【作者】 翟倩倩；

【导师】 吕全军； 鲁凤民；

【作者基本信息】 郑州大学 ， 营养与食品卫生， 2011， 硕士

【摘要】 肝细胞癌(Hepatocellular carcinoma, HCC)是全世界范围内最高发的恶性肿瘤之一,是原发性肝癌的主要类型,占其85%-90%。我国是肝癌的高发区,并且发病率有日渐升高之势。肝细胞癌发生最常见的病因包括HBV、HCV或两者的慢性感染,酗酒和摄入高含量黄曲霉素。肝细胞癌的发生是个长期的、复杂的、多因素相互作用并共同参与的连续过程。其分子生物学机制尚不完全明确。研究发现,Wnt信号通路在成人组织中可能异常活化,从而参与多种肿瘤的发生发展过程。SOX基因家族是Wnt信号通路末端的转录调控因子,是该通路发生不同水平激活的最后共同通道,可通过不同的调节机制影响Wnt通路靶基因的转录和表达,在肿瘤的发生发展中起重要作用。SOX7是SOX家族F亚组的关键成员之一,通过与转录因子TCF/LEF结合的方式抑制下游靶基因转录。已有研究报道,SOX7的异常表达与许多人类肿瘤的发生、发展有关。李正国等发现在48%(10/21)的前列腺癌组织和11种前列腺癌细胞系中SOX7 mRNA表达水平下调。张瑜等人指出在80%(16/20)结肠癌组织和9种结肠癌细胞系中SOX7均低表达。SOX7在原发性乳腺癌、原发性肾癌和原发性肺癌中的mRNA表达水平也明显下调。但是SOX7与HCC关系的研究却很少。目的1.研究SOX7在肝癌细胞系,HCC癌组织和匹配癌旁组织中的mRNA表达水平及其与临床病理参数的关系。2.研究SOX7 mRNA低表达肝癌细胞系和肝癌组织中SOX7 DNA启动子区的甲基化状态。方法1肝癌细胞系北京大学病原微生物学系提供的肝癌细胞系：HepG2,Snu449, SMMC-7721, Snu182, SK-Hep-1, Huh-1, Huh-7, Plc-PRF-5和Hep3B。2研究对象的选择、组织样本的采集及临床病理等相关因素的收集选取2009年5月至2010年4月在河南省肿瘤医院就医的肝癌患者作为本研究的研究对象,采集患者手术后肝癌组织及其匹配的癌旁组织标本40对。所有标本均经病理学鉴定。标本离体后放入-80℃冰箱中保存。收集患者的年龄,门静脉癌栓,肝内播散,肝硬化程度,微血管浸润,肝外转移,肿瘤大小,肿瘤部位等相关因素的资料。3细胞、组织中SOX7 mRNA表达水平的测定采用荧光定量PCR方法测定肝癌患者癌组织及匹配的癌旁组织中SOX7基因mRNA的表达水平,以HMBS作为内参基因。通过测定样本中目的基因模板的Ct值(循环阈值)来反映模板的含量。对每一个样本,用△Ct(CtsOX7-CtHMBs)值表示目的基因与内参基因HMBS间Ct的差值,2-Δct反应样本SOX7 mRNA的表达水平。4样本中SOX7启动子区甲基化水平的测定通过双酶切定量PCR检测Hep3B, SK-Hep-1, Plc-PRF-5, Snu449, Snul82, HepG2, Huh-1和19例SOX7 mRNA低表达肝癌患者癌组织及其匹配的癌旁组织中SOX7甲基化水平。每一标本的HM≥10%定义为发生甲基化,0%<HM<10%定为非甲基化。5统计学分析运用SPSS16.0统计分析软件对数据进行分析。采用Wilcoxon符号秩和检验比较40例肝癌患者癌组织与癌旁组织中的SOX7基因mRNA表达水平,Fisher确切概率法分析表达与临床病理等相关因素的关系。以α=0.05作为检验水准。结果1荧光定量PCR结果1.1 SOX7基因在肝癌细胞系中的mRNA表达水平以正常肝组织中SOX7 mRNA的表达水平为基础值,SOX7 mRNA在HepG2, Snu449, Snu182, SMMC-7721, SK-Hep-1, Huh-1, Huh-7, Plc-PRF-5和Hep3B中的表达有显著的下调趋势。1.2 SOX7 mRNA在肝癌组织及匹配的癌旁组织中的表达水平荧光定量PCR结果显示,与匹配癌旁肝组织相比,在40例样品中,19例肝癌组织中出现SOX7 mRNA表达下调。统计分析表明,肝癌组织中SOX7 mRNA的表达水平低于癌旁组织中SOX7 mRNA的表达水平(z=-1.978,P<0.05)。1.3 SOX7 mRNA在肝癌组织中的表达水平与临床病理等相关因素之间的关系SOX7 mRNA的表达与肝癌患者的一些病理因素有关。大于45岁患者SOX7mRNA表达水平明显低于小于等于45岁年龄组患者的表达水平(χ2=7.866,P<0.05)；肿瘤临床分期越高,SOX7 mRNA表达水平越低(χ2=8.319,P<0.05)；而门静脉癌栓、肝内播散、肝硬化程度与SOX7 mRNA的表达水平无关。1.4定量PCR检测的甲基化结果结果显示,SOX7 DNA启动子区在4株肝癌细胞系Hep3B、SK-Hep-1、HepG2、Huh-1中发生了甲基化,而在SOX7 mRNA表达下调的19例肝癌组织中只发现3例有SOX7 DNA启动子区发生甲基化。结论1. SOX7 mRNA表达水平在肝癌细胞系比正常肝组织表达下调,在肝癌组织比匹配的癌旁组织表达下调。2. SOX7 mRNA表达水平与年龄、临床分期密切相关。3. SOX7 DNA启动子区在肝癌细胞系Hep3B、SK-Hep-1、HepG2、Huh-1发生甲基化,而在SOX7 mRNA表达下调的肝癌组织中发现SOX7 DNA启动子区甲基化现象并不明显。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignant tumors, which is the main type of parimay liver cancer. It accounts for 85-90 percent of primary liver cancer. The incidence of HCC is higher and rapidly increasing in China. The most common causes of HCC include HBV, HCV or both chronic infection, alcohol and high intake of foods containing aflatoxin intake. The occurrence of HCC is a long-term, complex, multi-factor interaction and mutual participation in the continuous process. The molecular biologic mechanism of HCC is not completely clear. Wnt signaling pathway playing an important role in the cancer development was focused by scientists in recent years. Wnt signaling pathway has been reported to be crucial in mammalian embryonic development as a morphogen, which modulated cell proliferation and differentiation. Recently, many studies on tumorigenesis and progression suggested that Wnt signaling pathway plays the crucial role in the cancer development. As the terminal modulator of Wnt pathway, SOX genes which are common pathway of Wnt signaling pathway may play key roles in tumorigenesis and progression by modulating many downstream target genes. SOX7 is the key member of SOX F subgroup, which modulates the target genes through combined with transcription factor TCF/LEF. The recent researches show that the abnormal expression of SOX7 is involved in the tumorigenesis and development of many kinds of human tumors. Li found that the expression level of SOX7 mRNA is down-regulated in 48%(10/21) prostate tissues and 11 kinds of prostate cancer cells. Yu etc pointed out that the expression level of SOX7 mRNA in 80%(16/20) colon cancer tissues and 9 colon cancer cell lines. The expression level of SOX7 mRNA was also obviously down-regulated in primary breast cancer, the primary kidney and primary lung cancer. The relationship between SOX7 expression level and HCC has little studies.Objectives1 To investigate the expression of SOX7 mRNA in Human hepatocarcinoma cell lines and paired primary HCC and their matched adjacent hepatic tissues, and whether the expression levels of SOX7 was correlated with clinical and pathological parameters of HCC patients.2 To study the methylation of SOX7 in Human hepatocarcinoma cell lines and HCC tissues and their matched adjacent hepatic tissues.Methods1 Hepatocarcinoma cell linesHepG2, Snu449, SMMC-7721, Snu182, SK-Hep-1, Huh-1, Huh-7, Plc-PRF-5 and Hep3B were provided by Department of the Pathogenic Microbiology, Peking University.2 Subjects and samplesThe subjects were HCC patients treated in Henan Tumor Hospital from 2009 to 2010. The cancer tissues and matched adjacent tissues of the subjects were collected after operation. All samples were stored in the icebox at-80℃within half an hour. The patients’clinical data such as age, portal vein tumor thrombus, intrahepatic metastasis, microvascular invasion, level of cirrhosis, extrahepatic metastasis, tumor size and tumor location were collected. These data were statistically analyzed.3 Expression of SOX7 mRNA in samplesReal-time PCR was used to determine the mRNA expression level of SOX7 in the hepatocarcinoma cell lines, HCC tissues and matched adjacent tissues. Ct value represents the amounts of mRNA. For each sample,△Ct (Ctsox7-CtHMBs) value represents the difference mRNA expression level between SOX7 and HMBS, which is the internal reference gene.4 Methylation level of SOX7 in samplesWe quantified the methylation level of SOX7 promoter in 7 hepatocarcinoma cell lines,19 cases of paired HCC tissues by using methylation PCR assay. The sample was defined as methylation positive when the level of high methylation was more than 10%, else as methylation negative.5 Data analysis We used Fisher’s Exact Test. Spearman Rank Correlation analytical method has used to compare the expression level of SOX7 mRNA between HCC tissues and matched adjacent tissues. The statistical significance level was 0.05. All statistical analyses were performed using the program SPSS 16.0.Results1 The expression level of SOX7 mRNA in Hepatocarcinoma cell linesThe expression level of SOX7 mRNA was significantly lower than that in normal adjacent liver tissues.2 Expression of SOX7 mRNA in paired HCC tissuesThe expression of SOX7 mRNA is down-regulated in 19 (47.5%) cases of HCC tissues compared with the matched adjacent tissues.The expression level of SOX7 mRNA was significantly lower than that in matched adjacent tissues (z=-1.978, P< 0.05).3 Relationship between expression level of SOX7 mRNA in HCC and the patients clinical pathological characteristicsThe difference of SOX7 mRNA expression level of the different age groups is significant. The expression level of SOX7 mRNA was significantly down-regulation in high stage tumors compared to low stage tumors (χ2=8.319, P<0.05). There were no association between the expression level of SOX7 mRNA and intrahepatic metastasis, portal vein tumor thrombus and level of cirrhosis.4 Methylation results of quantitative PCRMethylation of SOX7 DNA promoter was found in the liver cancer cell lines Hep3B, SK-Hep-1, HepG2, Huh-1 and only 3 cases of 19 liver tissues in which SOX7 mRNA was down regulated.Conclusions1 The expression of SOX7 mRNA level in 7 HCC cell lines was significantly lower than that in the normal liver tissues. The expression level of SOX7 mRNA was significantly lower than that in matched adjacent tissues.2 The difference of SOX7 mRNA expression level among the different age groups is significant. The expression level of SOX7 mRNA was significantly down-regulation in high stage tumors compared to low stage tumors.3 Methylation of SOX7 DNA promoter was found in the liver cancer cell lines Hep3B, SK-Hep-1, HepG2, Huh-1 and less cases in which SOX7 mRNA was down regulated.更多还原