Chim
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001773:Blood Cells / Q000187:drug effects / Q000378:metabolism; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D064550:Death-Associated Protein Kinases / Q000235:genetics* / Q000378:metabolism; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000187:drug effects; D005260:Female; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D016133:Polymerase Chain Reaction
chemical_list: C522425:MIRN34 microRNA, human; D035683:MicroRNAs; D000077209:Decitabine; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D001374:Azacitidine