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展开更多
In this study, RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells. The expression levels of their mRNA and protein were greatly decreased by up t...In this study, RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells. The expression levels of their mRNA and protein were greatly decreased by up to 75% and 65% respectively after T24 cells were trans- fected with lipofectamine2000 for 72 h, indicating RNA interference is an effective tool in gene knockdown. Proliferation and apoptosis of T24 cells were detected by MTT, and annexin-V-FITC and propidium iodide staining flow cytometry, respectively. It was found that loss of the DNMT1 or DNMT3b expression could inhibit the cell growth and promote the cell apoptosis to some extent. However, combined treatment with shRNA targeting both DNMT1 and DNMT3b mRNA could obviously enhance the above effects. It was concluded that simultaneously silencing both genes could result in strong suppressing effect on tumor proliferation and promoting cell apoptosis than separate use, suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in the CpG island methylation in human bladder tumorigenesis.展开更多
目的探讨稳定低表达DNA甲基转移酶3b(DNMT3b)基因对人膀胱癌细胞生长和凋亡的影响。方法包装表达DNMT3b si RNA和阴性对照si RNA的慢病毒,通过慢病毒感染的方法获得稳定低表达DNMT3b的膀胱癌细胞BIU-87及对照细胞。MTT和流式细胞仪检测D...目的探讨稳定低表达DNA甲基转移酶3b(DNMT3b)基因对人膀胱癌细胞生长和凋亡的影响。方法包装表达DNMT3b si RNA和阴性对照si RNA的慢病毒,通过慢病毒感染的方法获得稳定低表达DNMT3b的膀胱癌细胞BIU-87及对照细胞。MTT和流式细胞仪检测DNMT3b稳定低表达对细胞增殖和凋亡的影响;体内裸鼠成瘤实验观察DNMT3b稳定低表达对肿瘤的抑制效果;荧光定量PCR和Western blot检测DNMT3b稳定低表达对细胞生长和凋亡相关基因表达的影响;甲基化特异性PCR检测对细胞生长和凋亡相关基因甲基化的影响。结果荧光定量PCR和Western blot检测结果显示,DNMT3b m RNA和蛋白水平在BIU-87稳定细胞株中稳定低表达;DNMT3b稳定低表达可以抑制BIU-87细胞的生长,并且抑制裸鼠皮下瘤体的生长;DNMT3b稳定低表达可以促进BIU-87细胞的凋亡;DNMT3b稳定低表达可以增加凋亡及细胞生长相关基因DAPK,Bax和RASSF1A m RNA和蛋白水平的表达;DNMT3b稳定低表达可以减少凋亡及细胞生长相关基因DAPK,Bax和RASSF1A启动子区域的甲基化水平。结论 DNMT3b稳定低表达可能通过调控凋亡相关基因以及细胞生长相关基因启动子区域的甲基化水平来影响基因的表达,从而抑制BIU-87细胞的生长,诱导细胞凋亡。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
基金supported by a grant from a Science and Technology Research Program of Hubei Province (No.2006AA301B56-1)
文摘In this study, RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells. The expression levels of their mRNA and protein were greatly decreased by up to 75% and 65% respectively after T24 cells were trans- fected with lipofectamine2000 for 72 h, indicating RNA interference is an effective tool in gene knockdown. Proliferation and apoptosis of T24 cells were detected by MTT, and annexin-V-FITC and propidium iodide staining flow cytometry, respectively. It was found that loss of the DNMT1 or DNMT3b expression could inhibit the cell growth and promote the cell apoptosis to some extent. However, combined treatment with shRNA targeting both DNMT1 and DNMT3b mRNA could obviously enhance the above effects. It was concluded that simultaneously silencing both genes could result in strong suppressing effect on tumor proliferation and promoting cell apoptosis than separate use, suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in the CpG island methylation in human bladder tumorigenesis.
文摘目的探讨稳定低表达DNA甲基转移酶3b(DNMT3b)基因对人膀胱癌细胞生长和凋亡的影响。方法包装表达DNMT3b si RNA和阴性对照si RNA的慢病毒,通过慢病毒感染的方法获得稳定低表达DNMT3b的膀胱癌细胞BIU-87及对照细胞。MTT和流式细胞仪检测DNMT3b稳定低表达对细胞增殖和凋亡的影响;体内裸鼠成瘤实验观察DNMT3b稳定低表达对肿瘤的抑制效果;荧光定量PCR和Western blot检测DNMT3b稳定低表达对细胞生长和凋亡相关基因表达的影响;甲基化特异性PCR检测对细胞生长和凋亡相关基因甲基化的影响。结果荧光定量PCR和Western blot检测结果显示,DNMT3b m RNA和蛋白水平在BIU-87稳定细胞株中稳定低表达;DNMT3b稳定低表达可以抑制BIU-87细胞的生长,并且抑制裸鼠皮下瘤体的生长;DNMT3b稳定低表达可以促进BIU-87细胞的凋亡;DNMT3b稳定低表达可以增加凋亡及细胞生长相关基因DAPK,Bax和RASSF1A m RNA和蛋白水平的表达;DNMT3b稳定低表达可以减少凋亡及细胞生长相关基因DAPK,Bax和RASSF1A启动子区域的甲基化水平。结论 DNMT3b稳定低表达可能通过调控凋亡相关基因以及细胞生长相关基因启动子区域的甲基化水平来影响基因的表达,从而抑制BIU-87细胞的生长,诱导细胞凋亡。
