Critical role of Delta DNMT3B4/2 in regulating RASSF1A promoter-specific DNA methylation in non-small cell lung cancer

Background Delta DNMT3B (anew DNMT3B subfamily) expression is initiated through a novel promoter. We identified at least 7 transcription variants of Delta DNMT3B as a result of alternative pre-mRNA processing. The aim of this study was to detect the expression pattern of Delta DNMT3B variants in non-small cell lung cancer (NSCLC) and to explore the role of Delta DNMT3B variants in regulating the promoter-specific DNA methylation. Methods Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual Delta DNMT3B variants according to their splicing patterns. The expressions of seven Delta DNMT3B variants were measured in 13 cell lines, 109 NSCLC patients, and the corresponding normal lung tissues using reverse transcription-PC R (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 Delta DNMT3B 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 Delta DNMT3B were designed and transfected by lipofectmane 2000 into H1299 and H358 cell lines. RASSF1A promoter methylation from cells treated by siRNA-Delta DNMT3B4/2 was detected using MSP and Bisulfite sequencing, and Western blotting was used to detect the protein expression of DNMT3B and Delta DNMT3B. Cell growth and cell cycle distribution were measured by applying real-time cell growth analysis and flowcytometry, respectively. Results Delta DNMT3B variants, not DNMT3B, were the predominant transcripts in both NSCLC cell lines and primary tumors. The expression of Delta DNMT3B4 strongly correlated to the promoter methylation status of RASSF1A in a primary NSCLC. The knockdown of Delta DNMT3B4/2 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 Delta DNMT3B4/2 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.Medicine, General & InternalSCI(E)PubMed中国科技核心期刊(ISTIC)6ARTICLE171712-172112