【作者】 张磊；

【导师】 赵平； 蔡建强； 林东昕；

【作者基本信息】 中国协和医科大学 ， 肿瘤学， 2010， 博士

【摘要】 背景和目的肝细胞肝癌(HCC)是常见的恶性肿瘤之一。我国HCC病人多有肝炎背景。乙型肝炎病毒感染导致的HCC具有非常明显的家族聚集性和遗传易感性,其发病率呈患者一级亲属、二级亲属递减,且均高于群体发病率。因此从基因水平研究肝癌的易感机制十分必要。从基因转录水平对HCC的发病机制进行研究目前已经有很多报道。近年来从基因组结构变异水平对拷贝数变异(copy number variations, CNVs)与疾病的关联进行研究逐渐成为热点。CNVs是指与基因组参考序列相比,基因组中≥1 kb的DNA片段插入、缺失和／或扩增。人类基因组CNVs图谱提示在研究与疾病相关的基因变异时必须考虑CNVs,否则会错过重要的信息。因此本研究的主要目的是在基因组结构水平上找到与肝炎病毒所致HCC发病机制密切相关的CNVs,并结合临床病理学资料进行预后分析,以期为HCC诊治和预后提供新的思路。方法我们通过SNP芯片对25例HCC患者的样本进行配对的全基因组检测,初步获得与HCC发病机制相关的基因CNVs结果。继之结合相关资料,进一步筛选出与肝炎所致HCC相关的候选基因,通过实时定量PCR检测后者在肝癌组织和癌旁组织中的拷贝数,明确候选基因是否存在CNVs。同时我们收集患者的临床病理学资料,并结合相关基因进行预后分析,进一步明确相关基因的CNVs是否与重要的临床病理因素相联系。结果比较患者的肝癌组织与其血液标本的基因表达谱,共得到1854个差异基因,其中上调基因1242个,下调基因612个,提示仅有少数基因参与肿瘤细胞的癌变过程。我们继之筛选出与JAK-STAT通路相关的五个基因：STAT3.STAT2、STAT4、JAK1、IL-6以及近来较多关注的Lin-28B进行研究。实时定量PCR检测结果显示HCC组织中STAT3的拷贝数显著高于相应的癌旁组织(P＜0.05)；HCC组织中STAT2、STAT4、JAK1、IL-6和Lin-28B的拷贝数与相应癌旁组织相比无显著增高(P＞0.05)。继之,我们比较每例患者HCC组织与癌旁组织的JAK1拷贝数差异,并据此将患者分为两组,一组患者HCC组织中JAK1的拷贝数比癌旁组织高,另一组相比较低。生存分析提示JAK1对患者术后的累积生存率没有显著影响(P＞0.05)。并且,术前的AFP水平、术前ALT水平、术前AST水平、肿瘤直径、分化程度、肿瘤个数在两组间的分布无统计学差异(P＞0.05)。对另一基因STAT4进行分析,与JAK1的统计结果相同(P＞0.05)。40例HCC患者的1、3年生存率分别为87.2%、67.9%。单因素分析发现影响患者术后累积生存率的因素包括术前AFP水平、肿瘤个数以及分化程度(P＜0.05)；多因素分析提示影响患者术后累积生存率的独立危险因素包括肿瘤个数及分化程度(P＜0.05)结论STAT3的拷贝数在HCC组织及癌旁组织中存在显著差异,提示STAT3的CNVs在HCC的发病机制中有重要作用；JAK1和STAT4的CNVs对于HCC患者的术后累积生存率以及相关的临床病理因素没有显著影响；肿瘤个数及分化程度是影响HCC预后的独立因素,术前AFP水平是影响预后的重要因素。更多还原

【Abstract】 Background & Aims Hepatocellular Carcinoma (HCC) is one of the most common malignant tumor in the world. Most patients with HCC have chronic hepatitis in China. Significantly, HCC which results from chronic B hepatitis virus (HBV) has familial aggregation and genetic susceptibility, and the incidence of HCC which are correlative with the first and the second degree relative decreased progressively, furthermore, the incidence were high than population risk respectively. Therefore, it is necessary to study the susceptible mechanism of HCC related to genome-wide abnormalities. Now there were lots of studies about the susceptible mechanism of HCC at transcriptional level. Recently, the studies focus on the genome-wide study of association between CNVs and diseases at the level of DNA structural variation. CNVs are the result of duplications, deletions, insertions, inversions and complex combinations of rearrangements, and is defined as a chromosomal segment that is 1kb or larger in length, whose copy number varied in comparison to a reference genome. In a recent study CNVs have been mapped, and scientists found that genomic regions containing CNV may harbor important genes and gene regulatory elements, and may have considerable influence on disease susceptibility. Thus, we attempt to find CNVs related to carcinogenesis of HCC result from hepatitis virus infection at the level of genomic structure abnormalities, and analyze the clinico-pathological data of HCC patients after surgical resection, which may provide new path to favor the diagnosis, treatment and prognosis of HCC.Methods We screened CNVs in 25 patients with HCC by the high-resolution SNP arrays, and gained preliminary data. Then, we selected certain gene which is important in the carcinogenesis of HCC result from hepatitis virus infection, and identified the existence of CNVs by detecting the copy number of selected genes in the HCC tissue and peri-carcinomatous tissue. At the same time, we collected the clinic-pathological data of HCC patients to analyze the relationships between the prognosis of patients and the CNVs of certain genes.Results There were 1854 CNVs by screening the HCC tissue and peri-carcinomatous tissue using SNP arrays. We find 1242 CNVs with up-regulation and 612 CNVs with down-regulation, which indicates only a little of genes take part in the progression of malignant transformation. We selected Lin-28B gene which was noticeable in the present studies and five genes relevant to JAK-STAT pathway including STAT3、STAT2、STAT4、JAK1、IL-6. The results of real-time PCR showed that the copy number of STAT3 was higher in HCC tissue than in peri-carcinomatous tissue(P<0.05), and the copy numbers of STAT2、STAT4、JAK1、IL-6、Lin-28B had no significant difference between two groups (P>0.05). Then we divided patients into two groups. The copy numbers of JAK1 were higher in HCC tissue than in peri-carcinomatous tissue in group 1 and lower in group 2. Survival analysis showed that JAK1 didn’t impact the prognosis of HCC patients after operation (P>0.05). Furthermore, tumor size, differentiation and the preoperative level of AFP, ALT, and AST had no significant difference between two groups (P>0.05).The analysis of STAT4 showed the same results as that of JAK1 (P >0.05). The overall 1-,3-year survival rates were 82.7%and 67.9%, respectively. Univariate analysis showed that tumor number, differentiation and preoperative level of AFP were significant factors affecting the survival(P<0.05). Multivariate analysis demonstrated that tumor number and differentiation were the independent factors affecting the survival(P<0.05).Conclusion The copy number of STAT3 was higher in HCC tissue than in peri-carcinomatous tissue, which shows that the CNVs of STAT3 play an important role in the carcinogenesis of HCC result from hepatitis virus infection. The CNVs of JAK1 and STAT4 didn’t impact the prognosis and some clinico-pathological factors of HCC patients after operation. Tumor number and differentiation were the independent factors affecting the survival. The preoperative level of AFP is an important prognostic factor for the surgical management.更多还原