Critical role of ΔDNMT3B4/2 in regulating RASSF1A promoterspecific DNA methylation in non-small cell lung cancer
Year: 2008
Type: article
Abstract: Background ΔDNMT3B (a new DNMT3B subfamily) expression is initiated through a novel promoter. We identified at least 7 transcription variants of ΔDNMT3B as a result of alternative pre-mRNA processing. The aim of this study was to detect the expression pattern of ΔDNMT3B variants in non-small cell lung cancer (NSCLC) and to explore the role of ΔDNMT3B variants in regulating the promoter-specific DNA methylation. Methods Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual ΔDNMT3B variants according to their splicing patterns. The expressions of seven ΔDNMT3B 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 ΔDNMT3B 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 ΔDNMT3B were designed and transfected by lipofectmane 2000 into H1299 and H358 cell lines. RASSF1A promoter methylation from cells treated by siRNA-δDNMT3B4/2 was detected using MSP and Bisulfite sequencing, and Western blotting was used to detect the protein expression of DNMT3B and ΔDNMT3B. Cell growth and cell cycle distribution were measured by applying real-time cell growth analysis and flowcytometry, respectively. Results ΔDNMT3B variants, not DNMT3B, were the predominant transcripts in both NSCLC cell lines and primary tumors. The expression of ΔDNMT3B4 strongly correlated to the promoter methylation status of RASSF1A in a primary NSCLC. The knockdown of ΔDNMT3B4/2 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 p16INK4a promoter in the NSCLC cell lines. Conclusions These results demonstrate an important role of ΔDNMT3B4/2 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.
Source: Chinese Medical Journal
Authors: Shu-hang WANG, Nin-hong LIU, Jie WANG, Hua BAI, Li MAO
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,000
OpenAlex ID: W4297752808