PTEN与TGFBR1互为ceRNA调控口腔鳞癌自噬的分子机制

CeRNA(competing endogenous RNA) is a kind of endogenous RNAs that share multiple MREs(miRNA response elements) and regulate their targets in a microRNA(miRNA) competing based mechanism. In this project,we hypothesize that PTEN and TGFBR1, two key tumor suppressor gene in oral squamous cell carcinoma(OSCC) may have the potential interaction as ceRNA, which regulate autophagy and carcinogenesis of OSCC by regulate a panel of shared miRNAs. This project include three parts:Firstly,we testify the hypothesis by bioinformatic analysis and data mining.The expression pattern of PTEN and TGFBR1 shared miRNA in human OSCC cell lines, transgenic mice OSCC and human OSCC samples were detected by in situ hybridization and qPCR.Biological validation of PTEN and TGFBR1 regulated miRNA were performed using knock down and RNA-IP method.Furthermore,functional PTEN and TGFBR1 3'UTR transfection and DICER knock down assay were performed to confirm the ceRNA interaction of PTEN and TGFBR1. Secondly,we explore the molecular mechanism of PTEN and TGFBR1 shared miRNA's influence on autophagy and OSCC initiation.We will focus potent shared oncomiR such as miR-130a and miR-181a on their target autophagy related gene(ATGs) using qPCR, confocal microscope and living cell imaging in OSCC cell lines as well as PTEN/TGFBR1 knock out mice. In vitro foci formation assay and in vivo carcinogenesis were performed using shared miRNA inhibitor or mimics. Thirdly, we will further design combined exogenous competing miRNA sponges to explore experimental therapeutics of OSCC by inhibiting PTEN and TGFBR1 shared oncomiR in vitro and in vivo. Achievments of this project will better understand the multiple layer regulation of key tumor suppressor gene in autophagy and carcinogenesis of OSCC and develop new strategies for OSCC treatment.

CeRNA(competing endogenous RNA) 是一种通过竞争共享miRNA而调控靶基因的内源性RNA。通过前期研究,本项目提出假说："口腔黏膜上皮及口腔癌中抑癌基因PTEN及TGFBR1互为ceRNA,通过竞争共享miRNA调控对方mRNA的表达，进而调控自噬信号通路及自噬相关基因的表达，影响口腔癌的自噬及癌发生"。拟从以下方面开展工作：①利用口腔癌细胞系、基因敲除动物模型和人类组织标本分析和验证PTEN及TGFBR1共享miRNA，并通过转染彼此 3'UTR及敲减DICER的策略验证PTEN及TGFBR1的ceRNA关系。②明确共享miRNA调控自噬相关基因对自噬及上皮恶性转化的分子机制。③设计外源竞争性miRNA海绵，探索体外及转基因动物口腔癌的实验性治疗。研究结果将加深对口腔癌mRNA-miRNA-mRNA分子网络调控机制的理解，为口腔癌的治疗提供新的靶点及策略。

自噬在癌症发生发展中具有重要的调控作用，PTEN和TGFBR1的缺失是口腔鳞癌发生过程中的两个重要的分子事件，但PTEN和TGFBR1能否通过互为ceRNA调控口腔鳞癌自噬尚不明确。在本项目中，我们探索了PTEN和TGFBR1所调控的自噬过程对口腔鳞癌发生发展所起的作用。除此之外，我们还继续探索了PTEN和TGFBR1敲除对口腔鳞癌免疫逃逸及免疫原性细胞死亡的作用。在完成的实验中，我们可以得出以下结论：①生物信息学分析筛选出与HNSCC生存预后较差相关的PTEN和TGFBR1共享micoRNA分子has-mir-543；②口腔鳞癌中自噬相关分子（LAMP1, LC3, TFEB）的表达与免疫原性细胞死亡相关分子（Calreticulin, HMGB1）以及肿瘤间质中PD-L1的表达具有正相关；③PTEN与TGFBR1双基因敲除，能显著上调小鼠口腔鳞癌微环境中的负性免疫检查点分子（CTLA-4, PD1, CD47, B7-H3等）；④通过靶向负性免疫检查点分子（CTLA-4, PD1, CD47, B7-H3等）能有效减少PTEN/TGFBR1 2cKO小鼠口腔鳞癌微环境中的免疫抑制细胞（MDSC, TAM, Treg），增强抗肿瘤免疫能力；⑤在PTEN/TGFBR1 2cKO小鼠中，利用工程化血小板纳米材料能有效提高口腔鳞癌光热治疗的疗效。