title: Demethylation of miR-9-3 and miR-193a genes suppresses proliferation and promotes apoptosis in non-small cell lung cancer cell lines.
abstract: BACKGROUND
  MicroRNAs miR-9-3 and miR-193a have recently been found to be hypermethylated in a variety of non-small cell lung cancer (NSCLC) cells and primary human tumors. The objectives of this study were to investigate the role of demethylation of miR-9-3 and miR-193a genes in regulating proliferation and apoptosis in NSCLCs, and to decipher the potential mechanisms underlying the properties.
  
  
  METHODS
  MTT and population doubling time by flow cytometry were used to assess cell proliferation. Enzyme-Linked Immunosorbent Assay and caspase-3 activity assay were employed to evaluate apoptosis. Real-time RT-PCR and Western blot were used to quantify gene expression at mRNA and protein levels, respectively. Methylation-specific PCR was utilized to assess methylation status.
  
  
  RESULTS
  We found that demethylation agent 5-Aza-2'-deoxycytidine (5-AzaC) reduced cell numbers and prolonged population doubling time (PDT), and promoted doxorubicin-induced apoptosis in seven NSCLC cell lines with different methylation statuses on miR-9-3 and miR-193a promoter regions: NCI-H1993/NCI-H1915 (miR-9-3(+)/miR-193a(+)), NCI-H1975/NCI-H200 (miR-9-3(+)/miR-193a(-)), A427/NCI-H2073 (miR-9-3(-)/miR-193a(+)), and NCI-H1703 (miR-9-3(-)/miR-193a(-)). Treatment with 5-AzaC concomitantly upregulated expression of miR-9-3 and miR-193a, and downregulated their respective target genes NF-κB and Mcl-1. The effects of 5-AzaC were abolished by concomitant knockdown of miR-9-3 and miR-193a using the complex antisense technique, whereas forced ectopic expression of miR-9-3 and miR-193a mimicked the effects of 5-AzaC. We further observed that the strength of proliferation inhibition and apoptosis promotion elicited by 5-AzaC was in the order of NCI-H1993/NCI-H1915 > A427/NCI-H2073 > NCI-H1975/NCI-H200 > NCI-H1703.
  
  
  CONCLUSIONS
  Methylation-silencing of miR-9-3 and miR-193a may be an important epigenetic mechanisms favoring NSCLC cell growth and survival for carcinogenesis and cancer progression, and demethylation to reactivate expression of miR-9-3 and miR-193a genes contributes, at least partially, to the anti-cancer properties of 5-AzaC and thereby may be worthy of future studies for the possibility of being a new therapeutic strategy for the treatment of human NSCLCs.
authors.affiliation: Department of General Oncology, Chinese PLA General Hospital, Beijing, China.
authors.forename: Jinliang
authors.initials: J
authors.lastname: Wang
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.forename: Lu
authors.initials: L
authors.lastname: Han
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Li
authors.forename: Haitao
authors.initials: H
authors.lastname: Tao
authors.forename: Sujie
authors.initials: S
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Hu
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D000077209:Decitabine; D015536:Down-Regulation; D004317:Doxorubicin / Q000633:toxicity; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000378:metabolism*; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016415:Sequence Alignment; D015854:Up-Regulation
chemical_list: D000903:Antibiotics, Antineoplastic; C579095:MCL1 protein, human; C546818:MIRN193 microRNA, human; C541874:MIRN92 microRNA, human; D035683:MicroRNAs; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D000077209:Decitabine; D004317:Doxorubicin; D001374:Azacitidine