基因启动子甲基化与肺癌的相关性研究
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摘要
肺癌是世界范围内发病率和死亡率最高、治疗效果相对较差的癌症。肺癌严重危害人类健康,已成为癌症死亡的首位病因。由于肺癌患者在出现症状首次就诊时,已有80 %因为出现转移或存在禁忌症而丧失手术机会。肺癌的总体5年生存率为15%,在我国低于10%。各期肺癌5年生存率:IA 67%、IB 57%、ⅡA 34%、ⅡB 24%、ⅢB 6%～8%、Ⅳ期为3%。因此,早期诊断肺癌对于提高5年生存率意义重大。
DNA甲基化作为表观遗传学的一部分,可调控基因表达。已发现诸多基因启动子区域的甲基化异常与癌症相关,如细胞周期调控基因、DNA修复基因、血管形成基因、细胞凋亡相关基因、细胞黏附迁移相关基因、激素受体基因等。因此,甲基化正在成为一类新的生物标志物,成为肿瘤有效诊断的标志分子,也正在成为有希望的肿瘤治疗的候选靶标。实验研究显示去甲基化治疗,可以使因为异常高甲基化而表达沉默的p16等控制细胞增值、分化和凋亡的基因重新恢复表达,从而可能通过抑制肿瘤细胞生长、增加细胞间的黏附、恢复对化疗药的敏感性等,达到肿瘤治疗的目的。本课题拟通过检测肺癌相关基因启动子甲基化分析启动子甲基化与基因表达在肺癌细胞的生长、分化、增殖和凋亡中的关系研究,检测人外周血标本中肺癌相关基因启动子甲基化频率,探讨基因甲基化与肺癌发病的关系,评价甲基化标志作为肺癌早期诊断和作为治疗靶标的可行性。
本课题选取NDRG2、P16、CDH1、RARbeta2及WIF-1五种候选抑癌基因,培养A549、GLC-82两种肺癌细胞,ECV-304、GES-1两种非肿瘤细胞作为对照,从细胞中提取基因组DNA,经过亚硫酸盐处理,使用PYRO ASSAY DESIGN设计的PCR引物进行目标基因启动子区域扩增,然后使用焦磷酸测序技术对基因甲基化程度进行分析。发现五种基因在四种细胞中都有不同程度的甲基化现象,其中NDRG2、P16、RARbeta2基因的甲基化频率在肺癌细胞组与非肿瘤细胞组之间有明显差异,而CDH1与WIF-1两种基因无显著差异。
为探讨基因启动子区域甲基化状态与基因表达之间的关系,我们采用甲基化转移酶抑制剂5-杂氮2-脱氧胞苷(5-Aza-2-CdR)对两种肺癌细胞进行不同浓度和时间的处理,发现处理后NDRG2、P16、RARbeta2三种基因恢复表达,提示这三种基因在肺腺癌细胞中的表达沉默与启动子高甲基化相关。
目前大部分甲基化基因与癌症相关性的研究都是基于临床活检的,基于体液(血浆/血清、痰、尿、腹水等)的甲基化分析很少。本实验对肺癌病人及正常人外周血提取的基因组DNA中五种基因启动子区域的甲基化程度进行了分析,其中NDRG2、P16、CDH1三个基因肺癌组的甲基化频率高于正常组,并具有统计学意义,与文献的结果一致。而WIF-1基因肺癌组的甲基化频率虽然略高于正常组,但两组间并无统计学差异,不排除检测样本量小、检测位点不特异以及不同检测方法带来的偏差。
Lung cancer is the number one cancer killer in the world,and its recovery rate is relatively low. Lung cancer is the first leading cause of cancer-related deaths. There were 80% paitents lose the chance of operation as a result of the transfer or contraindication when they have the onset of symptoms with lung cancer the first visit.The overall 5-year survival rate of lung cancer is 15%, less than 10% in China. The 5-year survival rate of lung cancer: IA 67%, IB 57%,ⅡA 34%,ⅡB 24%,ⅢB 6% ~ 8%,Ⅳof 3%. Therefore, early diagnosis of lung cancer 5-year survival rate for the increase is significant.
DNA methylation as part of epigenetics can control gene expression. There are many genes that are found to be methylated at promoter region,and they are all associated with cancer, such as cell cycle genes, DNA repair genes, angiogenesis genes, apoptosis related genes, cell adhesion and migration-related genes, hormone receptor gene et al.Therefore, methylation is to become a new class of biomarkers and has become an effective diagnostic marker of cancer, is also becoming a promising potential target for cancer treatment. Many studies have shown that demethylation treatment can restore expression of some silencing gene because of DNA hypermethylation such as p16 that can control cell proliferation, differentiation and apoptosis genes ,and it will inhibit tumor cell growth, increased cell adhesion to restore sensitivity to chemotherapy, to achieve the purpose of cancer treatment. So,our purpose is to investigate the relationship of lung cancer related gene promoter methylation with lung cancer cell growth, differentiation, proliferation and apoptosis,and development, to evaluate the feasibility of methylation as early diagnosis and therapeutic targets.
This topic selected NDRG2, P16, CDH1, RARbeta2, and WIF-1 candidate tumor suppressor gene, cultured lung cancer cells A549, GLC-82, control cell ECV-304, GES-1, extracted genomic DNA from cells, used pyrosequencing to detected some methylation loci on five genes after bisulfite treatment. The result showed that five genes had different degrees of DNA methylation in the four cells, the MTI of NDRG2, P16, RARbeta2 in lung cancer cells was highed than conreol cells .and the MTI of WIF-1 and CDH1was no significant difference between two groups.
To investigate the relationship between gene promoter region methylation status and gene expression, we used 5-Aza-CdR to teartment lung cancer cells with two different concentrations and different time. The result showed that the expression of NDRG2, P16 and RARbeta2 was restored, suggested that the silence these three genes is associated with promoter hypermethylation in lung cancer cells.
At present, most cancer research about gene methylat is based on clinical biopsy, but based on body fluids (plasma / serum, sputum, urine. Ascites, etc.) research is very little. In this study, we extracted genomic DNA from peripheral blood of lung cancer patients, used pyrosequencing to detected gene methylation.The result showed that the MTI of the NDRG2, P16 and CDH1 gene is higher in lung cancer patients group than conctrol group, and the statistic difference was significant(p<0.05), consisted with the literature. The MTI of WIF-1 is also higher in lung cancer patients group than conctrol group but the statistic difference was significant, maybe it is the bias from the detection of small sample size,detection loci and different methods.
DNA甲基化作为表观遗传学的一部分,可调控基因表达。已发现诸多基因启动子区域的甲基化异常与癌症相关,如细胞周期调控基因、DNA修复基因、血管形成基因、细胞凋亡相关基因、细胞黏附迁移相关基因、激素受体基因等。因此,甲基化正在成为一类新的生物标志物,成为肿瘤有效诊断的标志分子,也正在成为有希望的肿瘤治疗的候选靶标。实验研究显示去甲基化治疗,可以使因为异常高甲基化而表达沉默的p16等控制细胞增值、分化和凋亡的基因重新恢复表达,从而可能通过抑制肿瘤细胞生长、增加细胞间的黏附、恢复对化疗药的敏感性等,达到肿瘤治疗的目的。本课题拟通过检测肺癌相关基因启动子甲基化分析启动子甲基化与基因表达在肺癌细胞的生长、分化、增殖和凋亡中的关系研究,检测人外周血标本中肺癌相关基因启动子甲基化频率,探讨基因甲基化与肺癌发病的关系,评价甲基化标志作为肺癌早期诊断和作为治疗靶标的可行性。
本课题选取NDRG2、P16、CDH1、RARbeta2及WIF-1五种候选抑癌基因,培养A549、GLC-82两种肺癌细胞,ECV-304、GES-1两种非肿瘤细胞作为对照,从细胞中提取基因组DNA,经过亚硫酸盐处理,使用PYRO ASSAY DESIGN设计的PCR引物进行目标基因启动子区域扩增,然后使用焦磷酸测序技术对基因甲基化程度进行分析。发现五种基因在四种细胞中都有不同程度的甲基化现象,其中NDRG2、P16、RARbeta2基因的甲基化频率在肺癌细胞组与非肿瘤细胞组之间有明显差异,而CDH1与WIF-1两种基因无显著差异。
为探讨基因启动子区域甲基化状态与基因表达之间的关系,我们采用甲基化转移酶抑制剂5-杂氮2-脱氧胞苷(5-Aza-2-CdR)对两种肺癌细胞进行不同浓度和时间的处理,发现处理后NDRG2、P16、RARbeta2三种基因恢复表达,提示这三种基因在肺腺癌细胞中的表达沉默与启动子高甲基化相关。
目前大部分甲基化基因与癌症相关性的研究都是基于临床活检的,基于体液(血浆/血清、痰、尿、腹水等)的甲基化分析很少。本实验对肺癌病人及正常人外周血提取的基因组DNA中五种基因启动子区域的甲基化程度进行了分析,其中NDRG2、P16、CDH1三个基因肺癌组的甲基化频率高于正常组,并具有统计学意义,与文献的结果一致。而WIF-1基因肺癌组的甲基化频率虽然略高于正常组,但两组间并无统计学差异,不排除检测样本量小、检测位点不特异以及不同检测方法带来的偏差。
Lung cancer is the number one cancer killer in the world,and its recovery rate is relatively low. Lung cancer is the first leading cause of cancer-related deaths. There were 80% paitents lose the chance of operation as a result of the transfer or contraindication when they have the onset of symptoms with lung cancer the first visit.The overall 5-year survival rate of lung cancer is 15%, less than 10% in China. The 5-year survival rate of lung cancer: IA 67%, IB 57%,ⅡA 34%,ⅡB 24%,ⅢB 6% ~ 8%,Ⅳof 3%. Therefore, early diagnosis of lung cancer 5-year survival rate for the increase is significant.
DNA methylation as part of epigenetics can control gene expression. There are many genes that are found to be methylated at promoter region,and they are all associated with cancer, such as cell cycle genes, DNA repair genes, angiogenesis genes, apoptosis related genes, cell adhesion and migration-related genes, hormone receptor gene et al.Therefore, methylation is to become a new class of biomarkers and has become an effective diagnostic marker of cancer, is also becoming a promising potential target for cancer treatment. Many studies have shown that demethylation treatment can restore expression of some silencing gene because of DNA hypermethylation such as p16 that can control cell proliferation, differentiation and apoptosis genes ,and it will inhibit tumor cell growth, increased cell adhesion to restore sensitivity to chemotherapy, to achieve the purpose of cancer treatment. So,our purpose is to investigate the relationship of lung cancer related gene promoter methylation with lung cancer cell growth, differentiation, proliferation and apoptosis,and development, to evaluate the feasibility of methylation as early diagnosis and therapeutic targets.
This topic selected NDRG2, P16, CDH1, RARbeta2, and WIF-1 candidate tumor suppressor gene, cultured lung cancer cells A549, GLC-82, control cell ECV-304, GES-1, extracted genomic DNA from cells, used pyrosequencing to detected some methylation loci on five genes after bisulfite treatment. The result showed that five genes had different degrees of DNA methylation in the four cells, the MTI of NDRG2, P16, RARbeta2 in lung cancer cells was highed than conreol cells .and the MTI of WIF-1 and CDH1was no significant difference between two groups.
To investigate the relationship between gene promoter region methylation status and gene expression, we used 5-Aza-CdR to teartment lung cancer cells with two different concentrations and different time. The result showed that the expression of NDRG2, P16 and RARbeta2 was restored, suggested that the silence these three genes is associated with promoter hypermethylation in lung cancer cells.
At present, most cancer research about gene methylat is based on clinical biopsy, but based on body fluids (plasma / serum, sputum, urine. Ascites, etc.) research is very little. In this study, we extracted genomic DNA from peripheral blood of lung cancer patients, used pyrosequencing to detected gene methylation.The result showed that the MTI of the NDRG2, P16 and CDH1 gene is higher in lung cancer patients group than conctrol group, and the statistic difference was significant(p<0.05), consisted with the literature. The MTI of WIF-1 is also higher in lung cancer patients group than conctrol group but the statistic difference was significant, maybe it is the bias from the detection of small sample size,detection loci and different methods.
引文
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