Background △DNMT3B (a new DNMT3B subfamily) expression is initiated through a novel promoter. We identified at least 7 transcription variants of △DNMT3B as a result of alternative pre-mRNA processing. The aim of t...Background △DNMT3B (a new DNMT3B subfamily) expression is initiated through a novel promoter. We identified at least 7 transcription variants of △DNMT3B as a result of alternative pre-mRNA processing. The aim of this study was to detect the expression pattern of ,△DNMT3B variants in non-small cell lung cancer (NSCLC) and to explore the role of △DNMT3B variants in regulating the promoter-specific DNA methylation. Methods Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual △DNMT3B variants according to their splicing patterns. The expressions of seven △DNMT3B 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 RASSFIA promoter methylations in the tumors was detected using a methylation specific PCR (MSP). The relationships of the expression patterns of the △DNMT3B variants were analyzed by observing the status of p16 and RASSFIA promoter methylations in the tumors. The siRNA and the anti-sense oligo-dioxynucleotide specifically targeting the junction of exon 5 and 7 of △DNMT3B were designed and transfected by lipofectmane 2000 into H1299 and H358 cell lines. RASSFIA promoter methylation from cells treated by siRNA-△DNMT3B4/2 was detected using MSP and Bisulfite sequencing, and Western blotting was used to △DNMT3B. Cell growth and cell cycle distribution were measured flowcytometry, respectively. detect the protein expression of DNMT3B and by applying real-time cell growth analysis and Results △DNMT3B variants, not DNMT3B, were the predominant transcripts in both NSCLC cell lines and primary tumors. The expression of △DNMT3B4 strongly correlated to the promoter methylation status of RASSFIA in a primary NSCLC. The knockdown of △DNMT3B4/2 by RNA-interference or anti-sense approaches resulted in a complete demethylation of RASSFIA promoter with the reactivation of a RASSFIA 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 ,△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.展开更多
基金This study was supported by grants from the National Natural Science Foundation of China (No. 30572104, 30772472), the Capital Medical Developing Foundation (No. 2005-2022), and the 985 Project (No. 985-2-013-39).
文摘Background △DNMT3B (a new DNMT3B subfamily) expression is initiated through a novel promoter. We identified at least 7 transcription variants of △DNMT3B as a result of alternative pre-mRNA processing. The aim of this study was to detect the expression pattern of ,△DNMT3B variants in non-small cell lung cancer (NSCLC) and to explore the role of △DNMT3B variants in regulating the promoter-specific DNA methylation. Methods Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual △DNMT3B variants according to their splicing patterns. The expressions of seven △DNMT3B 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 RASSFIA promoter methylations in the tumors was detected using a methylation specific PCR (MSP). The relationships of the expression patterns of the △DNMT3B variants were analyzed by observing the status of p16 and RASSFIA promoter methylations in the tumors. The siRNA and the anti-sense oligo-dioxynucleotide specifically targeting the junction of exon 5 and 7 of △DNMT3B were designed and transfected by lipofectmane 2000 into H1299 and H358 cell lines. RASSFIA promoter methylation from cells treated by siRNA-△DNMT3B4/2 was detected using MSP and Bisulfite sequencing, and Western blotting was used to △DNMT3B. Cell growth and cell cycle distribution were measured flowcytometry, respectively. detect the protein expression of DNMT3B and by applying real-time cell growth analysis and Results △DNMT3B variants, not DNMT3B, were the predominant transcripts in both NSCLC cell lines and primary tumors. The expression of △DNMT3B4 strongly correlated to the promoter methylation status of RASSFIA in a primary NSCLC. The knockdown of △DNMT3B4/2 by RNA-interference or anti-sense approaches resulted in a complete demethylation of RASSFIA promoter with the reactivation of a RASSFIA 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 ,△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.