肝癌中抑癌基因PCDH8甲基化及其临床病理联系的研究

发布时间：2018-03-13 01:02

本文选题：肝癌　切入点：抑癌基因　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:探索新的有价值的肝癌(Liver Cancer)分子生物学标志物,研究肝癌中PCDH8基因启动子的甲基化状态及其蛋白的表达,并且分析PCDH8甲基化与肝癌临床病理各参数的关联。研究方法:(1)运用甲基化特异性PCR(Methylation-specific PCR,MSP)法检测42例肝细胞肝癌(HCC)、8例肝内胆管细胞癌(CC)和50例正常肝组织中3个抑癌基因PCDH8、HIN1、RAR-β2启动子区的甲基化状态。(2)运用免疫组织化学(immunohistochemistry,IHC)技术检测肝癌组织和正常肝组织中PCDH8蛋白的表达水平。(3)评估PCDH8启动子甲基化与肝癌患者临床病理特征的关系。(4)运用单变量和多变量Cox比例分析来确定肝癌独立预后因素,评估PCDH8甲基化与肝癌患者的总生存期的关系,评估甲胎蛋白(Alpha fetoprotein,AFP)水平、肿瘤的大小(体积或最大径)、肿瘤的分化程度(或异型性)等与肝癌患者的总生存时间的相关性。结果:(1)在肝癌中,PCDH8存在着较高的甲基化率(37/42(88.1%)HCC,7/8(87.5%)CC),但是只有16/50(32%)的正常肝组织发生甲基化,PCDH8基因启动子的甲基化率在肝癌患者和正常肝组织之间具有显著性差异,差异具有统计学意义(P0.001)。而其他抑癌基因HIN1和RAR-β2的甲基化率在肝癌患者和正常肝组织之间无显著差异,无统计学意义。(2)肝癌组织PCDH8蛋白表达减少。大部分的正常肝组织PCDH8蛋白表达呈强阳性(43/50,86%),但是在肝癌组织中,大部分病例PCDH8蛋白表达缺失(35/42HCC病例,83.3%;7/8 CC病例,87.5%)。(3)PCDH8启动子甲基化与其蛋白表达缺失显著相关。在正常肝组织的组织切片中,43/50例检测到PCDH8蛋白的表达。然而在50例肝癌组织(42例HCC,8例CC)中,有42例PCDH8蛋白的表达缺失,并且其中的40例发生PCDH8基因启动子甲基化。因此PCDH8启动子发生甲基化时,其蛋白表达往往缺失,启动子甲基化与其蛋白表达缺失存在着显著相关关系,差异具有统计学意义(P=0.004)。(4)肝癌组织中PCDH8的异常的甲基化状况与甲胎蛋白(alpha fetoprotein,AFP)水平显著相关(P=0.008)。肝癌中PCDH8基因启动子甲基化和其他临床病理特征,如年龄、性别、肿瘤的大小、肿瘤的分化程度等均没有显著相关关系(P0.05)。(5)肝癌病例中发生PCDH8甲基化的患者,其总体中位生存时间为15个月,而PCDH8未甲基化患者往往预后较好(该组患者在随访结束时均存活),两组差异经统计学分析P=0.041,具备差异意义。此外,肿瘤大小、肿瘤分化和AFP水平也与肝癌患者的生存时间相关。COX分析显示PCDH8甲基化可能不是一个独立的影响预后的因子(P0.05),同样肿瘤的大小、肿瘤的分化程度也不是独立的预测因子(P0.05)。多因素Cox比例分析显示,只有AFP水平这一项指标可以作为一个独立的肝癌预后因子。结论:肝癌中PCDH8存在着较高的甲基化率,而且PCDH8往往因为基因启动子的甲基化而失活,其蛋白表达受到启动子甲基化的调控。PCDH8基因启动子甲基化在肝癌的发生发展过程中起到了非常重要的作用,可以作为一个有价值的诊断生物标志物用于早期发现肝癌以及预测临床不良预后。肝癌组织中PCDH8的异常甲基化状态与AFP水平显著相关。肝癌中PCDH8未甲基化患者往往预后较好,但PCDH8可能不是一个独立的影响预后的因子,只有AFP水平可以作为一个独立的预后因素。
[Abstract]:Objective: To explore the new value of hepatocellular carcinoma (Liver Cancer) molecular markers of hepatocellular carcinoma, PCDH8 gene promoter methylation status and protein expression, and correlation analysis of PCDH8 methylation and clinicopathological parameters. Methods: (1) using methylation specific PCR (Methylation-specific PCR, MSP) were detected in 42 cases of hepatocellular carcinoma (HCC), 8 cases of intrahepatic cholangiocarcinoma (CC) in 3 tumor suppressor genes PCDH8, HIN1 and 50 cases of normal liver tissues, the methylation status of RAR- beta 2 promoter region. (2) by immunohistochemistry (immunohistochemistry, IHC) the expression level of PCDH8 protein in detection of hepatocellular carcinoma and normal hepatic tissue. (3) to evaluate the relationship between PCDH8 promoter methylation and clinicopathological characteristics of HCC patients. (4) using univariate and multivariate analyses Cox ratio to determine the factors of liver cancer and independent prognostic assessment, PCDH8 methylation The relationship between overall survival of patients with liver cancer, evaluation of alpha fetoprotein (Alpha fetoprotein, AFP), tumor size (volume or size), the degree of tumor differentiation (or atypia) and total survival time of patients with liver cancer correlation. Results: (1) in liver cancer, PCDH8 there is a higher rate of methylation (37/42 (88.1%) HCC, 7/8 (87.5%) CC), but only 16/50 (32%) methylation in normal liver tissues, PCDH8 gene promoter methylation rate has significant difference between HCC and normal liver tissues, the difference was statistically significant (P0.001) and the rest of the tumor suppressor gene HIN1 and RAR- beta 2 methylation rate had no significant difference between HCC and normal liver tissues was not statistically significant. (2) the decreased expression of PCDH8 protein in hepatocellular carcinoma. The expression of most of the normal liver tissue PCDH8 protein showed strong positive (43/50,86%), but in large hepatocellular carcinoma In some cases the expression loss of PCDH8 protein (35/42HCC cases, 83.3% CC cases; 7/8, 87.5%). (3) PCDH8 promoter methylation and protein expression. Significant correlation in normal liver tissues, the expression of 43/50 were detected PCDH8 protein. However, in 50 cases of hepatocellular carcinoma group (8 cases in 42 cases HCC, CC), the expression of PCDH8 protein in 42 cases, and 40 cases of PCDH8 gene promoter methylation. Therefore PCDH8 promoter methylation and its protein expression is often lack of promoter methylation and protein expression have significant correlation, the difference was statistically significant (P=0.004). (4) PCDH8 in hepatocellular carcinoma tissues and abnormal methylation status of alpha fetoprotein (alpha fetoprotein, AFP) significant level (P=0.008). The promoter methylation and other clinicopathological features of hepatocellular carcinoma PCDH8 gene, such as age, gender, tumor size, tumor The degree of differentiation were not significantly correlated (P0.05). (5) PCDH8 methylation in patients with liver cancer patients, the median overall survival time was 15 months, while PCDH8 patients without methylation (often a better prognosis in this group of patients were alive at the end of follow-up), two groups with statistical difference analysis of P=0.041, with significant differences. In addition, tumor size, tumor differentiation and the level of AFP is correlated with the survival time of liver cancer.COX analysis showed that PCDH8 methylation may not be an independent prognostic factor (P0.05), the same tumor size, tumor differentiation is not an independent predictor (P0.05). Multivariate Cox proportional analysis showed that only AFP level in this index can be used as an independent prognostic factor of HCC. Conclusion: hepatocellular carcinoma PCDH8 exists methylation rate is higher, but often because of PCDH8 gene promoter methylation and loss Live, its protein expression was regulated by promoter methylation of.PCDH8 gene promoter methylation in the occurrence and development of hepatocellular carcinoma has played a very important role, can be used as a valuable diagnostic biomarker for early detection of HCC and predict clinical adverse prognosis. PCDH8 was significantly correlated in HCC tissues and abnormal methylation the state and the level of AFP. PCDH8 in hepatocellular carcinoma patients without methylation are good prognosis, but PCDH8 may not be an independent prognostic factor, only AFP level could be used as an independent prognostic factor.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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