title: Epigenetic inactivation of miR-9 family microRNAs in chronic lymphocytic leukemia--implications on constitutive activation of NFκB pathway.
abstract: BACKGROUND
  The miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL).
  
  
  METHODS
  Methylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction.
  
  
  RESULTS
  The promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2'-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04).
  
  
  CONCLUSIONS
  Of the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL 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: Chi Shan Bonnie
authors.initials: CS
authors.lastname: Kho
authors.forename: Kit Fai
authors.initials: KF
authors.lastname: Wong
authors.forename: Kwan Yeung
authors.initials: KY
authors.lastname: Wong
authors.forename: Manuela
authors.initials: M
authors.lastname: Ferracin
authors.forename: George A
authors.initials: GA
authors.lastname: Calin
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; D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012189:Retrospective Studies; D015398:Signal Transduction
chemical_list: C541874:MIRN92 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B