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

通过破坏粘附连接相关的 RNAi 机制，口腔病原体介导促肿瘤转化

• 批准号: 10752248
• 负责人: Christina Rachel Kingsley
• 金额: $ 5.07万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752248
• 关键词: 3-Dimensional; Adherens Junction; Binding; Biological Markers; Biology; Cancer Patient; Carcinoma; Cell model; Cell-Cell Adhesion; Cells; Chad; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Complex; Confocal Microscopy; Data; Databases; Dental; Development; Diagnosis; Disease; Doctor of Philosophy; E-Cadherin; Epithelial Cells; Epithelium; Fellowship; Fostering; Fusobacterium nucleatum; Future; Homeostasis; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Inflammation; Intervention; Knock-out; Knowledge; Link; Malignant Neoplasms; Mediating; Messenger RNA; MicroRNAs; Microbe; Microprocessor; Molecular; Mouth Neoplasms; Oncogenes; Oncogenic; Oral; Oral cavity; Oral mucous membrane structure; Outcome; Pathologist; Pathway interactions; Patients; Periodontal Diseases; Persons; Physiological; Physiology; Protein Analysis; Publishing; Quantitative Reverse Transcriptase PCR; RNA; RNA Interference; RNA analysis; RNA-Induced Silencing Complex; Regulation; Risk; Risk Factors; Sampling; Scientist; Site; Stratified Epithelium; Survival Rate; Testing; The Cancer Genome Atlas; Therapeutic; Tissues; Training; United States; Up-Regulation; Work; cancer risk; cell behavior; cell transformation; crosslink; inflammatory marker; innovation; malignant mouth neoplasm; novel; oral bacteria; oral cavity epithelium; oral pathogen; oral tissue; oral tumorigenesis; pathogen; pathogenic bacteria; periodontopathogen; posttranscriptional; promoter; protein expression; recruit; success; successful intervention; therapeutic RNA; transcriptome sequencing; tumor progression; tumorigenic; two-dimensional

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.

摘要：美国每年约有 53,000 人被诊断患有口腔癌，其中 5- 由于诊断和干预较晚，年生存率仍然很低。最近的证据表明 牙周病是一个潜在的危险因素，而且也与不良结果相关。有趣的是， 牙周病和口腔癌有一个共同的现象：上皮屏障完整性被破坏， 炎。更具体地说，牙周病原体具核梭杆菌与 屏障破坏和口腔癌。尽管如此，尚不清楚口腔上皮屏障是否会受到破坏 具核梭杆菌是促肿瘤口腔转化的机制启动子，或者只是一个 它的后果。为了回答这个问题，我们在这里询问对上皮细胞至关重要的细胞复合物 屏障完整性，称为粘附连接。更具体地说，我们发现粘附连接 成分 PLEKHA7 是 E-钙粘蛋白和 p120 结合伴侣，可招募 RNA 的核心成分 干扰机器，包括微处理器、RNA 诱导沉默复合物和 mRNA 组 和 miRNA 来维持上皮稳态。培养的上皮细胞中 PLEKHA7 的消耗导致错误 RNAi 机制的定位、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，RNAi 干扰机制的这种破坏，特别是 与屏障完整性相关，迄今为止尚未在口腔癌中进行研究，暴露出显着的差距 知识。我们假设 PLEKHA7 招募并调节口腔上皮粘附的 RNAi 机制 连接以维持体内平衡，并且这种机制在口腔病原体存在的情况下被破坏， 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检验我们的假设 两个具体目标：1) 确定口腔上皮粘附连接是否招募 RNAi 机制来调节 使用二维和三维上皮细胞分析 miRNA 及其靶标 mRNA 的功能和水平 细胞模型并通过检查患者的组织； 2) 判断口腔牙周病原菌是否为梭杆菌 nucleatum 通过 miRNA 失调促进致癌和促炎标记物的上调， 使用具核梭杆菌共培养、免疫荧光、蛋白质和 RNA 分析。这个项目 意义重大，因为它将确定上皮促肿瘤转化的新分子机制 口腔粘膜，增进了我们对该疾病的了解。这项研究具有创新性，因为它将病原体、口腔 上皮屏障功能以及口腔癌的局部 RNAi 和 miRNA 调节。我们预计 研究将具有影响力，并有助于有效口腔癌生物标志物的未来开发，这些生物标志物是 及时、成功的干预以及基于 RNA 的潜在发展至关重要 疗法。总而言之，该奖学金将提供口腔上皮生物学的基础培训，以促进 将 DMD/PhD 实习生培养成为独特且急需的学术牙科科学家。