AIM: To investigate the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR),a DNA methyltransferase(DNMT) inhibitor,on the growth and survival of the Chinese retinoblastoma(RB) cell line HXO-RB44. ·METHODS: The DNA meth...AIM: To investigate the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR),a DNA methyltransferase(DNMT) inhibitor,on the growth and survival of the Chinese retinoblastoma(RB) cell line HXO-RB44. ·METHODS: The DNA methylation status of the Ras association domain family(RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction(MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining,respectively,when cells were treated with 5.0μmol/L of 5-Aza-CdR. The effect of 5.0μmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and flow cytometry. ·RESULTS: 5-Aza-CdR efficiently induced cell cycle arrest at G0 /G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0μmol/L 5-Aza-CdR. Accordingly,RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA,with a plateau on day four. 5-Aza-CdR at 5.0μmol/L completely demethylated the RASSF1A promoter in HXORB44 cells on day four,and as a result,RASSF1A expression increased significantly from day 4 to day 7.·CONCLUSION: 5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.展开更多
背景与目的:抑癌基因Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)启动子及第1外显子区CG位点高甲基化导致该基因沉默与多种恶性肿瘤的发生发展相关。本研究旨在探讨宫颈癌细胞系RASSFIA基因启动子及第1外显子区甲基...背景与目的:抑癌基因Ras相关区域家族1A(Ras association domain family 1A,RASSF1A)启动子及第1外显子区CG位点高甲基化导致该基因沉默与多种恶性肿瘤的发生发展相关。本研究旨在探讨宫颈癌细胞系RASSFIA基因启动子及第1外显子区甲基化状态以及甲基化转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-2’deoxycytidine,5-Aza-dc)作用对RASSFIA基因表达的影响。方法:采用5μmol/L(低浓度)和10μmol/L(高浓度)的5-Aza-dc作用于HeLa、Caski、HT-3以及C-33A等4种宫颈癌细胞系,分别采用甲基化特异PCR(methylation-specific PCR,MSP)和亚硫酸盐基因组测序法(bisulfite genome sequencing,BGS)检测5-Aza-dc处理前后RASSF1A基因启动子及第1外显子区甲基化状态,RT-PCR检测干预前后RASSF1A基因mRNA的转录表达。结果:HeLa和Caski两种HPV阳性细胞系RASSF1A基因启动子及第1外显子区均呈低甲基化状态,mRNA表达阳性。低浓度和高浓度5-Aza-dc作用后,mRNA表达未见明显改变(F HeLa=3.003,P=0.125;F Caski=0.045,P=0.956)。HT-3和C-33A两种HPV阴性宫颈癌细胞系RASSF1A基因启动子及第1外显子区则表现为高度甲基化状态,mRNA表达受到抑制。低浓度和高浓度5-Aza-dc作用后,HT-3和C-33A细胞系RASSF1A基因启动子及第1外显子区CG位点甲基化程度降低,检测到其mRNA表达,高浓度5-Aza-dc作用组表达水平明显高于低浓度组和细胞对照组(F HT-3=18.002,P=0.03;F C-33A=17.179,P=0.03),LSD-t检验显示差异有统计学意义(P<0.05)。结论:HPV阳性和HPV阴性宫颈癌细胞系中RASSFIA基因启动子及第1外显子区甲基化状态不同;RASSF1A基因启动子及第1外显子区的高甲基化可抑制该基因表达;5-Aza-dc处理可使RASSF1A基因启动子及第1外显子区去甲基化,重新激活基因的表达,这种作用在一定范围内有剂量依赖性。展开更多
A total of 110 primary NSCLCs （non-small cell lung cancers） were recruited in this study to characterize the pattern of 3p21 LOH together with the RASSF1A methylation status and their clinical implication. 3p21 LOH ...A total of 110 primary NSCLCs （non-small cell lung cancers） were recruited in this study to characterize the pattern of 3p21 LOH together with the RASSF1A methylation status and their clinical implication. 3p21 LOH by 8 microsatellite markers, RASSF1A methylation status by methylation-specific PCR （MSPCR） as well as bisulfite genomic sequencing （BGS）, and RASSF1A expression level by real-time quantitative PCR was performed. 3p21 LOH is frequent in NSCLC with a mean frequency of （41.2±3.7）%. Significant associations between 3p21 LOH and gender, smoking history, histological type, and tumor size were observed. Cases with LOH have a slightly lower RASSF1A expression than cases without LOH but not statistically significant. Comparison of RASSF1A methylation that resulted from the three analyses shows significant correlations from one another. Higher frequency of methylation was observed in larger tumors and in smokers compared with smaller tumors and non-smokers, respectively. A significant correlation was also observed in extent between methylation and RASSF1A expression, illustrating that epigenetic mechanism could affect gene expression. The significant clinicopathological relations of 3p21 LOH may be of great use for both early detection and therapeutic interventions.展开更多
基金Supported by the National Natural Science Foundation of China(No.3087282No.81072221)
文摘AIM: To investigate the effect of 5-Aza-2’-deoxycytidine(5-Aza-CdR),a DNA methyltransferase(DNMT) inhibitor,on the growth and survival of the Chinese retinoblastoma(RB) cell line HXO-RB44. ·METHODS: The DNA methylation status of the Ras association domain family(RASSF1A) promoter in the presence of 5-Aza-CdR at different concentrations was analyzed by methylation-specific polymerase chain reaction(MSP). RASSF1A mRNA and protein levels were measured by semiquantitative RT-PCR and immunohistochemistry staining,respectively,when cells were treated with 5.0μmol/L of 5-Aza-CdR. The effect of 5.0μmol/L 5-Aza-CdR on the proliferation and viability of HXO-RB44 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and flow cytometry. ·RESULTS: 5-Aza-CdR efficiently induced cell cycle arrest at G0 /G1 and apoptotic death in HXO-RB44 cells. MSP analysis showed that unmethylated RASSF1A DNA increased and methylated RASSF1A decreased in a dose-dependent manner in a range of 0.5-5.0μmol/L 5-Aza-CdR. Accordingly,RASSF1A expression was reactivated at both mRNA and protein levels. Incubation time of 5-Aza-CdR treatment also functioned as a factor for the demethylation status of RASSF1A promoter DNA,with a plateau on day four. 5-Aza-CdR at 5.0μmol/L completely demethylated the RASSF1A promoter in HXORB44 cells on day four,and as a result,RASSF1A expression increased significantly from day 4 to day 7.·CONCLUSION: 5-Aza-CdR inhibits the growth of the HXO-RB44 RB cell line and induces apoptosis by demethylating the RASSF1A gene.
基金Supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region (HKU7310/01M, 7486/03M, 7468/04)
文摘A total of 110 primary NSCLCs （non-small cell lung cancers） were recruited in this study to characterize the pattern of 3p21 LOH together with the RASSF1A methylation status and their clinical implication. 3p21 LOH by 8 microsatellite markers, RASSF1A methylation status by methylation-specific PCR （MSPCR） as well as bisulfite genomic sequencing （BGS）, and RASSF1A expression level by real-time quantitative PCR was performed. 3p21 LOH is frequent in NSCLC with a mean frequency of （41.2±3.7）%. Significant associations between 3p21 LOH and gender, smoking history, histological type, and tumor size were observed. Cases with LOH have a slightly lower RASSF1A expression than cases without LOH but not statistically significant. Comparison of RASSF1A methylation that resulted from the three analyses shows significant correlations from one another. Higher frequency of methylation was observed in larger tumors and in smokers compared with smaller tumors and non-smokers, respectively. A significant correlation was also observed in extent between methylation and RASSF1A expression, illustrating that epigenetic mechanism could affect gene expression. The significant clinicopathological relations of 3p21 LOH may be of great use for both early detection and therapeutic interventions.
