【作者】 王佳辰；

【导师】 王家祥；

【作者基本信息】 郑州大学 ， 外科学， 2007， 硕士

【摘要】 肝癌是人体常见的致命性肿瘤，预后差，严重威胁人类的健康，因此深入研究肝癌的发生发展机制，进而加强肝癌的防治具有重要意义。随着分子生物学的发展，已逐渐认识到表遗传学(Epigenetics)调节与肿瘤基因的转录调控密切相关；因为这种表遗传学调节不改变基因编码序列，并且是可逆的，因而越来越受到重视。研究表明，表遗传学或／和遗传学改变均可引起肿瘤的发生是一个涉及多种肿瘤基因功能改变的多步骤过程，其中肿瘤抑制基因的功能失活为细胞的恶性转化所必需。由DNMTs催化完成的DNA甲基化是DNA的一种天然修饰方式，是表遗传学主要内容之一，其在基因的表达调控、胚胎发育调节、基因组印记、X染色体失活及肿瘤的发生等许多方面发挥着重要的作用。在DNMTs家族中，DNMT3b的作用最为重要。DNMT3b主要表达于未分化的胚胎干细胞，催化DNA的从头甲基化，在正常体细胞中表达很低。CPG岛是公认的基因表达调控区域。大约50％的CPG岛位于基因启动子区域。在正常细胞大部分的启动子区域CPG岛是未甲基化的。由DNMT3b催化的肿瘤抑制基因启动子区CPG岛的甲基化反应，使CPG岛甲基化状态改变导致该基因表达沉默，进而引起肿瘤的发生。我们的研究也发现，在肝细胞恶性转化过程中，FHIT基因(一重要的肿瘤抑制基因)的表达明显受抑制，用siRNA抑制DNMT3b表达后，FHIT基因又恢复表达，但这一过程中，FHIT基因启动子区域CpG岛并未被甲基化，由此提示在肝细胞恶性转化过程中DNMT3b除发挥DNA甲基转移酶作用外尚有转录调控因子的作用。事实上，DNMT3b具有PWWP和PHD结构域，而这些结构域与蛋白质-蛋白质间的相互作用有关，说明DNMT3b已经具备了作为信号传导分子的基础。以前的研究多集中于DNMT3b作为酶的功能，而其作为转录调控因子的功能目前尚未见报道。信号转导子和转录激活子(Signal transducers and activators of transcription，STATs)是一重要的核转录因子．STATs家族成员参与了许多细胞因子、生长因子的信号传导，在调节人体免疫反应、炎症反应和细胞的生长、分化、凋亡等方面发挥着重要作用，与人类恶性肿瘤的发生、发展和演进有关。据文献报道，STATs及其下游基因Bcl-2、CyclinD1、c-myc、VEGF、P21等在肝癌组织中异常表达，而STATs在肿瘤的发生发展中，启动子区甲基化状态不变。有研究表明：STATs及其下游基因在肿瘤的发生、发展过程中起到重要作用。如：Bcl-2、CyclinD1、c-myc、VEGF都是重要的癌基因。它们在肝细胞肝癌的发生、增殖、分化、凋亡、血管生成和侵袭转移等多方面都发挥了重要作用。如果能够证实DNMT3b与STATs信号传导通路相关联，则提示DNMT3b-STATs信号传导通路的存在，这就说明DNMT3b即可以作为DNA甲基转移酶催化基因的甲基化，影响基因的表达，又可以作为转录调控因子发挥作用。这就进一步揭示了DNMT3b功能的多样性，为肿瘤的基因治疗提供新的靶点，为肝癌的防治提供新思路。方法1、传代培养SMMC7721细胞；2、将终浓度为50nM的DNMT3bsiRNA转染到肝癌细胞SMMC7721中：3、在SMMC7721细胞转染DNMT3bsiRNA 48h后，收集转染细胞和未转染细胞提取细胞质蛋白或总蛋白，用Western blotting检测DNMT3b的表达改变；4、在SMMC7721细胞转染DNMT3bsiRNA前及转染24、48h后，用细胞计数板计数转染细胞和未转染细胞的细胞数量；5、用Western blotting检测STATs及其下游基因c-myc，VEGF，CyclinD1等的表达改变情况；6、处理所得数据，得出结论。结果1、在SMMC7721细胞转染DNMT3bsiRNA 48h后，通过Western Blotting检测DNMT3b的表达发现：转染细胞的DNMT3b表达水平比未转染细胞明显降低。2、转染细胞的生长速度与未转染细胞相比明显受到抑制，统计学上有显著性差异(P＜0.05)，同时有大量死亡细胞存在。3、转染细胞的STAT1、STAT3、STAT5及其下游基因VEGF、c-myc、CyclinD1的表达水平与未转染细胞相比均降低。结论1、DNMT3bsiRNA的建立可以方便地研究DNMT3b的功能。通过DNMT3bsiRNA特异性的抑制DNMT3b的表达。2、在SMMC7721细胞体系中存在DNMT3b-STATs信号传导通路，抑制DNMT3b的表达可以同时下调STAT1、STAT3、STAT5及其下游基因VEGF、c-myc、CyclinD1等的表达。3、DNMT3b的表达改变，可以引起细胞周期蛋白CyclinD1的表达改变，使肿瘤细胞停滞在G1期，抑制肝癌细胞的增殖，促进细胞的凋亡。更多还原

【Abstract】 Human hepatocellular carcinoma is one of common fatal cancers and has poor prognosis. It seriously threaten human healthc,so deeply studying on the development of hepatocellular carcinoma and strengthening the liver cancer prevention have great significance.With the development of molecular biology,we have gradually come to realize that epigenetic regulation and transcriptional regulation of cancer gene are closely related. This adjustment does not change epigenetic gene coding sequence and is reversible, so the scientis pay more attention to this adjustment. Research shows that epigenetic and / or genetic changes may cause cancer. This is a multi-tumor gene function in the multi-step process. The inactivation of tumor suppressor gene function is necessary for the malignant transformation of cells. DNA methylation which is catalyzed by various methyltransferase enzymes (DNMTs)is a natural DNA modification method, is one of the main content of epigenetics. DNA methylation play an important role in many aspects such as regulating gene expression, regulating embryonic development, genomic imprinting, X chromosome inactivation and the incidence of tumors.In DNMTs family, DNMT3b is the most important one. DNMT3b expressed in the Undifferentiated embryonic stem cells catalyzes the DNA methylation, but in normal cells the expression is very low. CpG islands are recognized as an important regulatory mechanism for the gene expression. About 50% of CpG islands are located in promoterregions. Most of promoter-associated CpG islands are unmethylated in normal cells.The switching from unmethylated to methylated CpG islands is recognized as an essential contributor to gene silencing which associated with a loss of gene function .This process is catalyzed by DNMT3b and eventually lead to the occurrence of cancer. Our study also found that in the process of malignant transformation of hepatic cells, the expression of FHIT gene (an important tumor suppressor gene) is significantly inhibited. Using siRNA to inhibit the expression of the DNMT3b, the FHIT gene’s expression is also restored. But in this process,the CpG island of FHIT gene’s promoter region was not methylated. The results suggest that in the process of malignant transformation of hepatic cells DNMT3b not only play the role of DNA methyltransferase, but also play the role of transcriptional regulation factors. In fact, DNMT3b has PWWP and PHD domains. These domains are relevant with the protein-protein interactions. DNMT3b have had the basis of signal transduction molecules. Previous studies focused on DNMT3b as enzyme, but its function as a transcriptional regulatory factor has not been reported until now.Signal transducers and activators of transcription (STATs) are the important nuclear transcription factor. STATs family have many members. They play an important role, such as the signal transduction of many cytokines and growth factors and in the regulation of human immune response inflammatory response and cell growth, differentiation, apoptosis. STATs also have the functions of correlation with the growth, proliferation and evolution in human cancers. It also has been reported that the expression of STATs and its downstream gene such as Bcl-2, Cyclin D1, c-myc, VEGF, P21 are abnormal in hepatocellular carcinoma tissue, but the CpG island of STATs gene promoter region are invariably. Researches show that STATs and the downstream genes are great signficance in the development process in tumors. Bcl-2, Cyclin D1, c-myc and VEGF are very important cancer genes. They are great significance in many aspects of the hepatocellular carcinoma such as proliferation, differentiation, apoptosis, angiogenesis, invasion and metastasis and other functions.If we can certificate DNMT3b is associated with STATs signal transduction pathway, this suggested that DNMT3b-STATs signaling pathway is the existence and DNMT3b not only can be used as DNA methyltransferase to catalyze methylation of the gene, affect gene expression but also can play a role as a transcriptional regulatory factor. This will further reveals the diversity of the function of DNMT3b and gives us a new target for cancer gene therapy and provide new ideas for the prevention of hepatocellular carcinoma.Methods:1. Culture SMMC7721 cell line;2. SMMC7721were transfected with DNMT3bsiRNA transfection of hepatoma cells, the final concentration is 50nM;3. After transfected with or without DNMT3bsiRNA at 48h, collection transfected cells and untransfected cells and then cytoplasmic extract the total protein. Western blotting detection DNMT3b expression changes;4. Transfected with or without DNMT3bsiRNA before and after 24 or 48h, Use cell counting plate to count transfected cells and untransfected cell;5. Using Western blotting to detect the expression of STATs and its downstream genes,such as VEGF, Cyclin D1 expression, etc;6. Dealing with the data and coming to a conclusion.Results:1. After transfection with the DNMT3bsiRNA 48h, we use western blotting to detect the expression of DNMT3b in SMMC7721 cell line.we found that DNMT3b expression in transfected cells was significantly lower than untransfected cells and normal cells.2. Compared with the untransfected cells, the growth rate of transfected cells were inhibited. The difference was significant (P <0.05), while there are a large number of dead cells.3. Compared with the untransfected cells, the expression levels of STAT1, STAT3, STAT5 and its downstream Cyclin D1, c-myc, VEGF gene were also decreased ikin the transfected cells.Conclusion:1. The establishment of DNMT3bsiRNA can make us easily study the function of DNMT3b. 2. Finding that the signal transduction pathways DNMT3b-STATs exist in SMMC7721 cell line. Down regulation the expression of DNMT3b can inhibit the expression of STAT1, STAT3, STAT5 and theirs downstream gene Cyclin D1, c-myc, VEGF.3. Finding that down regulation the expression of DNMT3b, can induce the changes in the expression of Cyclin D1 protein, make tumor cells stagnated in G1 phase and inhibit the cell proliferation and apoptosis in hepatocellular carcinoma.更多还原