【作者】 谢华平；

【导师】 田德安；

【作者基本信息】 华中科技大学 ， 消化内科， 2012， 博士

【摘要】 肿瘤,是全球重要的公共卫生问题。全球每年肿瘤年发病率约为202.8／100,000,年死亡率约为127.9/100,000。全美国每4个死亡中就有一个是由肿瘤所引起。中国恶性肿瘤呈逐年上升趋势,目前已成为第一位死因。早在20世纪初,科学家就已经发现,染色体结构异常是肿瘤发病的关键因素。费城染色体的研究表明：染色体结构异常能引起基因表观遗传学改变,促进肿瘤发生、发展。拷贝数变异,包括扩增、缺失等,是染色体结构改变的重要组成部分。基于基因芯片的拷贝数变异检测,无法将拷贝数变异断点定位至碱基水平。为研究拷贝数变异可能带来表观遗传学改变,需要选择适当方法进行断点定位分析。本研究主要目的为：以染色体9p21大片段缺失为研究对象,以乳腺癌细胞系MCF-7和胰腺癌细胞系PANC-1为模板,探索缺失断点定位的实验方法,分析拷贝数变异对基因转录的影响。实验方法培养乳腺癌细胞系MCF-7和胰腺癌细胞系PANC-1,提取DNA及RNA。利用多重连接探针扩增技术(Multiplex Ligation-based Probe Amplification, MLPA),查明MCF-7和PANC-1细胞系中染色体9p21缺失情况,判断缺失位点可能区间,并用常规PCR加以确认。以MCF-7细胞系DNA为模板,探讨长片段PCR以及反向PCR进行缺失断点定位可行性,并明确MCF-7细胞系中9p21缺失断点准确位置。根据缺失断点定位信息,分析可能的基因转录子改变,运用cDNA快速扩增技术以及TA克隆测序,检测可能形成的新融合转录子。实验结果1、MLPA检测发现乳腺癌细胞系MCF-7和胰腺癌细胞系PANC-1中,均存在累及MTAP、CDKN2A等基因大片段缺失的情况。MCF-7细胞系的端粒侧断点位于MTAP基因内,着丝粒侧断点位于CDKN2A基因内；PANC-1细胞系染色体9p21大片段缺失的端粒侧缺失断点位于MTAP基因内,着丝粒侧的缺失断点因超出MLPA检测范围而未可知。2、长片段PCR以及反向PCR均证实,MCF-7细胞系9p21缺失位点位于chr9：21819532至chr9：21989622之间,缺失片段大小约为170kb;断点端粒侧位于MTAP基因的外显子4后,着丝粒侧位于CDKN2A (p14)基因的内含子1内近外显子1β处。3、在MCF-7细胞系中,检获到一个新MTAP转录融合子,由MTAP基因前4个外显子与非编码区chr9：22087143-22087648融合形成,含967个碱基、翻译125个氨基酸序列。在PANC-1细胞系中,检获到两个新MTAP转录融合子,其中一个与非编码区chr9：22384168-22384481融合,含876个碱基、翻译138个氨基酸序列；另外一个与非编码区chr9：22674440-22674564融合,含598个碱基、翻译149个氨基酸序列。实验结论MLPA技术是检测染色体9p21大片段缺失的有效方法,能准确判断大片段缺失边界范围,适合于aCGH检测后大样本筛查。乳腺癌细胞系MCF-7和胰腺癌细胞系PANC-1均存在着累及MTAP和CDKN2A基因大片段缺失。长片段PCR、反向PCR是缺失断点定位的良好方法,证实MCF-7细胞系中存在一个起于MTAP基因内、止于CDKN2A基因内、大小约为170kb的缺失片段。由于染色体9p21的缺失,乳腺癌细胞系MCF-7中存在一个缺失、融合形成新MTAP转录子；胰腺癌细胞系PANC-1中,存在两个缺失、融合形成新MTAP转录子。这些新MTAP转录融合子是否参与肿瘤发生、发展,还有待进一步研究。更多还原

【Abstract】 Background and AimsCancer is one main public health problem in the whole world. The global annual incidence of cancer is about202.8/100,000while its global annual morbility is about127.9/100,000. In the United States, one of every four deaths is caused by cancer. Tumor incidence grows gradually every year in China, which has already been the first cause of death. In the early20th centry, scientists had found that chromosomal structural variations are key factors of cancer initiation and development. The Philadelphia Chromosome further confirms that chromosomal rearrangements can result in epigenetic alterations and promote the initiation and development of cancers. Copy number variations, mainly including deletions and amplifications, are main parts of chromosomal changes. Copy number variations detected based on microarrays can not be charaterized in base pair level. In order to analyze possible epigenetic alterations caused by copy number variations, it is necessary to modify useful methods for breakpoint mapping. Focused on large deletions on chromosome9p21, this study is aimed to find out efficient methods to detect breakpoints as well as to analyze the possible changes on transcription level in breast cancer cell line MCF-7and pancreatic cancer cell line PANC-1.MethodsCell line DNA and RNA were extracted from cultured breast cancer cell line MCF-7and pancreatic cancer cell line PANC-1. MLPA (Multiplex Ligation-based Probe Amplification) was used to detect large deletions on chromosome9p21in these two cell lines, with deletion borders furtherly confirmed by conventional PCR. The long range and inverse PCR were used to detect deletion breakpoints on chromosome9p21in these two cell lines. RACE (Rapid Amplification of cDNA ends), TA cloning and sequencing were used to find out possible new transcripts.Results1、The large deletions on chromosome9p21, with invovlment in MTAP and CDKN2A genes, were found in both MCF-7and PANC-1cell lines. The homozygous deletions in MCF-7was found starting within MTAP gene and ending in CDKN2A gene while the homozygous deletions in PANC-1was found starting within MTAP gene, with the centromeric border beyond the detection range of MLPA method.2、The long range and inverse PCR confirmed the deletions spanning from chr9:21819532to chr9:21989622in MCF-7, with a deletion size of170kb, starting within the Intron4of MTAP on the tolemeric side and ending within the Intron1near Exon1β of CDKN2A on the centromeric side.3、In breast cancer cell line MCF-7, A new MTAP fusion transcript identified turned out to be fused between the first4exons of MTAP and non-coding region chr9:22087143-22087648, containing967base pairs on cDNA level and translating into a protein of125amino acids. In pancreatic cancer cell line PANC-1, two new MTAP fusion transcripts were identified. One transcript turned out to be fused between the first4exons of MTAP and non-coding region chr9:22384168-22384481, containing867base pairs on cDNA level and translationg into one protein of138amino acids. The other one turned out to be fused between the first exons of MTAP and non-coding region chr9:22674440-22674564, containing598base pairs on cDNA level and translating into149amino acids.ConclusionsThe MLPA method is an effient way to detect large deletions correctly, which is suitable to confirm abnormal regions detected by array-based Comparative Genomic Hybridization. Both the breast cancer cell line MCF-7and pancreatic cancer cell line PANC-1contain large deletions involved in MTAP and CDKN2A on chromosome9p21. The long range/inverse PCR confirmed the large deletion in MCF-7spanning from chr9:21819532to chr9:J21989622, with a deletion size of170kb, starting within the Intron4of MTAP and ending within the Intron1near Exon1β of CDKN2A. Due to large deletions on chromosome9p21, one new MTAP fusion transcript and two new MTAP fusion transcripts were found in MCF-7, PANC-1, respectively.It needs further study whether these new MTAP fusion transcripts are involved in the initation and development of breast/pancreatic cancer or not.更多还原