Oral pathogen - mediated pro-tumorigenic transformation through disruption of an Adherens Junction - associated RNAi machinery

摘要：在美国，每年约有53,000人被诊断患有口腔癌，而每年有5,000人被诊断患有口腔癌。 由于诊断和干预的延迟，一年存活率仍然很低。最近的证据表明 牙周病是一个潜在的危险因素，它也与不良的预后有关。有趣的是， 牙周病和口腔癌有共同的现象：上皮屏障完整性破坏和 发炎。更具体地说，牙周病原体核梭杆菌与 无论是屏障破坏还是口腔癌。尽管如此，尚不清楚口腔上皮屏障的破坏是否由 核梭杆菌是促癌口腔转化的机械性促进剂，或仅仅是一种 由此产生的后果。为了回答这个问题，我们在这里询问了一个细胞复合体，它是上皮细胞必不可少的 屏障完整，称为粘着结。更具体地说，我们发现粘连连接 组件PLEKHA7是E-钙粘蛋白和p120结合伙伴，招募RNA的核心组件 干扰机制包括微处理器、RNA诱导的沉默复合体和几组mRNAs 和miRNAs来维持上皮细胞的动态平衡。PLEKHA7从培养的上皮细胞中耗尽导致错误的- RNAi机制的本地化，降低miRNA沉默活性，增加癌基因表达，以及 促肿瘤细胞转化。然而，这种对RNAi干扰机制的破坏，特别是 与屏障完整性有关，到目前为止还没有在口腔癌中进行研究，暴露了在 知识。我们假设PLEKHA7招募并调节口腔上皮细胞黏附的RNAi机制 这种机制在口腔病原体存在的情况下会被破坏， 反过来，促进促肿瘤和炎症标志物的表达。我们将通过以下方式验证我们的假设 具体有两个目的：1)确定口腔上皮细胞黏附连接是否招募RNAi机制进行调节 利用二维和三维上皮细胞研究miRNAs及其靶向mRNAs的功能和水平 细胞模型和检查患者的组织；2)确定口腔牙周病原体梭杆菌 核酸盐通过miRNA失调促进致癌和促炎标记物的上调， 使用与核梭杆菌共培养，免疫荧光，蛋白质和RNA分析。这个项目 具有重要意义，因为它将识别一种新的上皮致瘤转化的分子机制 口腔粘膜，促进了我们对这种疾病的理解。这项研究是创新的，因为它将病原体、口腔 上皮屏障功能，以及口腔癌局部RNAi和miRNA调控。我们预计， 这项研究将对口腔癌的有效生物标志物的未来发展产生影响和贡献，这些生物标志物包括 对于及时和成功的干预以及对以RNA为基础的潜在发展至关重要 治疗学。总之，该奖学金将提供口腔上皮生物学的基础培训，以培养 将DMD/PhD实习生培养成一名独特且急需的牙科学术科学家。

Abstract: Approximately 53,000 people in the United States are diagnosed with oral cancer yearly, and the 5- year survival rate remains poor due to late diagnosis and intervention. Recent evidence has suggested periodontal disease as a potential risk factor, and it is also associated with poor outcomes. Interestingly, periodontal disease and oral cancer share common phenomenon: disruption of epithelial barrier integrity and inflammation. More specifically, the periodontal pathogen Fusobacterium nucleatum has been associated with both barrier disruption and with oral cancer. Still, it is unclear whether disruption of the oral epithelial barrier by Fusobacterium nucleatum is a mechanistic promoter of pro-tumorigenic oral transformation, or just a consequence of it. To answer this question, we interrogate here a cellular complex that is essential for epithelial barrier integrity, called the adherens junction. More specifically, we have found that the adherens junction component PLEKHA7, which is an E-cadherin and p120 binding partner, recruits core components of the RNA interference machinery including the microprocessor, the RNA induced silencing complex, and sets of mRNAs and miRNAs to maintain epithelial homeostasis. PLEKHA7 depletion from cultured epithelial cells results in mis- localization of the RNAi machinery, decreased miRNA silencing activity, increased oncogene expression, and pro-tumorigenic cell transformation. However, such disruption of the RNAi interference machinery, particularly associated with barrier integrity, has not been studied to date in oral cancer, exposing a significant gap in knowledge. We hypothesize that PLEKHA7 recruits and regulates the RNAi machinery at oral epithelial adherens junctions to maintain homeostasis, and that this mechanism is disrupted in the presence of oral pathogens, which in turn promote expression of pro-tumorigenic and inflammatory markers. We will test our hypothesis through two Specific Aims: 1) determine whether oral epithelial adherens junctions recruit the RNAi machinery to regulate function and levels of miRNAs and of their target mRNAs, using two dimensional and three-dimensional epithelial cell models and by examining tissues from patients; 2) determine if the oral periodontal pathogen Fusobacterium nucleatum promotes upregulation of oncogenic and pro-inflammatory markers through miRNA dysregulation, using co-cultures with Fusobacterium nucleatum, immunofluorescence, protein, and RNA analyses. This project is significant, since it will identify a novel molecular mechanism of epithelial pro-tumorigenic transformation in the oral mucosa, advancing our understanding of the disease. The study is innovative, as it links pathogens, oral epithelial barrier function, and localized RNAi and miRNA regulation with oral cancer. We anticipate that the study will be impactful and contribute to future development of effective biomarkers oral cancer, which are critically needed for timely and successful intervention, as well as to the potential development of RNA-based therapeutics. Altogether, this fellowship will provide foundational training in oral epithelial biology to foster the development of the DMD/PhD trainee into a unique and critically needed academic dental scientist.