摘要
随着肿瘤机制研究的不断深入,表观遗传学(epigenetics)已日益受到重视。表观遗传学是指在基因组序列不变的情况下,可以决定基因表达与否并可稳定遗传下去的调控信息；而表观遗传组学(epigenomics)是指在基因组水平上对表观遗传学改变的研究。目前,表观遗传组学研究已成为肿瘤研究领域的新前沿。
DNA甲基化是哺乳动物基因组最为常见、研究最为深入的一种表观遗传学事件,参与调节多种生命活动。每种类型的肿瘤都有其特定的DNA甲基化模式,不同肿瘤或同一肿瘤的不同发生阶段中基因组DNA上CpG岛甲基化状态的差异,构成了肿瘤特定DNA甲基化谱。舌鳞状细胞癌(Tougue Squamous Cell Carcinoma, TSCC)是口腔鳞状细胞最为常见的恶性肿瘤,其发生发展的分子机制还未阐明。过去十几年中,在口腔鳞状细胞癌(Oral Squamous Cell Carcinoma, OSCC)中相关基因的DNA异常甲基化研究方面取得了一些进展,但大部分研究只是针对几个或一群基因采取候选基因研究方法,迄今还没有基于全基因组层面上直接、全面、系统分析TSCC中DNA甲基化情况的报道。舌鳞癌DNA甲基化谱建立不仅有助于全面揭示舌癌发生发展的分子机制,更重要的是可为舌鳞癌的早期诊断、治疗及预后评价等提供非常有价值的依据。
缺少高通量筛选技术可能是影响基因组水平研究DNA甲基化谱系的关键因素。DNA甲基化芯片的发展为推动表观遗传组学研究提供了新的契机。NibmbleGen公司制备的甲基化芯片是目前国际上覆盖人类基因组CpG岛最多、注释最全面的一种芯片,而且这种芯片具有较高灵敏度和特异性的特点。
基于以上因素,本课题采用最新发展的甲基化DNA免疫沉淀(Methylated DNA Immunoprecipitation, MeDIP)结合NimbleGen HG18 CpG Promoter芯片高通量分析舌鳞癌组织DNA甲基化情况,并结合芯片提供的信息,进一步在舌鳞癌组织和癌旁正常对照组织中分析基因的表达情况,初步筛选出舌鳞癌相关的基因。
[甲基化芯片检测舌鳞癌基因组DNA甲基化分布]
本课题收集了9例患者舌鳞癌肿瘤组织及其癌旁正常对照组织标本,并将9例标本分别抽提获得DNA,组成肿瘤和正常两组样品。采用MeDIP方法富集两组样品基因组DNA中甲基DNA片段,在严格的质控检验后,分别与两张甲基化芯片进行杂交,对杂交信号进行扫描以及初步数据处理,并对两组间数据进行比较分析。结果显示,有1269个DNA甲基化位点只存在于肿瘤组织中,其中涉及到330个基因,这些基因分布在不同的染色体上。定位在1号染色体上的甲基化差异基因最多,有28个,占肿瘤组织内甲基化差异基因的8.48%；其次,19号染色体上有27个,占差异总数的8.18%；而定位在18号染色体上的差异基因最少,只有4个,所占比率为1.21%;330个舌鳞癌组织中甲基化基因的甲基化区域有218个(66.1%)位于基因的启动子区,并有70%为CpG岛。
同样,在癌旁正常对照组织中,差异DNA甲基化的位点共有1385个,涉及基因有321个。基因分布于各染色体上,其定位和分布数量差别较大。其中,定位在19号染色体上的甲基化差异基因最多,高达40个,占癌旁组织中甲基化差异基因的12.46%；其次为X染色体和1号染色体,分别有32个和23个,占差异总数的9.97%、7.17%；而定位在14号染色体上的差异基因最少,只有2个,所占比率为0.62%。321个甲基化基因的甲基化区域有210个(65.4%)位于基因的启动子区,43.6%为CpG岛。
筛选得到的差异甲基化基因具有一定家族聚集性,四个角蛋白家族基因和六个跨膜蛋白家族基因均在肿瘤组织中发生甲基化；而7个黑色素瘤抗原家族成员则在癌旁正常对照组织中发生甲基化。
采用在线GO分析网站Gostat对芯片筛选出来的差异甲基化基因进行功能分类,发现差异基因参与了基因转录调控、生殖、发育、代谢、离子转运、细胞生长和增殖、细胞分化和凋亡、细胞通讯、信号转导、细胞粘附等广泛的生物学过程,同时还涉及多条与机体免疫及肿瘤发生有关的信号通路,如Cell Communication、Purine metabolism、Cell adhesion molecules (CAMs)、Natural killer cell mediated cytotoxicity、Neuroactive、ligand-receptor、interaction、Glycerophospholipid metabolism、leukocyte transendothelial migration、Fc epsilon RI signaling pathway等。由此可以推测,这些甲基化异常的基因可能与TSCC的发生发展密切相关。
[芯片结果的初步验证及肿瘤相关基因的初步筛选]
从海量的芯片杂交数据中挑选出肿瘤组织中发生异常甲基化的ITIH5基因和FBLN1基因以及癌旁正常对照组织中发生异常甲基化的RUNX3基因,采用MSPCR方法检测三个基因分别在肿瘤组织和癌旁正常对照组织中的甲基化改变情况。结果发现,在癌旁正常对照组织中检测到ITIH5、FBLN1两个基因分别存在25%和20%的DNA异常甲基化,但两个基因在肿瘤组织中均存在较高频率的DNA异常甲基化,分别为70%(14／20)、55%(11／20)。统计学分析证实,两个基因在肿瘤组织和对照组织中的甲基化程度存在显著差异(P=0.004<0.01,P=0.026<0.05)。另外,RUNX3基因的DNA异常甲基化在癌旁正常对照组织和肿瘤组织中发生的频率分别为55%和15%,两者之间同样存在显著差异(P=0.008<0.01).MSPCR初步验证结果与芯片结果相似,初步证明芯片结果的可靠性。
为了进一步筛选舌鳞癌相关的肿瘤基因,我们通过分析芯片结果,选择出与肿瘤发生密切相关的并在舌鳞癌组织中未深入研究的6个基因(BCL2L14、CDCP1、DIRAS、FBLN1、ITIH5、RUNX3)进行初步分析。采用RT-PCR方法对6个基因在舌鳞癌组织中的表达情况进行初步筛选,结果提示,DIRAS、FBLN1、ITIH5在舌鳞癌组织中表达下调(P<0.05),下调频率分别是45%(9／20)、40%(8／20)、55%(11／20)；而BCL2L14、CDCP1、RUNX3则在舌鳞癌组织中表达显著上调(P<0.05),上调频率分别是60%(12／20),50%(10／20),75%(15／20)。提示6个功能不同基因可能与舌鳞癌的发生发展有关。
进一步对FBLN1、ITIH5、RUNX3三个基因进行表达异常与甲基化关系分析,结果发现,FBLN1、ITIH5基因分别在87.5%(7／8)、90.9%(10／11)表达下调的舌鳞癌组织标本中存在DNA异常甲基化；同样,RUNX3基因则在66.7%(10／15)表达上调的肿瘤组织对应的癌旁对照组织中存在异常甲基化。说明DNA异常甲基化与三个基因在组织中的表达异常有关。
综上所述,通过本研究初步构建了人舌鳞癌组织与癌旁正常对照组织基因组DNA甲基化谱,得到了大量的DNA甲基化分布信息,为进一步建立更为精确的舌鳞癌DNA甲基化谱奠定了基础,也为阐明舌鳞癌发病的分子机制提供了可能。另外,本课题初步筛选出6个与舌鳞癌发生发展密切相关的基因,可作为以后更深入研究的靶标,为舌鳞癌临床诊断、治疗或预后等分子标志物的筛选提供了实验依据。
Epigenetic differences become an important issue for explaining cancer formation. Epigenetic modifications control gene expression in a hereditary way. Epigenomics describes the changes in epigenetic modifications on the genome. Recently, epigenomics becomes one of the cutting age fields in cancer research.
DNA methylation is the most common and well-studied genomic modifications. DNA methylations regulate a variety of biological events. Different types of cancers have specific DNA methylation patterns. Moreover, DNA methylation patterns change in different stages during cancer formation. Consequently, DNA methylation on CpG islands becomes a genomic identity for a particular cancer. Tongue squamous cell carcinoma (TSCC) is most common oral squamous cancer. But, the mechanism is still unclear. In the past decades, abnormal DNA methylations in several genes have been found to be related to oral squamous cell carcinoma (OSCC) formation. However, most researches focused on genome-wide DNA methylation pattern instead of exploring methylation changes of several genes promoter. There is no report for genome-wide analysis of DNA methylation in TSCC. It is promising to establish genomic DNA methylation map for understanding the mechanism of TSCC, prediagnosis of TSCC and treatment of TSCC.
Lack of high-throughput DNA methylation detection techniques is the major reason impeding the development of genomic DNA methylation map. The development of DNA methylation chip provides a powerful method for the research on epigenomics. NimbleGen has a DNA methylation chip best covering the number of CpG islands in human genome, best annotating the chip, and best sensitivity in the DNA methylation chip international market.
We will use methylated DNA immunoprecipitation (MeDIP) and NimbleGen HG18 CpG Promoter Chip to analyze DNA methylations in TSCC with this high-throughput method. Comparing the gene expression data between TSCC sample and adjacent normal tissue in DNA methylation Chip, we are able to identify TSCC related genes.
DNA methylation Chip detect TSCC genomic DNA methylation
In this study, we collected nine samples from TSCC and their adjacent counterparts as control. After obtaining the genomic DNA from those samples, we mixed nine genomic DNA from TSCC as tumor group and mixed nine genomic DNA from adjacent tissues as control group. By performing MeDIP, we respectively enrich the methylated DNA fragments from two groups followed by hybridizing to DNA mehtylation microarray individually. After data processing, there are 1269 DNA methylation sites only existing in tumor samples, not in normal adjacent samples. Those methylation sites accounts for 330 different genes, spreading in different chromosomes. There are 28 genes (8.48%) showing DNA methylation difference in chromosome 1. Secondly,27 genes (8.18%) in chromosome 19 and only 4 genes (1.21%) show difference in DNA methylation.218 out of the 330 genes (66.1%) have the DNA mehtylation in the promoter region and 70% of them are on the CpG islands.
Similarly, in the normal adjacent sample, there are 1385 DNA methylation sites only existing in control tissue residing in 321 different genes. The number and distribution in different chromosomes have larger difference compared to those in tumor sample. Chromosome 19 has the largest number of the genes showing difference in DNA methylation. There are 40 genes (12.46%) in chromosome 19. X chromosome and chromosome 1 have 32 (9.97%) and 23 (7.17%) genes respectively. Chromosome 14 has the least number of the genes showing difference in DNA methylaion, only 2 genes (0.62%).210 out of the 321 genes (65.4%) have the DNA methylation in the promoter resion and 43.6% of them are on the CpG islands.
The genes showing different DNA methylation in tumor and normal samples are reside in the same gene family. For example, four keratin protein family genes and six transmembrane protein family members only have DNA methylation in tumor samples. In the other hand, seven melanoma antigen family members only have DNA methylation in adjacent normal samples.
We use Gene Ontology analysis website (Gostat) to classify the genes screening out from the DNA methylation chip. These genes are involved in transcription regulation, reproduction, development, metabolism, ion transportation, cell growth, cell differentiation and apoptosis, signal transduction and cell adhesion processes. At the same time, they are also involved in several immune and cancer development related pathways, such as cell communication, purine metabolism, cell adhesion molecules, natural killer cell mediated cytotoxicity, neuroactive ligand-receptor interaction, glycerophospholipid metabolism, leukocyte transendothelial migration and Fc epsilon RI signaling pathway. Those differential genes in DNA methylation might involve in TSCC development.
Preliminary results of verifying the TSCC related genes selected from DNA methylation microarray data
We selected ITIH5 and FBLN1 which are the differential genes in results from DNA methylation Chip of tumor samples. Moreover, we picked RUNX3 which shows differential changes in DNA methylation in adjacent normal samples. MSPCR were performed to detect the changes in DNA methylation both in tumor and normal samples. Although promoter hypermethylation of ITIH5 and FBLN1 were detected in 25% and 20% of normal samples respectively, higher percentage of promoter hypermethylation were found in tumor samples,70%(14/20) and 55% (11/20) respectively. The percentage differences between tumor and normal sample in those two genes are statistically significant. (P=0.004<0.01, P=0.026<0.05). In addition, there are 55% and 15% of DNA methylation in RUNX3 gene in normal and tumor samples respectively. This difference is also statistically significant (P=0.008<0.01). Consequently, preliminary MSPCR results are consistent to DNA methylation chip data.
In order to further identify TSCC related genes, we analyzed DNA methylation Chip data and selected six genes (BCL2L14, CDCPl, DIRAS, FBLNl, ITIH5 and RUNX3) which are correlated with carcinogenesis but are not studied previously for further studies in TSCC. We used RT-PCR to analyze the expression level of those six genes in TSCC samples. RT-PCR data showed that the expression level of DIRAS (45% 9/20), FBLN1 (40% 8/20) and ITIH5 (55% 11/20) were down-regulated in TSCC samples (P<0.05). However, BCL2L14 (60% 12/20), CDCP1 (50% 10/20) and RUNX3 (75% 15/20) were up-regulated in TSCC samples. Therefore, those genes might be related to TSCC carcinogenesis.
We further correlate the gene expression level with the DNA methylation in FBLN1, ITIH5 and RUNX3 genes. The DNA of FBLN1 (87.5% 7/8) and ITIH5 (90.9% 10/11) genes were methylated in TSCC samples whose expression level of FBLN1 and ITIH5 were down-regulated. Similarly, the DNA of RUNX3 (66.7% 10/15) gene was methylated in the adjacent normal tissue instead of TSCC tumor samples which shows up-regulating RUNX3 expression. Together, abnormal DNA methylation is related to the differential expression of FBLN1, ITIH5 and RUNX3 in TSCC tissues.
In summary, this study established the whole genome-wide DNA methylation profile in TSCC tissues and their adjacent counterparts. Upon DNA methylation chip, it helps to develop precise genomic DNA methylation map and provides possible molecular mechanisms for TSCC carcinogenesis. In addition, six genes which are related to TSCC development are also found result from this study and might be able to become the biomarkers for diagnosis, treatment and prognosis of TSCC.
引文
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