HBx regulation of miRNA-mediated mRNA silencing in HCC development.

描述(申请人提供)：在全球范围内，肝细胞癌是第六种最常见的癌症。缺乏早期发现方法和有效的治疗选择也使其成为最致命的疾病之一。尽管肝细胞癌的发病率很高，但关于肝细胞癌发生的分子机制仍不清楚。然而，众所周知，慢性乙肝病毒感染是发生肝细胞癌的主要危险因素之一。三个主要因素被认为在乙肝相关性肝细胞癌的发展中起作用：导致肝细胞破坏和再生的慢性炎症；整合到宿主基因组中的HBVDNA的有害影响；以及乙肝病毒蛋白的表达的影响，特别是乙肝病毒X蛋白(HBx)。HBX是一种多功能非结构蛋白 研究表明，这可能具有直接或合作的致癌潜力。HBx还可以激活核因子-kB等转录因子。核因子-kB的正常激活会引起即刻-早期反应，导致促生存因子和炎症因子的强烈激活。核因子-kB还参与了多个microRNAs(MiRNAs)的表达调控，miRNAs是一种小的~22nt的RNA，可以作为mRNAs的转录后调节因子。核因子-B和miRNAs都与癌症有关，研究成功地利用miRNA图谱的变化作为某些疾病的生物标记物，如癌症和病毒感染。这项拟议的研究将探讨核因子-kB在HBX介导的肝细胞miRNA图谱改变中的作用，以及这些改变如何影响肝细胞癌的发展。除了在调节细胞过程如增殖和免疫反应方面的关键作用外，还将特别关注一组特定的miRNAs，这些miRNAs与乙肝病毒、HBx或与乙肝病毒相关的肝细胞癌的相关性。在目标1中，将通过miRNA微阵列和定量逆转录-聚合酶链式反应(qRT-PCR)研究来确定HBX诱导的miRNA表达水平的变化。然后，将通过使用核因子-kB超抑制物抑制核因子-B的激活来确定这种表达模式的与核因子-kB相关的变化。重要的是，这些研究将在新鲜分离的、培养的原代大鼠肝细胞中进行，这些细胞比永生化细胞系更有可能保持其生理miRNA图谱。I目标2，对miRNA结合的mRNAs进行转录组范围的分析，将使我们能够确定所有活跃的受miRNAs调控的mRNAs的图谱，这将被随后的靶mRNA和蛋白质分析所验证。在目标3中，在发生肝癌的HBx转基因小鼠模型中，将在体内证实miRNA表达的渐进性变化和所提出的mRNA靶点。操纵广泛调控的mRNA靶标将证实它们的作用，以及改变的miRNA调节在肝细胞癌发展中的作用。总之，这些研究将支持这样的假设，即核因子-kB影响HBX介导的肝细胞miRNA谱的变化，并且这些变化直接改变了参与肝细胞癌发展的重要细胞靶点的调节。这些研究将有助于确定与肝细胞癌发展有关的分子机制的长期目标，并有可能确定预防或检测乙肝病毒相关肝细胞癌的新的治疗靶点和诊断标记。 公共卫生相关性：炎症在乙肝病毒相关性肝细胞癌的发生发展中起着重要作用。乙型肝炎病毒X蛋白(HBx)激活了炎症的主要介质--核因子-kB，核因子-kB的激活和HBx的表达均参与了肝细胞癌的发生发展。我们将探讨核因子-kB在HBx介导的肝细胞miRNA水平变化中的作用以及这些变化对乙肝病毒相关性肝细胞癌发生发展的影响。

DESCRIPTION (provided by applicant): Globally, hepatocellular carcinoma (HCC) is the sixth most common cancer. A lack of early detection methods and effective treatment options also make it one of the most deadly. Despite its prevalence, much remains unknown about the molecular mechanisms involved in the development of HCC. It is known however, that chronic infection with the hepatitis B virus (HBV) is one of the leading risk factors for developing HCC. Three main factors are proposed to play a role in the development of HBV-associated HCC: chronic inflammation resulting in hepatocyte destruction and regeneration; deleterious effects of HBV DNA integration into the host genome; and the impact of expression of HBV proteins, specifically the hepatitis B virus X protein (HBx). HBx is a multifunctional non-structural protein that studies have shown can have either direct or cooperative oncogenic potential. HBx can also activate transcription factors such as NF-kB. Normal activation of NF-kB causes an immediate-early response resulting in strong activation of pro-survival and inflammatory factors. NF-kB also is involved in the regulation of expression of multiple microRNAs (miRNAs), which are small ~22nt RNAs that can act as post-transcriptional regulators of mRNAs. Both NF-B and miRNAs have been implicated in cancer, and studies have successfully utilized alterations in the miRNA profile as biomarkers of certain diseases such as cancers and viral infections. The proposed studies will examine the role of NF-kB in the HBx-mediated alteration of the hepatocyte miRNA profile, and how these changes influence the development of HCC. Particular attention will be paid to a specific panel of miRNAs based on their association with HBV, HBx, or HBV-associated HCC in addition to their critical role in regulating cellular processes such as proliferation and the immune response. In Aim 1, HBx- induced changes in miRNA expression levels will be determined by miRNA microarray and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) studies. NF-kB-related changes to this expression pattern will then be determined by inhibiting NF-B activation with a NF-kB super-repressor. Importantly, these studies will be done in freshly isolated, cultured primary rat hepatocytes, which are a more likely to retain their physiological miRNA profile than immortalized cell lines. I Aim 2, a transcriptome-wide analysis of miRNA-bound mRNAs will allow us to identify a profile of all mRNAs actively being regulated by miRNAs, which will be validated by subsequent target mRNA and protein analysis. In Aim 3, progressive changes to miRNA expression and proposed mRNA targets will be confirmed in vivo over the course of HCC development in an HBx-transgenic mouse model that develops HCC. Manipulation of extensively regulated mRNA targets will confirm their role, and the role of altered miRNA regulation, in the development of HCC. Together, these studies will support the hypothesis that NF-kB influences the HBx-mediated alteration of the miRNA profile in hepatocytes, and that these changes directly alter regulation of important cellular targets involved in the development of HCC. These studies will aid in the long-term goal of determining molecular mechanisms involved in the development of HCC, and potentially identify novel therapeutic targets and diagnostic markers for preventing or detecting HBV-associated HCC. PUBLIC HEALTH RELEVANCE: Inflammation plays a significant role in development of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). The HBV X protein (HBx) activates NF-kB, the main mediator of inflammation, and both NF- kB activation and HBx expression have been implicated in HCC development. We will investigate the role of NF-kB in HBx-mediated changes in hepatocyte miRNA levels and the influence of these changes on the development of HBV-associated HCC.