Role of MicroRNA 363 in HPV-positive oral cancer

描述(申请人提供)：最近，与人乳头瘤病毒(HPV)相关的口咽鳞状细胞癌(OPSCC)的发病率大幅上升，而其他病例是由酒精和/或烟草使用引起的。由于HPV感染可显著降低死亡率，因此了解HPV相关口腔癌的分子机制对于了解HPV调控的途径与HPV阴性口腔癌的关系至关重要。最近，一组HPV阳性的OPSCC患者被发现有烟草暴露和P53基因改变，这证明有必要详细研究HPVE6和P53介导的致癌途径。在HPV相关的OPSCC中，病毒E6和E7癌蛋白高水平表达。MicroRNAs(MiRNAs)是一种长约22nT的单链RNA，通常作为基因表达的负调控因子，其在多种人类癌症中的表达谱发生改变。关于miRNAs在OP癌发病机制中的作用，现有的信息有限。我们发表的使用OPSCC细胞系的研究表明，在HPV阳性和HPV阴性的OP癌中，miRNA的表达谱明显不同，包括在HPV阳性的细胞系中miR-363的过度表达和miR-218的低表达。与HPV-16阴性的OPSCC组织、正常口腔黏膜和正常口腔角质形成细胞(NOK)相比，MIR-363在HPV-16阳性的OP癌组织中也过表达。我们的数据还表明，HPV-16E6癌基因促进miR-363的过度表达，而降低miR-218的表达。我们假设miR-363和/或miR-218水平的差异改变了其靶基因的表达，从而改变了参与HPV阳性口腔癌发生的细胞通路。在目标1中，我们将测试miR-363和miR-218是否分别是影响重要细胞通路的致癌和肿瘤抑制因子miRNAs。我们将通过在HPV阳性和HPV阴性的OPSCC细胞系中过表达或下调miR-363和miR-218的表达，以及表达E6的NOKs和NOK来实现这一点，并研究它们对细胞增殖、迁移、侵袭、黏附和凋亡的影响。在目标2中，我们将确定E6影响miR-363和miR-218表达的调控机制，以及这些机制是否依赖于p53。我们将使用DNA甲基化和组蛋白修饰的抑制剂来测试表观遗传过程是否参与了E6介导的这些miRNAs的调节。如果是这样的话，我们还将研究这些miRNA启动子的甲基化状态和染色质状态。我们还将测试E6是否通过使P53途径失活来影响miR-363和miR-218的表达。我们的研究将为以下方面提供有价值的信息：miR-363和miR-218在HPV阳性OP癌中的作用，E6对这些miRNAs的调控性质，以及在HPV阳性和HPV阴性的OPSCC中miR-363和miR-218的差异调控的分子基础。这一信息可用于这些癌症的未来诊断、预后和治疗靶点。 公共卫生相关性：这项建议的目标是确定HPV阳性和HPV阴性口咽癌细胞系中microRNAs 363和218的细胞功能。我们还将确定HPV-16癌蛋白调节HPV阳性口咽癌中microRNAs 363和218表达的机制。

DESCRIPTION (provided by applicant): Recently, there has been a substantial increase in the incidence of human papillomavirus (HPV)-associated oropharyngeal (OP) squamous cell carcinoma (OPSCC), while other cases result from alcohol and/or tobacco use. Since HPV infection confers a significantly decreased mortality rate, understanding the molecular mechanisms responsible for HPV-associated oral carcinoma is crucial to understanding the pathways regulated by HPV versus those involved in HPV-negative oral cancer. Recently, a subpopulation of HPV-positive OPSCC patients with tobacco exposure and p53 genetic alterations has been identified, warranting detailed investigation of the HPV E6- and p53-mediated pathways contributing to carcinogenesis. In HPV-associated OPSCC, the viral E6 and E7 oncoproteins are expressed at high levels. MicroRNAs (miRNAs) are ~22 nt long single-stranded RNAs that generally function as negative regulators of gene expression, and their expression profiles are altered in a variety of human cancers. Limited information is available on the role of miRNAs in the pathogenesis of OP cancers. Our published studies using OPSCC cell lines show that miRNA expression profiles in HPV-positive and HPV-negative OP cancers are distinctively different from each other, including the overexpression of miR-363 and underexpression of miR-218 in HPV-positive cell lines. MiR-363 is also overexpressed in HPV-16 positive OP cancer tissues compared to HPV-negative OPSCC tissues, normal oral mucosa and normal oral keratinocytes (NOKs). Our data also show that the HPV-16 E6 oncogene promotes miR-363 overexpression while reducing miR-218 expression. We hypothesize that differences in miR-363 and/or miR-218 levels change the expression of their target genes and this alters the cellular pathways involved in HPV-positive oral carcinogenesis. In Aim 1, we will test whether miR-363 and miR- 218 are oncogenic and tumor suppressor miRNAs, respectively, that affect important cellular pathways. We will accomplish this by overexpressing or knocking-down the expression of miR-363 and miR-218 in HPV-positive and HPV-negative OPSCC cell lines, NOKs and NOK expressing E6, and study their effect on cell proliferation, migration, invasion, adhesion and apoptosis. In Aim 2, we will identify the regulatory mechanisms by which E6 affects miR-363 and miR-218 expression and whether these are p53-dependent. We will use inhibitors of DNA methylation and histone modification to test whether epigenetic processes are involved in E6-mediated regulation of these miRNAs. If this is the case, we will also study the methylation status and chromatin state of these miRNA promoters. We will also test whether E6 affects miR-363 and miR-218 expression through inactivation of the p53 pathway. Our studies should provide valuable information on the role of miR-363 and miR-218 in HPV-positive OP cancers, the nature of regulation of these miRNAs by E6, and the molecular basis for the differential regulation of miR-363 and miR-218 in HPV-positive and HPV-negative OPSCC. This information could be utilized for future diagnosis, prognosis and therapeutic targets for these cancers. PUBLIC HEALTH RELEVANCE: The goals of this proposal are to identify the cellular functions of microRNAs 363 and 218 in HPV-positive and HPV-negative oropharyngeal cancer cell lines. We will also identify the mechanisms by which the HPV-16 oncoprotein regulates the expression of microRNAs 363 and 218 in HPV-positive oropharyngeal cancers.