【作者】 张小英；

【导师】 孙树汉；

【作者基本信息】 第二军医大学 ， 遗传学， 2009， 博士

【摘要】 原发性肝细胞癌(HCC)是世界常见的恶性肿瘤之一,流行病学调查发现数百万HCC患者合并有乙型肝炎病毒(HBV)的感染,而HBV患者发生HCC的几率比非HBV患者高100倍,乙型肝炎病毒(HBV)感染被认为是HCC发生最主要的原因。乙型肝炎病毒x蛋白(HBx)由HBV编码,在HBV相关肝癌的发生、发展、转移中具有重要作用,但由于很少有理想的动物模型来论证HBx在HCC发生发展中的作用,因此HBx在肿瘤发生发展过程中的分子机制并不完全清楚。幸运的是,p21-HBx转基因小鼠已经建立起来,它是通过同源重组和干细胞培养技术将HBx基因插入p21基因的第二外显子区域而建立的转基因动物模型。据报道该模型的p21-/-小鼠在2年以上的时间没有一只小鼠发生肿瘤,提示该模型小鼠的p21缺失不会增加小鼠发生HCC肿瘤的易感性。并且,HBx基因受PGK启动子(强启动子)的调控,因此,HBx蛋白能在该模型中的肝脏持续稳定的表达24个月以上,并且60%的该转基因小鼠在18个月左右能发展HCC,有些甚至发生了肿瘤转移,这现象与人类感染HBV后发展成HCC的病史相似。因此,p21-HBx转基因小鼠为我们研究HBx致HCC发生、发展、转移中可能的机制提供了理想的动物模型。MicroRNAs (miRNAs)是一种大小约21-23个碱基的非编码单链小分子RNA,在生物体内发挥重要作用,包括细胞增殖,凋亡,应激反应和肿瘤发生。最近研究显示miRNA在多种肿瘤中的表达水平发生了变化,可能在某些肿瘤的发生中发挥了重要作用。研究认为miRNA水平的变化常导致其靶基因的表达异常,从而影响了肿瘤的进程。许多肿瘤都涉及到了miRNA的变化,并且其变化程度还与肿瘤的分型分级以及预后密切相关,这提示我们miRNA在肿瘤发生发展中所发挥了重要作用,其表达的模式可作为肿瘤诊断/预后的分子标记。但现在很少有文献系统的研究与HBx特异相关的miRNA在HBx导致的HCC发生、发展、转移中的作用。目前普遍认为HBx是病毒反式作用因子,它通过与宿主的转录因子(如AP-1、AP-2、NF-κB、ATF2、CREB等)结合而发挥其生理功能,这种结合可以发生在宿主DNA的任何区域,从而导致宿主基因的不稳定性,而基因的这种不稳定性如果发生在原癌基因、抑癌基因或者miRNA基因等部位,就会改变这些基因的表达,从而导致肿瘤的发生。这就提示了,虽然目前一些已知的基因和蛋白能提供很多有用的信息,但是对miRNA研究的新发现也能为我们理解HBx致HCC发生、发展、转移机制提供新视角。因此,发现一个或一组与HBx特异相关的miRNA对我们具有重要意义,它能让我们更好的理解HCC的发生发展机制,做到早期诊断,改善治疗干预手段,防止HCC转移和复发,提高肝癌患者的存活率。基于上述发现,展开研究,利用real-time PCR技术对p21-HBx转基因小鼠和野生型小鼠肝脏的miRNA进行了表达水平分析,功能分析,并在HCC患者标本中进行验证,初步探讨了与HBx功能相关的miRNAs在HCC发生发展中的作用。通过实验我们发现,与野生型小鼠相比,转基因小鼠肝脏中有7个miRNA的表达水平发生变化,其中,5个上调的miRNA(miR-212, miR-23a,miR-155,miR-17-5p和miR-20a),2个下调的miRNA(miR-152和miR-200a),它们的变化趋势随着肿瘤的发展而表现的更为明显。随后,利用HBx敲入(将pEGFP-HBx转入HepG2细胞)和敲出(用adr HBx干扰HepG2.2.15细胞中HBx的表达)的细胞模型发现,筛选出的这7个特异性miRNA的表达水平与HBx的表达水平密切相关,它们呈剂量依赖关系。体外功能实验证明它们能调节细胞增殖、细胞凋亡和细胞侵袭的潜能,并可能在HCC发生发展中发挥作用:过表达的miR-212,miR-23a,miR-155,miR-17-5p和miR-20a能抑制细胞凋亡,而过表达的miR-152和miR-200a则能促进细胞凋亡;过表达的miR-212,miR-17-5p和miR-20a能促进细胞增殖,而过表达的miR-200a则能抑制细胞增殖;另外,过表达的miR-212,miR-23a,miR-17-5p和miR-20a能显著增强细胞侵袭和迁移的潜能;而过表达的miR-152和miR-200a则明显抑制了细胞侵袭和迁移的潜能,这些miRNAs的表达模式均在HCC患者标本中得以验证。我们的实验结果表明这组miRNA参与了HBx介导的HCC发生发展过程,因此可能是HBV相关HCC的标志物。同时,实验结果显示miR-143在10月龄的转基因小鼠肝脏(正常,未发现病变)中的表达水平明显降低,而在22月龄的转基因小鼠肝脏(发生HCC)中的表达水平明显上升,尤其是那些伴随肺转移的转基因小鼠,上调水平更明显,接着,HBV相关HCC患者标本中miR-143的表达模式也进一步验证了上述结果:miR-143在HBV相关HCC标本中显著上调,尤其是具有转移特点的HBV相关HCC标本中显著上调。体外功能实验发现miR-143并不能影响细胞的增殖和凋亡,却能显著提高细胞的侵袭能力,并且其促进细胞侵袭和迁移的能力是通过HBx调控途径而实现。进一步用软件分析和CHIP实验发现NF-κB可以直接结合到人mir-143基因上游的5kb左右位点,并且直接调控miR-143的表达。随后,利用TargetScan分析软件和荧光素报告酶系统筛选出其下游靶基因为FNDC3B(fibronectin type III domain containing 3B)。体内外实验均证明,受NF-κB调控的miR-143通过下调靶基因FNDC3B,从而参与了由HBx介导的HCC肿瘤转移的过程,这一发现为HBV相关性HCC的肿瘤转移网络补充了一条重要的信号通路——HBx/NF-κB/ miR-143/ FNDC3B。与HBx特异相关的miRNA及其在原发性肝细胞癌中的功能机制研究将为阐明HBV相关性HCC的发生发展分子机制提供许多有意义的新思路,为理解受HBx影响的纷繁复杂的信号通路提供一定的帮助,并为进一步早期诊断HCC,治疗HCC,防止HCC的转移和复发提供靶点。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Epidemiological studies have shown that millions of people are chronically infected with hepatitis B virus (HBV), and an abundance of evidence has proved that HBV infection is one of the important causes of HCC, given that the incidence of HCC in chronically HBV infected individuals is approximately 100-fold higher than in the uninfected population. The HBV X protein (HBx), a protein encoded by HBV, is thought to play a key role in the molecular pathogenesis of initiation, development and metastasis of HBV related HCC. Since there are few ideal animal models that can be used to demonstrate the role of HBx in HCC development, the molecular mechanisms underlying HBx protein-mediated tumorigenesis are not fully understood. Fortunately, a p21-HBx transgenic mouse model has been established by introducing the HBx gene into the p21 locus. In this model established by Wang Y et al., none of mice homozygously null for p21 over a 2-year period developed HCC, suggesting that p21 deficiency does not directly increase the susceptibility to HCC. Furthermore, in this model, HBx gene is driven by its own promoter (PGK promoter, strong promoter) which could express persistently for at least 24 months in the liver. HCCs often develop when the transgenic mice are 18 months of age. What’s more, some of the transgenic mice can even develop to metastatic tumors. Thus, the HBx transgenic mouse model is an ideal model for screening for possible mechanisms by which HBx leads to the initiation/development/metastasis of HCC.MicroRNAs (miRNAs) are 21-23 nt long non-coding RNA sequences that are involved in various biological processes, including cell proliferation, cell death, stress resistance, and tumorigenesis. Recent studies indicate that miRNAs may play a role in several cancers. Altered miRNA levels can result in aberrant expression of gene products that may contribute to cancer biology, which suggests that expression profiling of miRNAs may significantly contribute to cancer development. Furthermore, it has also been shown that miRNA expression patterns have relevance to the biological and clinical behavior of human cancers. These and other data demonstrate that miRNAs play a substantial role in the pathogenesis of cancers, and that expression profiling of miRNAs can be used as potential diagnostic/prognostic markers. However, to date very limited information has been published about miRNAs that are specifically involved in the initiation/development/metastasis of HBx-related HCC. The HBx protein is a viral transcriptional transactivator. It can modulate many host cell functions through its integrations with a variety of host factors (such as AP-1, AP-2, NF-κB, ATF2, CREB). The presence of HBx gene fragments at many different locations within the host DNA can result in genetic instabilities. Genetic instabilities associated with integrations potentially alter the expression of oncogenes, tumor suppressor genes, and miRNAs that may contribute to tumorigenesis. This strongly indicates that although research on known genes and proteins has already yielded useful information, new discoveries about miRNA may also build insights into the initiation/development/metastasis of HCC in the context of HBx. Discovery of unique new target molecules is of paramount importance and will be essential if we are to understand the mechanisms and improve the prognoses associated with hepatic cancers as early as possible, therapeutic intervention and prevent from tumor invasion/metastasis and postsurgical recurrence. The result should be an increase in the likelihood of successful treatment.Based on these findings, we screened candidate specific miRNA molecules in the livers of p21-HBx transgenic mice compared with wide-type mice, using real-time PCR in order to elucidate the possible underlying mechanisms which lead from chronic HBV infection to HCC. The functional effects of these miRNAs were then examined to elucidate the possible mechanisms of HCC. Finally, liver samples of HCC patients were analyzed to validate the expression patterns of the unique miRNAs. The results demonstrated that five upregulated miRNAs including miR-212, miR-23a, miR-155, miR-17-5p and miR-20a and two downregulated miRNAs including miR-152 and miR-200a that were selectively expressed in HBx-expressing livers of p21-HBx transgenic mice. The expression levels of these unique miRNAs were corrected with the expression of HBx, and displayed the mode of dose-dependent. The five upregulated and two downregulated miRNAs were identified by in vitro analysis as being involved in regulating cell apoptosis, proliferation and migration. Overexpression of miR-212, miR-23a, miR-155, miR-17-5p and miR-20a can significantly reduced cell apoptosis, while overexpression of miR-152 and miR-200a can induced the cell apoptosis. Overexpression of miR-212, miR-17-5p and miR-20a can significantly increase cell proliferation, but overexpression of miR-152 and miR-200a can reduced the cell proliferation. Furthermore, overexpression of miR-212, miR-23a, miR-17-5p and miR-20a can significantly increase cell migration, but overexpression of miR-152 and miR-200a can reduced the cell migration. Our results were validated in ectopic HBx-expressing HCC patients. Our data strongly suggest that these unique miRNAs be involved in tumorigenesis in the context of HBx, and thus may be useful in the prognosis of HBV-related HCC of tumor metastasis in HBV-HCC.Meanwhile, we have found that miR-143 was significantly down-regulated in livers of ten-month old transgenic mice while dramatically up-regulated after the HCC developed in the transgenic mice especially when accompanied by tumor metastasis to the lung. Next, we use the HBV-HCC patients to validate the results in humans, and also find that miR-143 was dramatically up-regulated after the HCC developed in HBV-HCC patients, especially when accompanied by tumor metastasis. Functional study performed in vitro demonstrated that miR-143 has no role in regulating cell proliferation and apoptosis, but has signifigant affect on cell migration. Furthermore, we have found that miR-143 is directly regulated by the transcription factor NF-κB, and its direct target gene is FNDC3B (fibronectin type III domain containing 3B). The study in vitro and in vivo we performed has demonstrated that upregulated miRNA-143 induced by NF-κB enhances hepatocarcinoma metastasis by repression of fibronectin. And the novel HBx/NF-κB/ miR-143/ FNDC3B pathway is an important complement to the network.Altered expression profling of miRNA mediated by HBx may sheds new insight into elucidating the molecular mechanism of the development and progression of HBV-HCC and a target for early diagnose, therapy and inhibiting tumor metastasis and recurrence of HCC.更多还原