【作者】 陈红；

【导师】 罗阳；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2009， 博士

【摘要】 前言胃癌是源自胃粘膜上皮的恶性肿瘤,在我国发病率很高,死亡率占恶性肿瘤的第一位。胃癌是多种基因变化参与和积累的结果,揭示这些基因变化的规律,不仅可以阐明癌变的机制,更可以协助肿瘤的诊断、治疗和预后判断。肝细胞生长因子(hepatocyte growth factor, HGF)主要由间质细胞如成纤维细胞、巨噬细胞等产生,是一种上皮形态发生过程中的旁分泌调节物,具有多种生物学特性,能刺激多种上皮和内皮细胞进行有丝分裂、运动以及促进肾小管形态发生和血管内皮再生。HGF生物学活性由原癌基因c-met编码的跨膜受体蛋白所介导。研究发现HGF/c-Met通路异常活化在肿瘤生长、侵袭和转移中起重要作用。在许多上皮源性肿瘤中常可测得HGF过度表达,乳腺癌患者组织和血液中HGF表达水平显著高于非乳腺癌患者,出现淋巴结或肝转移者表达水平最高,而且HGF和c-Met表达水平与肿瘤的复发有关。对HGF异常表达的人类乳腺癌细胞系的HGF启动子区1kb范围内序列突变筛查研究发现,HGF的异位表达和可诱导表达的分子基础是HGF基因转录起始点上游一个cis作用元件DNA序列的改变。野生型cis作用元件是30个脱氧腺苷酸(A)形成的PolyA结构,将这个PolyA结构命名为DATE (deoxyadenosine tract element, DATE),当DATE发生截短改变,就会改变某些转录因子如C/EBP-β1、PAPP1和PAPP2对HGF启动子区DATE及临近结合位点的结合力,增加HGF启动子的活性,促进其在肿瘤细胞中表达。研究发现复发性胃癌患者血清HGF显著高于正常人和原发性胃癌者,且HGF水平与肿瘤浸润深度及血管受侵程度密切相关,在胃癌中,胃纤维母细胞通过激活HGF/c-Met系统来刺激肿瘤的生长和浸润。胃癌中常可见到c-Met的扩增和过度表达,因此,HGF/c-Met与胃癌的发生、发展、转移及预后密切相关。为探讨HGF的DATE截短改变是否与胃癌发生相关,我们检测了胃癌细胞系和80例胃癌患者HGF启动子区DATE截短改变情况,并进一步用RT-PCR检测HGF的DATE发生截短改变后,HGF在相应胃癌细胞系和胃癌组织中的表达,明确二者的相关性,为胃癌的诊断和治疗开辟一个新思路。材料与方法1、胃癌细胞系、胃癌组织和正常人外周血HGF的DATE截短筛查常规酚氯仿法提取胃癌细胞系、80例胃癌组织及相应癌旁组织和100例正常人外周血基因组DNA,通过PCR反应扩增HGF启动子区DATE序列,产物经8%的变性聚丙烯酰胺凝胶电泳检测,检测到DATE截短改变的标本进行TA克隆测序验证,HeLa细胞系DATE的PolyA数目是30,与正常人基因组DATE序列相同,将HeLa细胞DATE作为野生型,与野生型DATE相比,胃癌细胞系、胃癌组织和正常人DATE的PolyA缺失大于等于5A,定义为DATE出现截短改变。2、RT-PCR检测胃癌细胞系和胃癌组织中HGF表达5种胃癌细胞系和30例胃癌组织和相应癌旁组织用TRIzol法提取总RNA后,反转录合成cDNA, RT-PCR检测HGF表达,明确DATE截短改变与HGF表达的关系。3、DATE截短改变和微卫星不稳定性的相关性分析选取微卫星位点D5S396和D5S2056, PCR扩增发生DATE截短改变的6例胃癌组织和相应癌旁组织的2个微卫星位点,比较胃癌组织DATE截短改变和微卫星不稳定现象之间的相关性。实验结果1、胃癌细胞系HGF启动子区DATE截短改变和HGF表达关系本研究发现胃癌细胞系MGC803、MKN28和HGC27发生了DATE截短改变,基因型分别是30A/25A、25A/25A和27A/25A。胃粘膜上皮细胞系GES-1和胃癌细胞系SGC7901、BGC823和AGS DATE没有截短改变。RT-PCR检测HGF在胃粘膜上皮细胞系GES-1及胃癌细胞系SGC7901、MGC803、BGC823和AGS中表达,发现HGF只在DATE出现截短改变的MGC803中表达,DATE没有截短改变的细胞系中没有表达,这表明HGF启动子区DATE的截短改变促进了HGF在胃癌细胞系的表达。2、胃癌组织中HGF启动子区DATE截短改变和HGF表达关系本研究检测了80例胃癌组织及相应癌旁组织中HGF启动子区DATE的截短改变情况。发现15%(12／80)的胃癌患者存在DATE的截短改变。胃癌组织DATE的PolyA的截短变化范围从5A-20A。在12例DATE截短改变的胃癌样本中,7例(58%)DATE截短改变只发生在胃癌组织,而相应癌旁组织DATE没有截短改变,其余5例DATE截短改变同时存在于癌组织和癌旁组织,提示DATE的截短改变与胃癌发生密切相关。RT-PCR检测HGF在30例胃癌组织中表达情况,发现只在DATE截短改变的6例(占20%)胃癌组织中有HGF表达,而未出现截短改变的胃癌组织和癌旁组织未见HGF表达。且DATE截短最多的标本(其癌组织DATE基因型20A/10A), HGF表达最高,以上结果提示HGF表达水平与DATE截短程度相关。3、HGF启动子区DATE截短改变与胃癌发生相关性分析研究中发现DATE在相应癌旁组织中也出现截短改变,提示DATE的截短改变具有多态性。分析了100例中国正常人群外周血中HGF启动子区DATE截短改变情况,发现4%(4／100)的人群中出现DATE截短改变。比较胃癌患者和正常人群中DATE截短发生率,χ2检验显示两者差别有统计学意义(P<0.05),表明DATE截短与胃癌发生相关。4、DATE截短改变和微卫星不稳定性相关性分析为了确定DATE在肿瘤组织中的不稳定现象和肿瘤组织中微卫星不稳定性之间的相关性,PCR扩增已确定DATE截短改变的6例胃癌组织和癌旁组织的D5S396和D5S2056微卫星位点,发现5例在D5S396和D5S2056两个位点都出现了微卫星不稳定性,一例未检测到微卫星不稳定性,这例标本癌旁组织和癌组织DATE出现相同程度的截短改变。这表明通过分析胃癌组织DATE截短改变可以判定胃癌组织发生了微卫星不稳定性改变,以上提示HGF启动子区DATE可作为筛查胃癌组织微卫星不稳定性的一个简单而有效的指标。结论(1)胃癌中存在HGF启动子区DATE的截短改变,DATE的截短改变能促进HGF在胃癌中的表达,表明DATE的截短改变与胃癌发生密切相关。(2)胃癌中DATE的截短改变,能有效判定胃癌组织发生微卫星不稳定现象,可作为筛查胃癌组织微卫星不稳定性的一个简单而有效的指标。更多还原

【Abstract】 IntroductionGastric cancer is the second most common malignancy worldwide. It develops as a result of multistep process characterized by the accumulation of genetic alteration involving a variety of oncogenes and tumor suppressor genes. Therefore, better knowledge of the changes in genes occurred during gastric carcinogenesis may lead to improvements in diagnosis, treatment and prevention.HGF, hepatocyte growth factor, is an important paracrine mediator of mesenchymal-epithelial interaction, instructing epithelial cells to undergo processes such as cell proliferation, survival, migration, and morphogenesis. HGF exerts its effects via binding to and activating a tyrosine kinase transmembrane cell surface receptor known as Met. HGF gene expression is highly cell type specific and is restricted to mesenchymal cells such as fibroblasts.Normal epithelial cells do not express this gene, although all epithelial cells express the HGF receptor. Studies have established that HGF/Met signaling actively contributes to the process of neoplastic transformation, tumor metastasis, and tumor maintenance. In the case of breast adenocarcinomas, activation of autocrine HGF expression in the cancer cells and its overexpression are believed to contribute to neoplasia. Clinical studies have reported that high levels of HGF mRNA and protein within the breast carcinoma tissue are associated with poor survival in patients.Studies uncovered that the HGF transcript is expressed in human breast adenocarcinoma cells but not in the normal mammary ductal epithelium. In the case of gastric cancer, high levels of HGF are believed to contribute to the process of neoplastic transformation, tumor metastasis, and tumor maintenance.Study has been established to scan human carcinoma cell lines that aberrantly expressed the HGF gene for promoter mutation. The results led to the identification of a novel 30-bp cis-acting DNA element within the proximal promoter region of the human HGF gene. The element consists of a tract of 30 deoxyadenosines (30As; poly [dA]) which termed "deoxyadenosine tract element" (DATE). Functional studies revealed that wild-type DATE alters chromatin structure and silences the HGF promoter in normal epithelial cells through interactions with several nuclear factors and that DATE deletion mutagenesis (DATE truncation or shortening) distorts this DNA structure, resulting in differential binding of several factors to this element and its adjacent sites. This leads to recruitment of chromatin modifying and transcription factors such as C/EBP-βand poly (ADP-ribose) polymerase (PARP), theculmination of which is activation of the HGF promoter in the tumor cells.In this study,wei evaluated the DATE genotype in samples from 80 cases of human gastric carcinoma and matched normal adjacent tissue and detected HGF expression in tumor cells and tissues The results revealed that DATE is a target of deletion mutagenesis (DATE truncation) in gastric cancer and that truncated DATE significantly associates with high HGF gene expression in tumor cells, Notably, HGF gene expression is significantly higher in the mammary tissues of individuals with truncated DATE than in tissues of individuals with wild-type DATE, These results not only shed light on our understanding of the genetic basis of human gastric cancer tumorigenesis but also led to develop better diagnostic and therapeutic targets for patients with gastric cancer.Materials and methods1. Scanning human gastric carcinoma cell lines, tumor tissues and peripheral blood of normal individuals for DATE truncation.In order to examine DATE truncation, we amplified DATE in tumor tissues, cell lines and peripheral blood of normal individuals DNA by PCR, The amplified products were first analyzed in 8% denaturing polyacrylamide gel containing 8 M urea. In cases in which the DATE size variation seems small, DNA sequencing were performed.2. Detection of HGF gene expression in tumor cells and tissus by RT-PCR.In order to reveal that truncated DATE significantly associates with high HGF gene expression in tumor cells and tumor tissues, HGF gene expression was detected in tumor cells and thirty gastric cancer tissues and matching normal tissues by RT-PCR.3. Study of truncated DATE associated with microsatellite Instability.We used 2 poly(CA) microsatellites D5S396 and D5S2056 to establish the MSI status of six gastric cancer primary tumors with truncated DATE to find that truncated DATE associates with microsatellite instability.Results1. Truncation mutation of DATE in the HGF gene promoter region correlates with activation of HGF gene expression in human gastric carcinoma cell lines.HGF of HeLa cells had a stretch of 30As in their DATE identical to the normal human HGF promoter sequence, indicating that these cells had a wild-type DATE, whereas those exhibited a truncated mutant version of DATE in which the number of As was reduced by 5 bp or more. We screened carcinoma cell lines for truncation mutation of DATE. We found that some gastric cancer cell lines (MGC803, MKN28 and HGC27) had truncated mutation of DATE. In MGC803 cells, DATE length was truncated by 5As (thus, these cells had a DATE genotype of 25As in heterozygous manner) and HGC27cells were heterozygous for short alleles of 27As and 25As, while in MKN-28cells, both alleles were truncated by 5 nucleotides (25As) in DATE. On the other hand, GES-1, SGC7901, BGC823 and AGS cells were homozygous for 30As, 29As,29As, and 28As respectively. Detection of HGF gene expression in tumor cells (GES-1, SGC7901,MGC803,BGC823 and AGS) by RT-PCR, we found HGF gene expression was noted in MGC803 cells with truncated DATE but not in other cells with wild-type DATE.2. DATE is a target of deletion mutagenesis in human gastric cancer tissue, and its truncated version associates with aberrant HGF expression.We evaluated the DATE genotype in samples 80 cases of human gastric carcinoma and matched normal adjacent tissue, we found that approximately 15% (12/80) gastric cancer tissues possessed the truncated DATE variant. To be conservative in these analyses, we designated DATE as a truncated variant if it had 25As or fewer as we described above. The size of truncated DATE among different gastric tumor cases ranged from lOAs (loss of 20As) to 25As (loss of 5As). In 58% (7/12) of the cancer cases with shortened DATE, truncated DATE was present only in the tumor and not in the corresponding adjacent normal tissue, indicating that DATE is unstable and prone to deletion mutagenesis in tumor cells. HGF mRNA was highly expressed in cancer tissues with the truncated DATE, since it was readily detectable by 32 cycles of RT-PCR, but little or no HGF expression was detected in tissues with wild-type DATE under these conditions.3. Truncated DATE significantly associates with gastric cancer.Our data suggest that DATE is most likely polymorphic in nature since in some cases both tumor and the corresponding normal tissue had the truncated DATE variant. Therefore, to address this possibility, we determined the incidence of truncated DATE using peripheral blood lymphocyte genomic DNA from 100 healthy Chinese adult human subjects. We observed an incidence of truncated DATE (DATE with 25As or less) in the general population of approximately 4%(4/100). Comparison of the prevalence of truncated DATE in gastric cancer cases with that of normal subjects indicated that truncated DATE significantly correlates with breast cancer incidence (15%vs.4%, P=0.01).4. Study of truncated DATE associated with microsatellite Instability.We used 2 poly(CA) microsatellites D5S396 and D5S2056 to establish the MSI status of six gastric cancer primary tumors with truncated DATE, we found that 5 gastric cancer tissues showed MSI by the use of D5S396 and D5S2056 microsatellites, indicating that analysis of DATE was able to confirm the MSI status 5 of 6 gastric tumors(83.3% efficiency), therefore DATE can be an indicator of MSI in gastric cancer.Conclusion(1)Gastric cancer harbor a truncated DATE variant in their gastric tumors and that the truncated allele is associated with cancer incidence and aberrant HGF expression.(2)Analysis of DATE was able to confirm the MSI status and can be an indicator of MSI in gastric cancer.更多还原