类固醇激素受体辅激活子3（SteroidReceptorCoactivator，SRC-3）是一种具有多种生理功能的转录协同因子，它能够与核受体等多种转录因子相互作用而增强它们的转录活性。我们的前期研究发现在68％人肝细胞癌（hepatocellularcarcinoma，HCC）组织中存在着癌基因SRC-3的高表达，并且SRC-3能促进肝癌细胞的增殖、侵袭、成瘤，这表明SRC-3是在发生发展中起重要作用的癌基因。 乙型肝炎病毒X蛋白（hepatitisBvirusXprotein，HBx）是一种多效转录激活因子，能和细胞内多种与转录、基因调控有关的因子结合，广泛激活病毒与细胞的启动子。体内...Steroid Receptor Coactivator, SRC-3, is a transcriptional coactivator with multiple physiological functions. It can interact with a variety of transcription factors including nuclear receptor to enhance their transcriptional activity. Our previous study have shown that overexpression of SRC-3 is detected in 68% of human hepatocellular carcinoma （HCC） tissues, and SRC-3 can accelerate tumor cell pr...学位：理学硕士院系专业：生命科学学院生物学系_细胞生物学专业学号：2162007115192

李婷

Xiamen University Institutional Repository

 学校编码：10384                                 分类号    密级        学号：21620071151925                                        UDC                硕  士  学  位  论  文     乙型肝炎病毒 X 蛋白与 SRC-3 相互作用的研究 The Interaction Between Hepatitis B virus X Protein And SRC-3 李 婷 指导教师姓名：俞春东 教授、博导 专  业 名 称：细胞生物学 论文提交日期：2010 年 4 月 论文答辩时间：2010 年 6 月 学位授予日期：2010 年  月 答辩委员会主席：          评阅人：          2010 年  月厦门大学博硕士论文摘要库                        厦门大学博硕士论文摘要库  厦门大学学位论文原创性声明  本人呈交的学位论文是本人在导师指导下,独立完成的研究成果。本人在论文写作中参考其他个人或集体已经发表的研究成果，均在文中以适当方式明确标明，并符合法律规范和《厦门大学研究生学术活动规范（试行）》。 另外，该学位论文为（                            ）课题（组）的研究成果，获得（               ）课题（组）经费或实验室的资助，在（               ）实验室完成。（请在以上括号内填写课题或课题组负责人或实验室名称，未有此项声明内容的，可以不作特别声明。）  声明人（签名）：           年   月   日                 厦门大学博硕士论文摘要库                                             厦门大学博硕士论文摘要库  厦门大学学位论文著作权使用声明  本人同意厦门大学根据《中华人民共和国学位条例暂行实施办法》等规定保留和使用此学位论文，并向主管部门或其指定机构送交学位论文（包括纸质版和电子版），允许学位论文进入厦门大学图书馆及其数据库被查阅、借阅。本人同意厦门大学将学位论文加入全国博士、硕士学位论文共建单位数据库进行检索，将学位论文的标题和摘要汇编出版，采用影印、缩印或者其它方式合理复制学位论文。 本学位论文属于： （     ）1.经厦门大学保密委员会审查核定的保密学位论文，于   年  月  日解密，解密后适用上述授权。 （     ）2.不保密，适用上述授权。 （请在以上相应括号内打“√”或填上相应内容。保密学位论文应是已经厦门大学保密委员会审定过的学位论文，未经厦门大学保密委员会审定的学位论文均为公开学位论文。此声明栏不填写的，默认为公开学位论文，均适用上述授权。）                                声明人（签名）： 年   月   日     厦门大学博硕士论文摘要库                                             厦门大学博硕士论文摘要库目录 I 目录  摘 要.................................................... 1 Abstract................................................. 3 前 言.................................................... 5 1. 类固醇激素受体辅激活子 SRC-3 ............................. 5 1.1 类固醇激素受体辅激活子 SRC-3 的发现 ..........................................5 1.2 类固醇激素受体辅激活子 SRC-3 的结构 ..........................................5 1.3 类固醇激素受体辅激活子 SRC-3 基因的定位与表达 ......................6 1.4 类固醇激素受体辅激活子 SRC-3 的主要功能 ..................................6 1.5 类固醇激素受体辅激活子 SRC 家族在肝脏中的表达及调控 .........8 2. 乙型肝炎病毒 X 蛋白（Hepatitis B virus X protein， HBx） .9 2.1 乙型肝炎病毒 X 基因的结构 ............................................................10 2.2 乙型肝炎病毒 X 蛋白的表达与定位 ................................................11 2.3 乙型肝炎病毒 X 蛋白的功能 ............................................................12 2.3.1 乙型肝炎病毒 X 蛋白的反式激活作用 ...................................12 2.3.2 乙型肝炎病毒 X 蛋白激活细胞质内信号转导通路 ...............12 2.3.3 乙型肝炎病毒 X 蛋白对细胞周期的影响 ...............................13 2.3.4 乙型肝炎病毒 X 蛋白与细胞凋亡 ...........................................14 2.3.5 乙型肝炎病毒 X 蛋白与 DNA 修复.........................................14 2.3.6 乙型肝炎病毒 X 蛋白与 DNA 甲基化.....................................15 2.3.7 乙型肝炎病毒 X 蛋白促进细胞侵袭转移 ...............................15 2.4 乙型肝炎病毒 X 蛋白与肝细胞癌 HCC...........................................16 3. 本文的目的、内容和意义 ................................. 17 材料与方法.............................................. 18 1. 实验材料 ............................................... 18 1.1 主要试剂及药品 .................................................................................18 厦门大学博硕士论文摘要库目录 II 1.2 主要设备及器材 .................................................................................19 1.3 细胞株 .................................................................................................20 1.4 菌株 .....................................................................................................20 1.5 质粒载体 .............................................................................................20 2. 实验方法 ............................................... 21 2.1 大肠杆菌感受态制备 .........................................................................21 2.2 克隆构建 .............................................................................................21 2.2.1 设计引物 ....................................................................................21 2.2.2 PCR 扩增含 HBx 全长基因的片段 ..........................................22 2.2.3 PCR 扩增产物的琼脂糖凝胶电泳分析及回收纯化...............22 2.2.4 双酶切 PCR 产物和载体...........................................................22 2.2.5 连接含粘性末端的全长 DNA 和载体......................................23 2.3 质粒（连接产物）转化 .....................................................................23 2.4 质粒提取 .............................................................................................24 2.4.1 质粒小量提取 ............................................................................24 2.4.2 QIAGEN-tip 100 中量提取质粒 ..............................................24 2.5 细胞培养及药物处理 .........................................................................26 2.6 细胞转染 .............................................................................................26 2.6.1 脂质体转染 ................................................................................26 2.6.2 磷酸钙转染 ................................................................................27 2.7 蛋白提取与 Western blotting..............................................................27 2.7.1 蛋白提取 ....................................................................................27 2.7.2 BCA（bioinchoninic acid，二羧基二喹啉）测蛋白浓度 ......27 2.7.3 Western blotting..........................................................................28 2.8 免疫共沉淀 .........................................................................................29 2.9 核浆蛋白分离 .....................................................................................30 2.10 荧光素酶活性测定实验 .....................................................................31 2.11 总 RNA 提取，反转录（Reverse Transcription，RT）PCR 及实时定量荧光 PCR（Real-time PCR）...................................................................31 厦门大学博硕士论文摘要库目录 III 2.11.1 细胞样品 RNA 的提取..............................................................31 2.11.2 反转录 cDNA.............................................................................32 2.11.3 Real-time PCR............................................................................32 结果与分析.............................................. 34 1． HBx 基因的真核表达载体的构建及表达 ...................... 34 1.1 HBx 基因的扩增 ................................................................................34 1.2 重组质粒的双酶切鉴定 .....................................................................34 1.3 pCR3.1-HA-HBx 表达载体在真核细胞中的表达与定位................35 2． HBx 瞬时转染引起 SRC-3 蛋白水平的上调.................... 37 3． HBx 增强 SRC-3 蛋白稳定性................................ 39 4． HBx 与 SRC-3 之间存在相互作用............................ 41 5． HBx 与 SRC-3 的协同作用.................................. 43 讨论.................................................... 47 参考文献................................................ 50 致谢.................................................... 60                厦门大学博硕士论文摘要库目录 IV                    厦门大学博硕士论文摘要库Contents V Contents  Abstract (in Chinese) .................................... 1 Abstract (in English) .................................... 3 Introduction ............................................. 5 1. Steroid Receptor Coactivator - 3 (SRC-3) ................ 5 1.1 The discovery of SRC-3 ........................................................................5 1.2 The structure of SRC-3..........................................................................5 1.3 The positioning and expression of SRC-3.............................................6 1.4 The main function of SRC-3 .................................................................6 1.5 The expression and regulation of SRC family in the liver ....................8 2. Hepatitis B virus X protein，HBx ........................ 9 2.1 The structure of HBx...........................................................................10 2.2 The positioning and expression of HBx ..............................................11 2.3 The function of HBx............................................................................12 2.3.1 The transactivation of HBx.........................................................12 2.3.2 HBx activates cytoplastic signaling pathway .............................12 2.3.3 HBx affects the cell cycle ...........................................................13 2.3.4 HBx and cell apoptosis ...............................................................14 2.3.5 HBx and DNA repairing .............................................................14 2.3.6 HBx and DNA methylation ........................................................15 2.3.7 HBx accelerates cell invasion and migration .............................15 2.4 HBx and HCC .....................................................................................16 3. Purpose of this thesis ................................. 17 Materials and Methods ................................... 18 1. Materials .............................................. 18 1.1 Main reagents ......................................................................................18 1.2 Main equipment...................................................................................19 厦门大学博硕士论文摘要库Contents VI 1.3 Cell lines..............................................................................................20 1.4 Bacterial strain.....................................................................................20 1.5 Plasmids...............................................................................................20 2. Methods ................................................ 21 2.1 Competent cell.....................................................................................21 2.2 Clone construction...............................................................................21 2.2.1 Primer design..............................................................................21 2.2.2 PCR amplification of full-length gene fragments of HBx..........22 2.2.3 The analysis and purification of PCR products ..........................22 2.2.4 Double digestion of PCR product and the vector .......................22 2.2.5 Ligation.......................................................................................23 2.3 Transformation ....................................................................................23 2.4 Plasmids extraction..............................................................................24 2.4.1 Plasmid miniprep........................................................................24 2.4.2 Purification of the plasmid（QIAGEN-tip 100）......................24 2.5 Cell culture and drug treatment ...........................................................26 2.6 Cell transfection ..................................................................................26 2.6.1 Lipofectin transfection................................................................26 2.6.2 Calcium phosphate transfection..................................................27 2.7 Protein extraction and Western blotting ..............................................27 2.7.1 Protein extraction........................................................................27 2.7.2 Protein concentration measurement............................................27 2.7.3 Western blotting..........................................................................28 2.8 Co- immunoprecipitation ....................................................................29 2.9 Separation of protein in cytoplasm and nucleolus...............................30 2.10 Luciferase assay ..................................................................................31 2.11 RNA extraction, reverse transcription PCR and Real-time PCR ........31 2.11.1 RNA extraction with cell sample................................................31 2.11.2 Reverse Transcription cDNA......................................................32 2.11.3 Real-time PCR............................................................................32 厦门大学博硕士论文摘要库Contents VII Results................................................. 34 1． The construction of HBx eukaryotic expression vector and gene expression .................................................... 34 1.1 Amplification of HBx gene .................................................................34 1.2 Double digestion of HBx expression vector........................................34 1.3 The expression and localization of HBx in eukaryotic cell.................35 2． HBx upregulates SRC-3 protein level .................... 37 3． HBx enhances the protein stability of SRC-3 ............ 39 4． HBx interacts with SRC-3 directly ...................... 41 5． HBx cooperates with SRC-3 .............................. 43 Discussion.............................................. 47 Reference............................................... 50 Acknowledgement ......................................... 60                     厦门大学博硕士论文摘要库摘要 1 摘 要   类固醇激素受体辅激活子 3（Steroid Receptor Coactivator，SRC-3）是一种具有多种生理功能的转录协同因子，它能够与核受体等多种转录因子相互作用而增强它们的转录活性。我们的前期研究发现在 68％人肝细胞癌（hepatocellular carcinoma，HCC）组织中存在着癌基因 SRC-3 的高表达，并且 SRC-3 能促进肝癌细胞的增殖、侵袭、成瘤，这表明 SRC-3 是在发生发展中起重要作用的癌基因。 乙型肝炎病毒 X 蛋白（hepatitis B virus X protein，HBx）是一种多效转录激活因子，能和细胞内多种与转录、基因调控有关的因子结合，广泛激活病毒与细胞的启动子。体内外的研究表明，HBx 在肝癌的发生发展过程中具有重要作用。 为了更清楚地认识和了解HBx与SRC-3是否能够协同促进肝癌的发生发展，我们构建了HBx表达载体，并且在HEK293T细胞中表达。在本文的研究中我们发现，在HEK293T细胞中，过表达HBx可在不影响SRC-3 mRNA水平的情况下显著提高其蛋白水平，另外在HBx存在的情况下，SRC-3的蛋白降解减缓，这说明HBx蛋白的存在可能抑制了SRC-3的降解，增强了其蛋白质稳定性。根据免疫共沉淀的实验结果显示，HBx与SRC-3之间存在直接的蛋白相互作用。在肝癌细胞HepG2中，HBx能与SRC-3协同激活NF-κB、AP-1等转录因子以增强靶基因MMP-9等的表达。根据以上这些研究结果，我们认为HBx可能通过与SRC-3之间的相互作用来抑制SRC-3蛋白的降解以及通过与SRC-3协同激活在肝癌中起重要作用的转录因子，促进肝癌的发生发展。   关键词：肝细胞癌（hepatocellular carcinoma, HCC）；乙型肝炎病毒X 蛋白(Hepatitis B virus X protein，HBx)；类固醇激素受体辅激活子-3（SRC-3）    厦门大学博硕士论文摘要库摘要 2   厦门大学博硕士论文摘要库Abstract 3 Abstract  Steroid Receptor Coactivator, SRC-3, is a transcriptional coactivator with multiple physiological functions. It can interact with a variety of transcription factors including nuclear receptor to enhance their transcriptional activity. Our previous study have shown that overexpression of SRC-3 is detected in 68% of human hepatocellular carcinoma （HCC） tissues, and SRC-3 can accelerate tumor cell proliferation, invasion and tumorigenesis, suggesting that SRC-3 plays an important role in the progression of HCC.  Hepatitis B virus X protein (HBx) is a multifunctional transcriptional activator. It can bind to multiplicate intracellular factors involved in transcription and gene regulation, and activate a wide range of genes. Studies in vivo and in vitro have shown that, HBx has an important role in the development and progression of liver cancer.  To determine whether HBx can cooperate with SRC-3 to promote HCC progression, we constructed a HBx expression vectors and transfected in HEK293T cells. Overexpression of HBx can significantly increases the protein level of SRC-3 significantly, without affecting its mRNA level. The presence of HBx protein may inhibit the degradation of SRC-3 and thus enhance its protein stability. Co-immunoprecipitation experiment results showed that, HBx could interact with SRC-3. In HepG2 cells, HBx could cooperate with SRC-3 to activate AR, NF-κB, AP-1 and other transcription factors, to enhance target gene expression of MMP-9. Our results demonstrate that HBx can inhibit the degradation of SRC-3 protein and cooperate with SRC-3 to promote the development of HCC.    Key word: HCC(hepatocellular carcinoma), Hepatitis B virus X protein (HBx), Steroid Receptor Coactivator (SRC-3)  厦门大学博硕士论文摘要库Degree papers are in the “Xiamen University Electronic Theses and Dissertations Database”. Fulltexts are available in the following ways: 1. If your library is a CALIS member libraries, please log on http://etd.calis.edu.cn/ and submitrequests online, or consult the interlibrary loan department in your library. 2. For users of non-CALIS member libraries, please mail to etd@xmu.edu.cn for delivery details.厦门大学博硕士论文摘要库

The Interaction Between Hepatitis B virus X Protein And SRC-3

http://dspace.xmu.edu.cn/bitstream/handle/2288/46602/%e4%b9%99%e5%9e%8b%e8%82%9d%e7%82%8e%e7%97%85%e6%af%92%20X%e8%9b%8b%e7%99%bd%e4%b8%8eSRC-3%e7%9b%b8%e4%ba%92%e4%bd%9c%e7%94%a8%e7%9a%84%e7%a0%94%e7%a9%b6.pdf?sequence=1&isAllowed=y

The Interaction Between Hepatitis B virus X Protein And SRC-3

Abstract

Similar works

Full text

Available Versions

Xiamen University Institutional Repository