【作者】 张晓梅；

【导师】 杨云生； 郭明洲； 裴雪涛；

【作者基本信息】 中国人民解放军军医进修学院 ， 消化系病， 2010， 博士

【摘要】 背景肝细胞癌(hepatocellular carcinoma, HCC)是我国常见的恶性肿瘤之一严重危害人类健康。肝癌的发病机制很复杂,表观遗传学改变调控的wnt/β-catenin信号通路相关基因失活在肝癌发生中发挥了重要作用。Dact (Homo sapiens dapper, antagonist ofβ-catenin)是wnt/β-catenin信号通路的负性调节基因家族。有研究表明Dactl和Dact3的表达分别受DNA甲基化和组蛋白修饰等表观遗传学调控,具有抑制肿瘤发生的作用。但Dact2与肿瘤的关系研究文献报道很少,在肝癌中的表观遗传学变化及功能尚不清楚。目的1.检测Dact2在永生化肝细胞系和肝癌细胞系中的表达及甲基化改变。2.检测Dact2在正常肝组织、癌旁组织及不同分化程度肝癌组织中的表达及意义。3.检测Dact2在肝癌组织中的甲基化改变。4.探讨肝癌组织中DNA甲基化与Dact2表达之间的关系。5.检测Dact2对肝癌细胞生长的影响。6.探讨Dact2抑制肝癌细胞生长的可能机制。方法1. RT-PCR和Western blot检测细胞系中Dact2在甲基转移酶抑制剂5-aza-dc处理前后mRNA及蛋白的表达。2.甲基化特异性PCR (methylation specific PCR, MSP)检测细胞系和组织标本DNA中Dact2甲基化情况。3.免疫组织化学方法检测Dact2在正常肝组织、癌旁组织及不同分化程度肝癌组织中的表达分布。4.脂质体法转染Dact2真核表达载体,流式细胞分选结合G418筛选瞬时转染Dact2的细胞。5.克隆形成实验及BrdU掺入标记方法分析Dact2对细胞生长的影响6. Hoechst 33258染色分析Dact2对细胞凋亡的影响。结果1. Dact2在肝癌细胞系中的表达及甲基化改变本课题选用5株肝癌细胞系：HepG2、SNU449、SMMC7721、SNU182、PLC-PRF-5和一株永生化的肝细胞系Lo.2。在mRNA水平,Dact2在SNU449细胞系中无表达,在HepG2、SNU182、SMMC7721表达较弱,在Lo.2和PLC-PRF-5细胞中表达正常；5-aza-dc处理后,HepG2、SMMC7721、SNU182细胞中Dact2较未处理时表达上调,而LO.2、PLC-PRF-5和SNU449中Dact2无改变。选取HeoG2和SNU182两株细胞系检测Dact2蛋白的表达,结果显示Dact2蛋白在5-aza-dc处理后上调,与mRNA水平的表达改变一致。MSP检测结果显示lo.2和PLC-PRF-5中Dact2为非甲基化,HepG2、SNU449、SMMC7721、SNU182细胞系中存在甲基化改变,其中,SNU182、SNU449和SMMC7721细胞中同时有甲基化和非甲基化条带。Dact2启动子甲基化改变与基因表达呈对应关系。2. Dact2在肝癌组织标本中的表达及甲基化情况在表达部位上,Dact2在正常肝组织、癌旁组织和癌组织中表达均在细胞浆；在表达强度上,肝癌组织内Dact2表达较癌旁明显减低,两组比较有统计学意义(P<0.001)；在高分化和中分化肝癌组织中Dact2的表达显著高于低分化组(P＜0.05),而与患者的年龄、姓别、肿瘤大小及HBV相关性肝癌无显著相关。38例肝癌组织中的Dact2甲基化率为60.5%；在16例表达下调的肝癌标本中检测到Dact2甲基化的有11例(68.75%)。3.恢复Dact2表达对肿瘤细胞生长的影响克隆形成实验结果显示恢复Dact2表达后hepG2细胞的克隆数量明显少于对照转染的空载体,Dact2对细胞长期生长抑制作用大于空载体的2倍。Brdu掺入标记后转染Dact2的阳性细胞为39.63%,明显少于空质粒组48.32%,两组比较有显著差异(P<0.05)。4. Hoechst 33258染色分析Dact2对细胞凋亡的影响转染Dact2后HepG2的凋亡率为10.9%,而转染空质粒的凋亡率4.9%；SNU449细胞转染Dact2后细胞凋亡率为12.30%,转染空质粒的凋亡率12.10%,提示Dact2对细胞凋亡无明显影响。结论本课题检测了肝癌中Dact2的表达及DNA甲基化改变,5-aza-dc处理细胞系能恢复dact2的表达,表明在肝细胞癌中Dact2的表达受表观遗传学调控；恢复表达Dact2能够明显抑制肝癌细胞的增殖,但对凋亡无明显影响。以上结果为进一步研究Dact2在肝癌诊断和靶向治疗中的作用奠定了基础。更多还原

【Abstract】 BackgroundHepatocellular carcinoma (HCC) is one of the most common cancer in our country. Its pathogenesis remains unclear. Epigenetic silence of wnt/β-catenin pathway associated genes plays important roles during HCC carcinogensis. Recent studies have demonstrated that Dact1 and Dact3 (homo sapiens dapper, antagonist of P-catenin) were epigenetic regulator of wnt/β-catenin pathway in HCC and colorectal cancer respectively. However, Dact2 epigenetic changes in HCC remains unclear.Aims1. To detect Dact2 expression and DNA methylation in immortal liver cell line and HCC cell lines.2. To examine Dact2 expression in normal liver, HCC and adjacent normal tissues.3. To detect DNA methylation of Dact2 in HCC tissues.4. To investigate the relationship of Dact2 expression and DNA methylation.5. To study the effects of Dact2 on cell growth.6. To clarify the possible mechanisms of Dact2 on cell growth.Methods1. RT-PCR and Western blot were performed to detect Dact2 expression in immortal liver cell line and HCC cell lines before and after 5-aza-dc treatment.2. Methylation specific PCR (MSP) was used to detect DNA methylation of Dact2 in HCC cell lines and tissues.3. Immunohistochemisty was performed to examine Dact2 expression in normal liver, HCC and adjacent normal tissues.4. LipofectamineTM 2000 was used to transfect eukaryotic expression vector into HCC cell lines. FCM and G418 were used to select transient transfection cells.5. Colony formation assay and BrdU incorporation assay were used to detect cell growth of transfected cell.6. Apoptosis was analyzed by nuclear staining with Hoechst 33258.Results1. HepG2, SMMC7721, SNU449, SNU182, PLC-PRF-5 and LO.2 cell lines were supplied by Gastroenterology Department of PLA General Hospital. Dact2 expression was detected in Lo.2 and PLC-PRF-5, and down regulated in HepG2, SNU182 and SMMC7721. And Dact2 was silenced in SNU449. Promoter region methylation was revealed by MSP in HepG2, SNU449, SMMC7721 and SNU182 cells. Dact2 expression can be reversed in HepG2, SNU449, SMMC7721 and SNU182 cells after treatment with 5-aza-dc.2. Cytoplastic positive staing of Dact2 was found in normal, adjacent tissues and cancer tissues, but was downregulated in cancer tissues (P<0.001). Significant different of Dact2 expression was found among tumor differentiation levels (P<0.001). Dact2 methylation was exhibited in 23 (60.5%) of 38 HCC cases. Among 19 pairs of tissues,16 were found to lose Dact2 expression compared with their matched normal HCC tissues. Eleven of these 16 cases had Dact2 hypermethylation in tumors (68.75%).3. Reexpression of Dact2 resulted in significant suppression of long-term cell growth more than 2-folds by colonal formation assays. In BrdU incorporation assay, the percentage of positive cells nuclei is decreased to 36.49% compare with control group (51.96%) (P<0.05).4. Effects of Dact2 on HCC cell apoptosis. Dact2 has no influence on apoptosis in HepG2 and SNU449 cell lines.In conclusion, Dact2 expression is silenced by promoter region hypermethylation in HCC. Restoration of Dact2 expression was induced by 5-aza-dc treatment. Forced expression of Dact2 in methylated HepG2 cell line result in growth suppression, but no apoptosis changes was found in HepG2 cell.更多还原