口腔疣状癌基因组DNA甲基化谱研究
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摘要
口腔疣状癌(oral verrucous carcinoma, OVC)是一种不同于鳞癌的有着独特的病理学特征和生物学行为的独立性疾病,其发病机制尚不清楚。DNA甲基化芯片和生物信息学技术的发展提高了我们对疾病的生物学的研究和探讨疾病分子机制能力。本研究采用甲基化DNA免疫沉淀(Methylated DNA immunoprecipitation, MeDIP)结合NibleGen CpG promoter芯片技术探讨口腔疣状癌及其正常对照组织中基因组DNA甲基化情况,并采用多种生物信息学方法对甲基化基因进行分析,为进一步探讨OVC发生的分子机制和发现治疗靶基因奠定基础。
目的：分析口腔疣状癌DNA甲基化位点上基因,挖掘其隐含的生物学意义。
方法：收集5例口腔疣状癌癌组织及相应患者的正常粘膜组织,采用甲基化DNA免疫沉淀(Methylated DNA immunoprecipitation, MeDIP)分别和十张NibleGen CpG promoter芯片进行杂交,对杂交信号进行扫描以及原始数据进行归一化处理和比较分析。利用多种生物信息学工具和软件对筛选基因进行染色体定位分析、GO (Gene Ontology)分析、Pathway分析。
结果：芯片杂交结果符合质量控制要求,按照Peak Count>2为筛选标准,肿瘤中发生超甲基化位点的有1023个,这些位点包含了1248个基因,其甲基化位点在其启动子内或者上下游发生甲基化,489个(47.8%)甲基化位点发生在CpG岛上。发生去甲基化位点的有640个,包含了780个基因,474个(74.06%)甲基化位点位于基因的启动子区域；363个(56.71%)甲基化位点位于发生在CpG上。
染色体定位分析可见在肿瘤组织中分布于1,17号染色体上的发生甲基化位点最多, Y染色体上的甲基化位点最少；在正常组织中定位在19号染色体上的发生甲基化位点最多,其次为17号染色体；Y染色体上去甲基化位点最少。
GO分析显示口腔疣状癌差异甲基化基因细胞成分大部分为细胞核以及整合于细胞膜上。生物学过程主要参与信号传导,DNA依赖的转录调节,G-protein偶联蛋白受体信号通路,氧化还原等生物过程；分子功能上大部分基因为蛋白结合分子,钙锌结合分子,肽链内切酶的活性序列特异性DNA断裂及蛋白质GTP酶激活因子等分子功能相关；
Pathway分析结果显示口腔疣状癌甲基化基因主要参与神经活性配受受体相互作用,NK细胞介导的细胞毒性,细胞凋亡,细胞因子间相互作用、MARK信号通路等通路。
结论：
1.口腔疣状癌与对应的正常口腔粘膜组织DNA甲基化位点存在差异；
2.口腔疣状癌发生甲基化和去甲基化区域的大部分位于基因的启动子区并且大部分为CpG岛；
3.运用生物信息学工具可快速、平行地分析大量的基因芯片数据,实现对甲基化基因初步的功能归类,为口腔疣状癌的发病机制提供新的思路。
Oral verrucous carcinoma (oral verrucous carcinoma, OVC) is different from the squamous cell carcinoma, which has a unique pathological features and biological behavior. its pathogenesis is still unknown. The development of DNA methylated microarray and bioinformatics enable us to understand disease biology and molecular mechanisms of disease more efficient. In this study, we use the method of Methylated DNA immunoprecipitation and combine with NibleGen CpG promoter profiles to analyse genomic DNA methylated in oral verrucous carcinoma and normal tissue, meanwhile, we also use a variety of bioinformatics methods to initially screen disease genes, in order to further rexplore the molecular mechanism and potential markers/ targets for clinical utility in treatment of OVC.
Objective:analysis of DNA methylated gene with oral verrucous carcinoma, then apply the bioinformatics tools for analyzing the DNA methylated genes in OVC to obtain the implied biological significance.
Methods:Total methylated DNA immunoprecipitation (Met hylated DNA immunoprecipitation, MeDIP)was isolated from 5 OVC patients and corresponding normal mucosa and hybride with 10 NibleGen CpG promoter microarray,then scan the hybridization signals and get raw data and comparative analysis; Then several bioinformatic analysises were used in the second screening of genes in OVC, which included gene ontology(GO)analysis, pat-hway analysis, chromosomal assignment;
Results:The results of microarray hybridization results accorded with quality control. Using a the Peak Count> 2 for the criteria.1023 DNA areas which contain 1248 genes occur methylation.Their methylated sites locate within or upstream and downstream their promoter.There are 952 (93.06%)methylated sites which were located on the promoter regions of genes and 489(47.8%) methylated sites occurred in the CpG islands.Correspondingly,the occurrence of de-methylated sites are 640, contains 780 genes.There are 474 (74.06%) methylated sites whi-ch were located on the promoter regions of genes, and 363 (56.71%) methylated sites occurred in the CpG islands.
Chromosome 1 happen to methylate at most, a total of 125, followed by chromosomes 17, however, in the Y chromosome happen to methylat-ed sites for at least,a total of 7.Demethylated sites, in chromosome 19, up to a total of 90.In the Y chromosome methylated sites are at least,a total of 7.
GO classification of differentially expressed DNA methylated genes identified cellular component subgroups consisted of genes mainly located in nucleus, integral to membrane; the biological process subgroups involved genes in signal transduction, regulation of transcription, DNA-dependent, G-protein coupled receptor protein signaling pathway, oxidation reduction; the function of most genes related to protein binding, calcium zinc ion binding et al. Pathway analysis showed that differentially expressed DNA methylated genes in oral verrucous carcinoma mainly involvedin pathways including Neuroactive ligand-receptor interaction, Natural killer cell mediated cytotoxicity, Apoptosis, Cytokine-cytokine receptor interaction,MARK signaling pathway.Conclusion The use of bioinformatics tools to quickly analyze a large number of parallel gene chip data that initially classified the functions of genes,which provide a new way of thinking for the pathogenesis of oral verrucous carcino-ma.
Conclusion:
1. DNA methylated and demethylated sites a-re different in Oral verrucous carcinoma and corresponding normal oral mucosa;
2. The region of methylated and demethylated in oral verrucous carcinoma mostly locate in the gene promoter region and CpG islands;
3. Bioinformatic tools Can provide the quick and parallel analysis of massive data derived from genechips and enable the function classification of the differentially expressed genes, which provides new clues on the research of pathogenesis and epidemiology of OVC.
目的：分析口腔疣状癌DNA甲基化位点上基因,挖掘其隐含的生物学意义。
方法：收集5例口腔疣状癌癌组织及相应患者的正常粘膜组织,采用甲基化DNA免疫沉淀(Methylated DNA immunoprecipitation, MeDIP)分别和十张NibleGen CpG promoter芯片进行杂交,对杂交信号进行扫描以及原始数据进行归一化处理和比较分析。利用多种生物信息学工具和软件对筛选基因进行染色体定位分析、GO (Gene Ontology)分析、Pathway分析。
结果：芯片杂交结果符合质量控制要求,按照Peak Count>2为筛选标准,肿瘤中发生超甲基化位点的有1023个,这些位点包含了1248个基因,其甲基化位点在其启动子内或者上下游发生甲基化,489个(47.8%)甲基化位点发生在CpG岛上。发生去甲基化位点的有640个,包含了780个基因,474个(74.06%)甲基化位点位于基因的启动子区域；363个(56.71%)甲基化位点位于发生在CpG上。
染色体定位分析可见在肿瘤组织中分布于1,17号染色体上的发生甲基化位点最多, Y染色体上的甲基化位点最少；在正常组织中定位在19号染色体上的发生甲基化位点最多,其次为17号染色体；Y染色体上去甲基化位点最少。
GO分析显示口腔疣状癌差异甲基化基因细胞成分大部分为细胞核以及整合于细胞膜上。生物学过程主要参与信号传导,DNA依赖的转录调节,G-protein偶联蛋白受体信号通路,氧化还原等生物过程；分子功能上大部分基因为蛋白结合分子,钙锌结合分子,肽链内切酶的活性序列特异性DNA断裂及蛋白质GTP酶激活因子等分子功能相关；
Pathway分析结果显示口腔疣状癌甲基化基因主要参与神经活性配受受体相互作用,NK细胞介导的细胞毒性,细胞凋亡,细胞因子间相互作用、MARK信号通路等通路。
结论：
1.口腔疣状癌与对应的正常口腔粘膜组织DNA甲基化位点存在差异；
2.口腔疣状癌发生甲基化和去甲基化区域的大部分位于基因的启动子区并且大部分为CpG岛；
3.运用生物信息学工具可快速、平行地分析大量的基因芯片数据,实现对甲基化基因初步的功能归类,为口腔疣状癌的发病机制提供新的思路。
Oral verrucous carcinoma (oral verrucous carcinoma, OVC) is different from the squamous cell carcinoma, which has a unique pathological features and biological behavior. its pathogenesis is still unknown. The development of DNA methylated microarray and bioinformatics enable us to understand disease biology and molecular mechanisms of disease more efficient. In this study, we use the method of Methylated DNA immunoprecipitation and combine with NibleGen CpG promoter profiles to analyse genomic DNA methylated in oral verrucous carcinoma and normal tissue, meanwhile, we also use a variety of bioinformatics methods to initially screen disease genes, in order to further rexplore the molecular mechanism and potential markers/ targets for clinical utility in treatment of OVC.
Objective:analysis of DNA methylated gene with oral verrucous carcinoma, then apply the bioinformatics tools for analyzing the DNA methylated genes in OVC to obtain the implied biological significance.
Methods:Total methylated DNA immunoprecipitation (Met hylated DNA immunoprecipitation, MeDIP)was isolated from 5 OVC patients and corresponding normal mucosa and hybride with 10 NibleGen CpG promoter microarray,then scan the hybridization signals and get raw data and comparative analysis; Then several bioinformatic analysises were used in the second screening of genes in OVC, which included gene ontology(GO)analysis, pat-hway analysis, chromosomal assignment;
Results:The results of microarray hybridization results accorded with quality control. Using a the Peak Count> 2 for the criteria.1023 DNA areas which contain 1248 genes occur methylation.Their methylated sites locate within or upstream and downstream their promoter.There are 952 (93.06%)methylated sites which were located on the promoter regions of genes and 489(47.8%) methylated sites occurred in the CpG islands.Correspondingly,the occurrence of de-methylated sites are 640, contains 780 genes.There are 474 (74.06%) methylated sites whi-ch were located on the promoter regions of genes, and 363 (56.71%) methylated sites occurred in the CpG islands.
Chromosome 1 happen to methylate at most, a total of 125, followed by chromosomes 17, however, in the Y chromosome happen to methylat-ed sites for at least,a total of 7.Demethylated sites, in chromosome 19, up to a total of 90.In the Y chromosome methylated sites are at least,a total of 7.
GO classification of differentially expressed DNA methylated genes identified cellular component subgroups consisted of genes mainly located in nucleus, integral to membrane; the biological process subgroups involved genes in signal transduction, regulation of transcription, DNA-dependent, G-protein coupled receptor protein signaling pathway, oxidation reduction; the function of most genes related to protein binding, calcium zinc ion binding et al. Pathway analysis showed that differentially expressed DNA methylated genes in oral verrucous carcinoma mainly involvedin pathways including Neuroactive ligand-receptor interaction, Natural killer cell mediated cytotoxicity, Apoptosis, Cytokine-cytokine receptor interaction,MARK signaling pathway.Conclusion The use of bioinformatics tools to quickly analyze a large number of parallel gene chip data that initially classified the functions of genes,which provide a new way of thinking for the pathogenesis of oral verrucous carcino-ma.
Conclusion:
1. DNA methylated and demethylated sites a-re different in Oral verrucous carcinoma and corresponding normal oral mucosa;
2. The region of methylated and demethylated in oral verrucous carcinoma mostly locate in the gene promoter region and CpG islands;
3. Bioinformatic tools Can provide the quick and parallel analysis of massive data derived from genechips and enable the function classification of the differentially expressed genes, which provides new clues on the research of pathogenesis and epidemiology of OVC.
引文
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