目的:探讨5-氮-2-脱氧胞苷(5-Aza-CdR)和曲古抑菌素A(TSA)对人胃癌细胞系MGC-803生物学行为的影响.方法:人胃癌细胞系MGC-803按常规培养于含100g/L小牛血清的RPMI1640培养液中.实验分为5组:(1)空白对照组:不含细胞的培养液;(2)阴性对照...目的:探讨5-氮-2-脱氧胞苷(5-Aza-CdR)和曲古抑菌素A(TSA)对人胃癌细胞系MGC-803生物学行为的影响.方法:人胃癌细胞系MGC-803按常规培养于含100g/L小牛血清的RPMI1640培养液中.实验分为5组:(1)空白对照组:不含细胞的培养液;(2)阴性对照组:细胞只换液,不加药物;(3)5-Aza-CdR组:细胞接种24h后,加入10.0μmol/L的5-Aza-CdR;(4)TSA组:细胞接种24h后,加入300μg/LTSA;(5)5-Aza-CdR+TSA组:细胞接种24h后,加入10.0μmol/L5-Aza-CdR,24h后再加入300μg/LTSA.应用RT-PCR检测各组细胞培养72h后细胞Runx3 mRNA的表达,MTT比色法测定各组细胞分别培养24、48、72h后细胞增殖.结果:单独应用5-Aza-CdR和TSA作用于MGC-803细胞72h后,Runx3 mRNA相对表达量增加,联合用药组分别与5-Aza-CdR组及TSA组相比,差异有统计学意义(0.883±0.025 vs 0.760±0.286,0.735±0.018,均P<0.05).细胞生长抑制率在同一组别随着药物作用时间的延长而增加,并且呈正相关(r=0.738,P<0.05);而在同一时间不同组别比较联合用药组细胞生长抑制率较单用药物组明显增加(24h:57.3% vs 40.4%,39.0%;48h:70.0% vs 56.0%,51.3%;72h:86.3% vs 68.0%,65.8%,均P<0.05).细胞Runx3 mRNA的相对表达量及生长抑制率在药物组与对照组间比较,差异均有统计学意义(P<0.05).结论:与单用5-Aza-CdR或TSA相比,联合应用2种药物更显著的增强Runx3 mRNA的表达,抑制细胞的增殖.展开更多
Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast ca...Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast cancer lines it has been shown that shelterin proteins are dysregulated thereby affecting the telomere stability and contributing to the neoplastic conversion of the mammary epithelial cells. Interestingly, the regulation of some of the shelterin genes is thought to be controlled epigenetically. Methods and Results: In this study, we set out to measure the effect of increased shelterin gene expression on telomere length in breast cancer cell line 21NT treated with 5-aza-2-deoxycytidine (5-aza-CdR) using known telomere length assays. We measured telomere lengths using: Telomere Restriction Fragment length (TRF), absolute quantitative-PCR and cytogenetic Interphase Quantitative Fluorescent in situ Hybridization (iQ-FISH). We found that non-cytotoxic levels of 5-aza-CdR affect telomere lengths by causing a significant and stable increase in telomere lengths of the breast cancer cell line. The increase in telomere lengths was consistently observed when various telomere length methods were used. Conclusions: Further investigation is required to understand the underlying mechanism involved, and the significance of telomere length elongation in relation to clinical outcome when epigenetic modifying drugs are utilized.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
文摘目的:探讨5-氮-2-脱氧胞苷(5-Aza-CdR)和曲古抑菌素A(TSA)对人胃癌细胞系MGC-803生物学行为的影响.方法:人胃癌细胞系MGC-803按常规培养于含100g/L小牛血清的RPMI1640培养液中.实验分为5组:(1)空白对照组:不含细胞的培养液;(2)阴性对照组:细胞只换液,不加药物;(3)5-Aza-CdR组:细胞接种24h后,加入10.0μmol/L的5-Aza-CdR;(4)TSA组:细胞接种24h后,加入300μg/LTSA;(5)5-Aza-CdR+TSA组:细胞接种24h后,加入10.0μmol/L5-Aza-CdR,24h后再加入300μg/LTSA.应用RT-PCR检测各组细胞培养72h后细胞Runx3 mRNA的表达,MTT比色法测定各组细胞分别培养24、48、72h后细胞增殖.结果:单独应用5-Aza-CdR和TSA作用于MGC-803细胞72h后,Runx3 mRNA相对表达量增加,联合用药组分别与5-Aza-CdR组及TSA组相比,差异有统计学意义(0.883±0.025 vs 0.760±0.286,0.735±0.018,均P<0.05).细胞生长抑制率在同一组别随着药物作用时间的延长而增加,并且呈正相关(r=0.738,P<0.05);而在同一时间不同组别比较联合用药组细胞生长抑制率较单用药物组明显增加(24h:57.3% vs 40.4%,39.0%;48h:70.0% vs 56.0%,51.3%;72h:86.3% vs 68.0%,65.8%,均P<0.05).细胞Runx3 mRNA的相对表达量及生长抑制率在药物组与对照组间比较,差异均有统计学意义(P<0.05).结论:与单用5-Aza-CdR或TSA相比,联合应用2种药物更显著的增强Runx3 mRNA的表达,抑制细胞的增殖.
文摘Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast cancer lines it has been shown that shelterin proteins are dysregulated thereby affecting the telomere stability and contributing to the neoplastic conversion of the mammary epithelial cells. Interestingly, the regulation of some of the shelterin genes is thought to be controlled epigenetically. Methods and Results: In this study, we set out to measure the effect of increased shelterin gene expression on telomere length in breast cancer cell line 21NT treated with 5-aza-2-deoxycytidine (5-aza-CdR) using known telomere length assays. We measured telomere lengths using: Telomere Restriction Fragment length (TRF), absolute quantitative-PCR and cytogenetic Interphase Quantitative Fluorescent in situ Hybridization (iQ-FISH). We found that non-cytotoxic levels of 5-aza-CdR affect telomere lengths by causing a significant and stable increase in telomere lengths of the breast cancer cell line. The increase in telomere lengths was consistently observed when various telomere length methods were used. Conclusions: Further investigation is required to understand the underlying mechanism involved, and the significance of telomere length elongation in relation to clinical outcome when epigenetic modifying drugs are utilized.
