Role of MicroRNA 363 in HPV-positive oral cancer

描述（由申请人提供）：最近，人乳头瘤病毒（HPV）相关口咽（OP）鳞状细胞癌（OPSCC）的发病率大幅增加，而其他病例则由酒精和/或烟草使用引起。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阴性口腔癌相关的途径至关重要。最近，一个HPV阳性OPSCC患者与烟草暴露和p53基因改变的亚群已被确定，HPV E6和p53介导的途径有助于致癌的详细调查。在HPV相关的OPSCC中，病毒E6和E7癌蛋白以高水平表达。MicroRNA（miRNAs）是约22 nt长的单链RNA，通常作为基因表达的负调控因子，并且它们的表达谱在多种人类癌症中改变。关于miRNA在OP癌症发病机制中的作用的信息有限。我们发表的使用OPSCC细胞系的研究表明，HPV阳性和HPV阴性OP癌症中的miRNA表达谱彼此明显不同，包括HPV阳性细胞系中miR-363的过表达和miR-218的低表达。与HPV阴性OPSCC组织、正常口腔粘膜和正常口腔角质形成细胞（NOK）相比，miR-363在HPV-16阳性OP癌组织中也过表达。我们的数据还表明，HPV-16 E6癌基因促进miR-363过表达，同时降低miR-218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，这改变了HPV阳性口腔癌发生的细胞途径。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抑癌miRNA。我们将通过在HPV阳性和HPV阴性OPSCC细胞系、NOK和表达E6的NOK中过表达或敲低miR-363和miR-218的表达来实现这一目标，并研究它们对细胞增殖、迁移、侵袭、粘附和凋亡的影响。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E6介导的这些miRNA的调节。如果是这种情况，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p53通路的失活影响miR-363和miR-218的表达。我们的研究将提供有关miR-363和miR-218在HPV阳性OP癌症中的作用、E6对这些miRNA的调节性质以及miR-363和miR-218在HPV阳性和HPV阴性OPSCC中差异调节的分子基础的有价值的信息。这些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：该提案的目标是鉴定microRNA 363和218在HPV阳性和HPV阴性口咽癌细胞系中的细胞功能。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.