Regulation of ZEB1 by Porphyromonas gingivalis in gingival epithelial cells

项目摘要 口腔癌是世界上第11种被诊断为最常见的癌症，由于诊断较晚，它有 5年后存活率约为50%。转移是通过上皮间充质进行的。 过渡(EMT)，这是上皮细胞失去紧密连接和尖端/底端极性的过程 并转变为更具侵袭性的间充质表型，使癌细胞能够通过侵袭进入全身 血液中的血迹。EMT通过几种转录因子介导，但我们将重点关注ZEB1。 牙龈卟啉单胞菌是一种口腔专性厌氧菌，当存在时会改变宿主/微生物的关系。 从协同作用到更不利的环境，这会导致牙周病。牙龈假单胞菌一直是 显示可以侵袭牙龈上皮细胞，一旦进入细胞内就能抑制细胞凋亡，加速细胞周期， 最近，我们的实验室发现ZEB1基因表达上调。尽管我们开始理解 牙龈假单胞菌的潜在致癌作用，我们目前还不知道ZEB1是如何被牙龈假单胞菌调控的 牙龈上皮细胞。我们的初步数据表明，这种调控是通过SMAD信号发生的 并通过长的非编码RNA ZEB1-AS1进行调控。Smad信号是转化生长因子-b信号的一个分支 途径，在胚胎发育过程中负责从最初的 上皮组织。Smad信号通过转化生长因子-b受体对Smad2/3的磷酸化而发生，随后 Smad2/3和Smad4形成转录调控三聚体，最后核定位和 ZEB1基因上调。此外，ZEB1可以与Smad2/3和Smad4结合形成ZEB1/SMAD三聚体，从而 调控ZEB1转录。我们发现牙龈假单胞菌上调了调控的SMAD3，这表明 牙龈假单胞菌能够拦截这一途径，上调ZEB1的表达。我们还发现牙龈假单胞菌 上调ZEB1-AS1，已在肝癌和食管鳞癌中发现 直接上调ZEB1，我们的初步数据支持这一点，因为击倒ZEB1-AS1会导致 牙龈假单胞菌对牙龈上皮细胞中ZEB1调控的取消。我们将研究其机制。 ZEB1-AS1的调控包括通过与P300结合直接促进核内基因的表达， 和/或控制ZEB1mRNA的稳定性。EMT监管是一个复杂的过程，涉及到几个 宿主信号通路，所以我们也怀疑ZEB1-AS1可能通过潜在的方式与SMAD信号整合 充当SMAD3的脚手架。更好地了解牙龈假单胞菌介导的EMT将为 为未来的研究奠定基础，以针对这些诊断和治疗的调节机制，并最终 减少目前患者确诊后5年内50%的生存机会。

Project Summary Oral cancer is the 11th most frequently diagnosed cancer in the world, and due to its late diagnosis, it has an approximately 50% survival rate after 5 years. Metastasis is meditated through Epithelial to Mesenchymal Transition (EMT), which is the process by which epithelial cells lose their tight junctions and apical/basal polarity and shift to a more invasive mesenchymal phenotype giving the cancer cells systemic access through invasion of the bloodstream. EMT is mediated through several transcriptional factors, however we will focus on ZEB1. Porphyromonas gingivalis is an oral obligate anaerobe that when present shifts the host/microbe relationship from synergy to a more dysbiotic environment, which contributes to periodontal disease. P. gingivalis has been shown to invade gingival epithelial cells, and once inside the cells can inhibit apoptosis, ramp up the cell cycle, and more recently by our lab has been shown to upregulate ZEB1. Although we are beginning to understand the potential oncogenic role of P. gingivalis, we do not currently know how ZEB1 is regulated by P. gingivalis in gingival epithelial cells. Our preliminary data suggests that this regulation is occurring through SMAD signaling regulation and through the long non-coding RNA ZEB1-AS1. SMAD signaling is a branch of the TGF-b signaling pathway, which during embryonic development is responsible for producing mesenchymal cells from the initial epithelium. SMAD signaling occurs by phosphorylation of SMAD2/3 by the TGF-b receptor, followed by the formation of a transcriptional regulatory trimer by SMAD2/3 and SMAD4, and finally nuclear localization and upregulation of ZEB1. Additionally, ZEB1 can bind to SMAD2/3 and SMAD4 to form a ZEB1/SMAD trimer to regulate ZEB1 transcription. We have found that P. gingivalis upregulates the regulatory SMAD3, which suggests P. gingivalis is able to intercept this pathway to upregulate ZEB1. We have also found that P. gingivalis upregulates ZEB1-AS1, which has been shown in hepatocarcinoma and esophageal squamous cell carcinoma to directly upregulate ZEB1, and our preliminary data supports this as knocking down ZEB1-AS1 leads to abrogation of ZEB1 regulation by P. gingivalis in gingival epithelial cells. We will investigate mechanisms of regulation by ZEB1-AS1 including direct promotion of gene expression in the nucleus through binding to P300, and/or control of ZEB1 mRNA stability. EMT regulation is a complex process that involves integration of several host signaling pathways, so we also suspect that ZEB1-AS1 will be integrated with SMAD signaling by potentially acting as a scaffold for SMAD3. Developing a better understanding of P. gingivalis mediated EMT will lay the foundation for future studies to target these regulatory mechanisms for diagnosis and treatment, and ultimately reduce the 50% chance of survival current patients have 5 years after diagnosis.