【作者】 卢倩；

【导师】 董家鸿；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 【研究背景与目的】乙型肝炎病毒(hepatitis B virus,HBV)慢性感染是肝细胞癌(hepatocellular carcinoma,HCC)发病的独立高危因素。整合的HBV-DNA基因组编码的乙型肝炎病毒X蛋白(HBx)是一种重要的反式作用因子,能广泛激活多种原癌基因的转录表达,是肝细胞恶性转化的重要原因。HBx蛋白可干扰癌细胞间的粘附连接,促进细胞外基质降解,诱导肝癌细胞发生上皮间叶表型转化(epithelial-mensenchymal transition,EMT),促进肝细胞癌侵袭转移。上皮钙粘附素(E-cadherin)表达缺失是EMT的重要标志。组织病理和免疫化学染色分析发现,E-cadherin表达缺失的HCC分化程度低,恶性程度高,更易发生肝内转移和门静脉侵犯,E-cadherin的表达缺失与HCC临床预后密切相关。E-cadherin编码基因CDH1的转录沉默是导致其表达缺失重要机制。Snail1蛋白是锌指蛋白Snail超家族的成员,具有强大的EMT诱导能力。借由其羧基末端的锌指结构与CDH1核心启动子区的E-box基序结合,Snail1蛋白能有效遏制后者的转录表达,造成E-cadherin表达缺失,诱导EMT发生。基于以上的研究背景,我们推测:HBx蛋白有可能通过激活肝癌细胞的Snail1编码基因SNAI1转录,上调Snail1蛋白表达,进而诱导肝癌细胞发生EMT,提高其侵袭运动能力。我们希望能够通过本研究明确,在HBx促进肝细胞癌侵袭转移过程中,Snail1蛋白发挥了关键的介导作用,并初步探讨HBx对SNAI1基因的转录调控机制。【材料与方法】一、采用免疫组织化学染色的方法,检测74例肝细胞癌组织标本中HBx、Snail1、E-cadherin、N-cadhe、Vimentin的表达,并分析之间的相关性。收集上述病例的临床病理特征,分析HBx、Snail1表达与HCC临床病理之间的联系。初步明确:1、Snail1蛋白是否与肝细胞癌临床病理分期及恶性程度相关;2、HBx与Snail1表达之间是否存在相关性。二、构建SNAI1基因重组腺病毒表达载体Ad-SNAI1。利用Ad-SNAI1高效感染肝癌细胞株HepG2,诱导外源性Snail1蛋白过表达,观察Snail1蛋白对肝癌细胞株上皮表型及侵袭运动能力的影响。三、利用我所改建并保存的HBx基因重组腺病毒表达载体Ad-HBx感染无HBV感染背景的肝癌细胞株HepG2。观察外源性HBx基因的介入是否能诱导肝癌细胞株内源性Snail1蛋白表达增高,及其对肝癌细胞上皮表型及侵袭运动能力的影响。四、利用RNAi技术沉默肝癌细胞株内源性SNAI1转录表达,观察内源性Snail1表达缺失是否能逆转HBx对肝癌细胞株EMT的诱导。为此,首先设计并筛选出有效的RNA干扰序列,并委托上海吉玛制药技术有限公司合成pGPU6/GFP/Neo-shSNAI1。为了获得高效的转染效率以保证shSNAI1导入HepG2细胞,将pGPU6/GFP/Neo-shSNAI1质粒改造为重组腺病毒载体Ad-shSNAI1。Ad-shSNAI1和Ad-HBx共转染HepG2细胞株,观察细胞表型及侵袭运动能力的改变。五、构建5’端裁剪的SNAI1系列启动子pGL3-Basic报告基因质粒。以海肾荧光素酶质粒(pRL-TK Vector)为内参质粒,转染肝癌细胞株SMMC-7721。比照在共转染空腺病毒载体和Ad-HBx条件下,各启动子区的转录活性,确定HBx蛋白是否能激活SNAI1转录活性,并初步明确其调节区段。【结果】一、74例肝癌组织标本中,58例HBx表达阳性,47例Snail1蛋白表达阳性。45例同时表达HBx、Snail1,两者表达显著相关,p=0.002。HBx表达阳性与E-cadherin表达缺失、N-cadherin表达增强相关,Snail1表达阳性与E-cadherin表达缺失、N-cadherin、Vimentin表达增强相关。结合临床病理特点分析提示:HBx表达阳性与HCC TNM分期、肿瘤大小、门静脉侵犯显著相关,Snail1表达阳性与HCC TNM分期,肿瘤大小、门静脉侵犯、肝周侵犯和外周血HBV-DNA活跃复制相关。提示:Snail1可作为肝细胞癌具有较强侵袭转移能力的分子标志,Snail1的表达增高极有可能与HBV感染有关。二、成功构建SNAI1重组腺病毒载体Ad-SNAI1。Ad-SNAI能高效感染HepG2细胞,SNAI1mRNA和Snail1蛋白表达显著上升。HepG2细胞外源性Snail1蛋白的过表达,造成E-cadherin表达缺失,N-cadhrein、Vimentin表达明显上调;随机运动实验、侵袭实验和趋化运动实验证实,肿瘤细胞运动和侵袭能力显著增强。三、HepG2细胞株转入外源性HBx基因后,HBx蛋白能有效表达。RT-PCR,Western blot检测发现,HBx蛋白能诱导SNAI1 mRNA和Snail1蛋白表达水平明显上调。HepG2细胞E-cadherin表达受到显著抑制,N-cadherin、Vimentin表达上调,肿瘤细胞侵袭运动能力增强。四、成功筛选出针对SNAI1的有效RNAi靶序列,并成功的将hU6-shSNAI1片段克隆至腺病毒载体,改建成Ad-shSNAI1。Ad-shSNAI1感染靶细胞,证实能有效沉默目的基因转录表达。相同方法改建阴性对照载体Ad-shNC。将Ad-shSNAI1与Ad-HBx共转染HepG2细胞株。RT-PCR、Western blot检测证实,与阴性对照相比,RNAi组在mRNA水平和蛋白水平,E-cadherein表达都得以保留,N-cadhrein、Vimentin表达仍维持低水平。肿瘤细胞侵袭运动能力反而出现下降。HBx的促EMT和促侵袭转移能力被有效遏制。五、成功构建了三个5’端缺失、保留共同3’端的SNAI1启动子荧光素酶报告质粒。共转染Ad-HBx后,外源性HBx蛋白能显著增强SNAI1启动子活性。通过系列启动子缺失实验,进一步发现HBx在SNAI1基因启动子区的转录活性调控序列定位于-869~-514 (相对于转录起始位点)。HBx可能通过激活PI3K-Akt通路上调SNAI1启动子转录活性。【结论】一、肝癌组织中,HBx蛋白的表达与Snail1蛋白表达密切相关,二者的阳性表达均与临床肝细胞癌恶性程度高,侵袭能力强相关。二、外源性Snail1过表达可诱导肝癌细胞发生EMT,增强其侵袭能力。HBx蛋白能诱导肝癌细胞内源性Snail1过表达,促进EMT进程。三、干扰肝癌细胞SNAI1基因转录,沉默Snail1蛋白内源性表达,可有效逆转HBx蛋白对肝癌细胞株的EMT诱导,肝癌细胞仍维持上皮表型,侵袭运动能力反而受到抑制。证实Snail1是HBx诱导肝癌细胞EMT的关键中间分子。四、HBx可能通过激活PI3K-Akt通路上调SNAI1启动子转录活性,其调控区段位于-869~-514 (相对于转录起始位点)之间。更多还原

【Abstract】 BACKGROUND AND OBJECTSThe chronic infection of hepatitis B virus (HBV) is an independent risk factor of hepatocellular carcinoma (HCC). The hepatitis B virus X protein(HBx) encoded by HBV genome is an important transcriptional regulator which is closely involved in transactivating of oncogenes and plays an important role in malignant transformation of hepatic cells. HBx is also a key mediator of HCC invasion and metastasis in some way of disrupting the adherens junctions of cell to cell, promoting degradation of intercellular substance, inducing epithelial to mesenchymal transition (EMT) of tumor cells.The functional loss of E-cadherin is the hallmark of EMT. Previous studies have shown that HCC with lower E-cadherin expression that showed poor differentiation and more likely introhepatic metastasis and portal vein invasion. The expression of E-cadherin is also closely related to the prognosis of HCC. Transcriptional silence of CDH1 which encoding E-cadherin is the most important mechanism of down-regulation of E-cadherin.Recently, zinc-finger transcription factor Snail,which plays an important role in the formation of mesoderm,has been described to directly repress transcription of the CDH1 by binding to the E-boxes (CACCTG sequence) on its promoter. Because of the strong capacity of repress CDH1 transcription and induction of EMT, Snail is also involved in the progression of various epithelial carcinomas , trigerring invasion, metastasis, and dedifferentiation. Clinical research has showed that Snail is related to the invasion, metastasis and prognosis of HCC. But no data showed that if there was somehow relationship between HBx and Snail. It is still not known that what kind of role snail played in the progression of HCC with HBV infection.In this study, we supposed that HBx could up-regulate the expression of Snail1 protein by activating the transcriptional activity of SNAI1 gene. Moreover, the Snail1 up-regulation can induce EMT of HCC, trigerrin the invasion and metastasis of hepatocellular carcinoma. The emphasis is that Snail1 is the key mediator in the progression of HBx promoting the invasion and metastasis of HCC.MATERIALS AND METHODS1. HBx, Snail1, E-cadherin, N-cadhe and Vimentin were detected in 74 cases of HCC via immunochemistry staining. The correlations of the proteins and the the correlations between expression of proteins and clinical pathological properties were analyzed. We expected to identified that if the expression of Snail1 protein was correlated to the pathological staging of HCC and HBx protein expression.2. We cloned SNAI1 gene coding domain into pAdeasy-1 to form recombinant adenovirus Ad-SNAI1. Transfecting Ad-SNAI1 into HepG2 cells was performed to induce exogenous overexpression of Snail1. Then to investigate EMT markers and the invasive and migratory properties of HCC.3. HepG2 cells were transfected with recombinant adenovirus Ad-HBx. Snail1, EMT markers and the cells’property of invasion and migrasion were measured. So we can see whether HBx could induce endogenous overexpression of SNAI1,and furthermore, induce EMT and promote invasion and migration by this way.4. Silencing the transcription of SNAI1 was performed to investigate whether loss of Snail1 could inverse EMT of HCC induced by HBx. Therefore, we cloned the proper shRNA of SNAI1 to pGPU6/GFP/Neo vector. To improve the efficiency of target gene silencing, we subcloned the hu6 promoter and shRNA from the vector to adenovirus.Then Ad-shSNAI1 and Ad-HBx were cotransfected to HepG2 cells. And then, Snail1, EMT markers and the cells’property of invasion and migrasion were measured.5. A series of 5’-flanking SNAI1 promoter were amplificated from human liver DNA, and then subcloned to pGL3-Basic vector. Transfected the pGL3-Basic-SNAI1 promoters into SMMC-7721 cells together with or without Ad-HBx.The activities of promoters was mesuered and compared. pRL-TK vector was cotransfected as the inner control.RESULTS1. There are 58 in 74 cases that with positive HBx expression, 47cases for Snail1. The expression of HBx and SNAI1 were correlated significantly in 45 cases which both positive expression for HBx and Snail1 (p=0.002). SNAI1 positive expression was correlated with the absence of E-cadherin, up-regulation of N-cadherin and Vimentin. Clinical data Analysis showed: HBx positive expression was correlated with the clinical features including TNM staging, tumor size, portal invasion, peri-liver invasion and HBV-DNA in peripheral blood. So we considered Snail1 may be testified as a marker for the invasive and migratory properties of HCC, and the up-regulation of SNAI1 was concerned with HBx.2. Successfully subcloned the SNAI1 coding domain to adenovirus vector pAdeasy-1. The recombinant adenovirus named Ad-SNAI1. We got high expression level of the target gene with Ad-SNAI1 transfeced into HepG-2 cells. The expression of E-cadhrein was down-regulated accomplished with N-cadhrein and Vimentin up-regulated and improved invasion and migration of the hepatocarcinoma cells.3. Transfected with Ad-HBx, the SNAI1 mRNA and Snail were all overexpressed in HepG2 cell line. E-cadhrein was greatly down-regulated together with N-cadhrein and Vimentin up-regulation. The hepatocarcinoma cells also showed improver invasion and migration.4. siRNA sequence of SNAI1 was designed and tested. Then the hU6-shSNAI1 segament was subcloned to adenovirus vector to form Ad-shSNAI1. Ad- shSNAI1 was cotransfected with HBx to HepG2 cell line. The epithelial phenotype was maintained with slight upregulation E-cadherin and downregulation of mensenchymal markers. Decreased invasion and migration of HepG2 cell lines was also observed. It proved that EMT and promoted invasion and migration induced by HBx could be reversed thoroughly by Snail1 silencing.5. Successfully subcloned the 5’-flanking SNAI1 promoters to pGL3Basic vector. HBx could improve the transcriptional activity of SNAI1 promoter. The segment (-869~-514) ( count from the transcriptional initiation site) played an important role in the procedure of the transcriptional regulation of SNAI1 by HBx.CONCLUSIONS1. The expression of SNAI1 was closely correlated with HBx. Both of them had related to the invasion and metastasis of HCC. 2. Ectogenetic Snail1 could induce EMT of HCC cells. Up-regulation of the Snail1 by HBx could induce EMT in HCC cells.3. Sanil1 silencing could efficiently revesed EMT of HCC cells induced by HBx, the cells maintained epithelial phenotype and the invasive & migratory abilities of HCC were decreased. It showed that Snail1 was the key mediator in the procedure of HBx induced EMT in HCC.4. HBx may upregulate the transcriptional activity of SNAI1 gene by activating PI3K-Akt pathway. The segment (-869~-514) ( count from the transcriptional initiation site) played an important role in the procedure of the transcriptional regulation of SNAI1 by HBx.更多还原