title: Critical role of deltaDNMT3B4/2 in regulating RASSF1A promoter-specific DNA methylation in non-small cell lung cancer.
abstract: BACKGROUND
  DeltaDNMT3B (a new DNMT3B subfamily) expression is initiated through a novel promoter. We identified at least 7 transcription variants of deltaDNMT3B as a result of alternative pre-mRNA processing. The aim of this study was to detect the expression pattern of deltaDNMT3B variants in non-small cell lung cancer (NSCLC) and to explore the role of deltaDNMT3B variants in regulating the promoter-specific DNA methylation.
  
  
  METHODS
  Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual deltaDNMT3B variants according to their splicing patterns. The expressions of seven deltaDNMT3B variants were measured in 13 cell lines, 109 NSCLC patients, and the corresponding normal lung tissues using reverse transcription-PCR (RT-PCR). The status of the p16 and RASSF1A promoter methylations in the tumors was detected using a methylation specific PCR (MSP). The relationships of the expression patterns of the deltaDNMT3B variants were analyzed by observing the status of p16 and RASSF1A promoter methylations in the tumors. The siRNA and the anti-sense oligo-dioxynucleotide specifically targeting the junction of exon 5 and 7 of deltaDNMT3B were designed and transfected by lipofectmane 2000 into H1299 and H358 cell lines. RASSF1A promoter methylation from cells treated by siRNA-deltaDNMT3B4/2 was detected using MSP and Bisulfite sequencing, and Western blotting was used to detect the protein expression of DNMT3B and ADNMT3B. Cell growth and cell cycle distribution were measured by applying real-time cell growth analysis and flowcytometry, respectively.
  
  
  RESULTS
  ADNMT3B variants, not DNMT3B, were the predominant transcripts in both NSCLC cell lines and primary tumors. The expression of deltaDNMT3B4 strongly correlated to the promoter methylation status of RASSF1A in a primary NSCLC. The knockdown of deltaDNMT3B4/2 by RNA-interference or anti-sense approaches resulted in a complete demethylation of RASSF1A promoter with the reactivation of a RASSF1A gene expression in less than 12 hours, but no effect resulted from the p16(INK4a) promoter in the NSCLC cell lines.
  
  
  CONCLUSIONS
  These results demonstrate an important role of deltaDNMT3B4/2 in the maintenance of promoter-specific DNA methylation in a cell type specific manner and provide a novel cell model for the study of the regulation of replication-independent DNA methylation.
authors.affiliation: Department of Thoracic Medical Oncology, Peking University School of Oncology, Beijing Cancer Hospital, Beijing Institute for Cancer Research, Beijing 100036, China.
authors.forename: Shu-Hang
authors.initials: SH
authors.lastname: Wang
authors.forename: Nin-Hong
authors.initials: NH
authors.lastname: Liu
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Hua
authors.initials: H
authors.lastname: Bai
authors.forename: Li
authors.initials: L
authors.lastname: Mao
mesh_terms: D001483:Base Sequence; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000502:physiology*; D019175:DNA Methylation; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D000097584:DNA Methyltransferase 3B
chemical_list: D004248:DNA (Cytosine-5-)-Methyltransferases; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D000097584:DNA Methyltransferase 3B; C411762:RASSF1 protein, human