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

描述（由申请人提供）：在全球范围内，肝细胞癌（HCC）是第六大常见癌症。缺乏早期检测方法和有效的治疗选择也使其成为最致命的疾病之一。尽管它的流行，许多仍然是未知的分子机制参与肝癌的发展。然而，已知慢性感染B型肝炎病毒（HBV）是发展HCC的主要危险因素之一。提出了三个主要因素在HBV相关HCC的发展中起作用：导致肝细胞破坏和再生的慢性炎症; HBV DNA整合到宿主基因组中的有害作用;以及HBV蛋白，特别是肝炎B病毒X蛋白（HBx）表达的影响。HBx是一种多功能的非结构蛋白 研究表明，它们可能具有直接或协同致癌潜力。HBx还可以激活转录因子，如NF-κ B。NF-kB的正常激活引起立即早期反应，导致促存活因子和炎性因子的强烈激活。NF-kB还参与多种microRNA（miRNAs）表达的调节，miRNAs是小的~ 22 nt RNA，可作为mRNA的转录后调节因子。NF-B和miRNA都与癌症有关，研究已经成功地利用miRNA谱的改变作为某些疾病如癌症和病毒感染的生物标志物。拟议的研究将检查NF-κ B在HBx介导的肝细胞miRNA谱改变中的作用，以及这些变化如何影响HCC的发展。将特别关注一组特定的miRNA，基于它们与HBV、HBx或HBV相关HCC的相关性，以及它们在调节细胞过程如增殖和免疫应答中的关键作用。在目标1中，将通过miRNA微阵列和定量逆转录聚合酶链反应（qRT-PCR）研究确定HBx诱导的miRNA表达水平变化。然后通过用NF-kB超阻遏物抑制NF-B活化来确定这种表达模式的NF-kB相关变化。重要的是，这些研究将在新鲜分离、培养的原代大鼠肝细胞中进行，这些肝细胞比永生化细胞系更有可能保留其生理学miRNA谱。在第2个目的中，miRNA结合mRNA的转录组范围分析将允许我们鉴定由miRNA积极调节的所有mRNA的概况，这将通过随后的靶mRNA和蛋白质分析来验证。在目标3中，将在发生HCC的HBx转基因小鼠模型中，在HCC发生过程中体内证实miRNA表达和所提出的mRNA靶点的进行性变化。对广泛调节的mRNA靶点的操纵将证实它们的作用，以及改变的miRNA调节在HCC发展中的作用。总之，这些研究将支持这样的假设，即NF-kB影响肝细胞中HBx介导的miRNA谱的改变，并且这些改变直接改变参与HCC发展的重要细胞靶点的调节。这些研究将有助于确定HCC发生的分子机制的长期目标，并可能确定预防或检测HBV相关HCC的新治疗靶点和诊断标志物。 公共卫生相关性：炎症在B型肝炎病毒（HBV）相关肝细胞癌（HCC）的发生中起重要作用。HBV X蛋白（HBx）激活NF-κ B（炎症的主要介质），NF-κ B激活和HBx表达均与HCC的发展有关。我们将研究NF-κ B在HBx介导的肝细胞miRNA水平变化中的作用，以及这些变化对HBV相关HCC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.