口腔黏膜白斑恶性变的表观遗传调控的机制及外泌小体靶向治疗研究

Oral leukoplakia is the most common premalignant disorder of the oral mucosa and its malignant transformation is associated with epithelial to mesenchymal transition (EMT). EMT mediates cancer cell invasion, metastasis, and drug resistance by enriching cancer stem cells, promoting angiogenesis and altering autophagy. We found that EMT features are seen in oral epithelial dysplasia and in its progression to cancer, accompanying significantly decreased expression of miR-185, overexpression of VEGF and overactive PI3K-AKT-mTOR signaling pathway. We also found that patients with oral epithelial dysplasia or oral cancer have significantly decreased miR-185 level in the saliva compared to normal control. Overexpression of miR-185 led to significantly decreased expression of its target genes AKT and VEGF but increased autophagic abilities in oral cancer cells. Given miR-185 is mainly packaged and cargoed by exosome, a kind of nanometer sized extracellular vesicles shuttled between cells and released into body fluids, we hypothesize that exosome encapsulated miR-185 mediates the process of EMT during the malignant transformation of oral leukoplakia. We plan to investigate whether direct delivery of high concentration exosomal miR-185 to oral cancer animal model could block EMT or increase tumor sensitivity to radiochemotherapy. In addition, we plan to verify whether saliva exosome carried miR-185 could be served as a non-invasive biomarker for early detection of oral dysplasia and furthermore prediction of resistance to radiochemotherapy in the treatment of oral cancer.

口腔黏膜白斑上皮间质转化（EMT）与肿瘤干细胞的产生，血管增生和细胞自噬功能紊乱相关是恶变的关键。我们发现miR-185在有异常增生的口腔白斑和口腔癌组织中显著降低，伴随EMT激活，VEGF增高，PI3K/AKT通路激活和细胞自噬降低。miR-185转染口腔癌细胞降低靶基因AKT﹑VEGF表达，提示口腔白斑恶变与miR-185低表达相关。细胞实验显示miR-185通过外泌体跨细胞转运，外泌体携带高拷贝miR-185可逆转EMT并降低细胞增殖。本项目拟通过口腔癌前病变动物模型和口腔癌细胞株，阐明装载高拷贝数miR-185外泌体逆转EMT、阻断白斑恶变的作用机制；探讨唾液外泌体/miR-185早期诊断口腔白斑恶变的可行性；探索通过外泌体载体投递miR-185﹑靶向调控多个目标基因治疗新模式。拟揭示外泌体/miR-185在口腔白斑EMT机制中的作用，为早期诊断和靶向治疗口腔黏膜癌前病变提供依据。

近年来随着外泌体在肿瘤诊疗领域的广泛应用，其不仅作为载体在分子的靶向治疗方面显现出诸多优势，还在肿瘤的早期诊断方面具有潜力。目前外泌体用于口腔癌治疗的研究尚少，仅限于体外研究。本课题组前期体外实验发现 miR-185 可通过抑制 Akt 降低人口腔鳞癌（OSCC）细胞的上皮间质转化（EMT）进而减少癌细胞增殖。因此，本研究建立口腔潜在恶性疾患与口腔癌动物模型，首先通过外泌体性状鉴定及体内外示踪研究证实 miR-185 EVs 可靶向作用口腔癌细胞，局部涂抹的方式可以使 miR-185 EVs 与地鼠颊囊黏膜稳定高效结合，给药途径有效；然后进一步探索基因工程法装载的含有高拷贝 miR-185 的骨髓间充质干细胞外泌体对口腔潜在恶性疾患及口腔癌的作用，并对其作用机制进行探讨。. 通过检测白斑和口腔癌患者唾液中的外泌体、miR-185表达水平，发现单纯增生、白斑异常增生、鳞状细胞癌患者唾液中外泌体浓度进行性降低。外泌体中miR-185的表达水平在白斑伴轻度异常增生、白斑伴重度异常增生/原位癌、鳞状细胞癌中进行性降低。表明唾液外泌体及外泌体中的miR-185可在一定程度上反应口腔白斑-口腔癌病理生理状态，初步探讨了唾液外泌体及其中的miR-185作为生物标志物判断口腔潜在恶性疾癌变的可能性。