Ephrin-As信号介导上皮-间质转化调节口腔鳞癌转移的实验研究

Most researches about the function of ephrin-A/EphA signaling in tumorigenesis focus on the ephrin-A1and EphA2. We know little about the function of the other ephrin-As in human tumors. In our preliminary study, when ephrin-A1and ephrin-A3 were transfected into mouse squamous cell carcinoma (SCC) cells in which the expression of ephrin-A1and ephrin-A3 was very low, the transfection of ephrin-A3 changed the SCC cell shape from mesenchymal morphology to a cuboidal and clustered appearance. Also the increase of E-Cadherin and reduction of Vimentin was detected by western blot in the SCC cells transfected with ephrin-A3. However, the function of ephrin-A1 transfected into the same cells was different with ephrin-A3. These data suggest a function role for ephrin-A3 in inhibiting EMT in SCC cells and the difference between ephrin-A1 and ephrin-A3 in regulating tumorigenesis. The research was designed to investigated the function of ephrin-A1 and ephrin-A3 in oral SCC. The EphA2 knockout mice and EFNA1,EFNA3,EFNA4 triple knockout mice, and human oral SCC cell lines and human SCC tissues were used in the experiment. The in vitro, in vivo assay and analysis of clinical SCC tumor samples were included in the research. We expect to reveal the molecular mechanism of the regulation of EMT and tumor metastasis by ephrin-As.

目前关于ephrin-A在肿瘤中的作用研究集中在ephrin-A1，对于其它ephrin-A配体的功能知之甚少。我们的前期研究发现，与ephrin-A1不同，ephrin-A3的表达可使小鼠鳞癌细胞由间质细胞形态变为方形的上皮样细胞形态，伴随E-Cadherin表达增高、Vimentin表达降低以及Akt1表达增高和磷酸化，提示ephrin-A3可能发挥抑制肿瘤上皮-间质转化(EMT)的作用。据此我们推测：ephrin-A1、ephrin-A3在口腔鳞癌中具有不同功能，ephrin-A3可能通过调控EMT调节口腔鳞癌转移。本课题以EphA2基因敲除小鼠和EFNA1、EFNA3和EFNA4基因共同敲除小鼠、以及人口腔鳞癌细胞系、人口腔鳞癌病理组织为研究对象，从体外、体内实验及临床病理研究等层面展开研究，期望能够揭示Ephrin-As调节口腔鳞癌EMT和转移的分子机制。

在恶性肿瘤发展过程中，上皮间质转化(EMT)可以使肿瘤细胞失去细胞间连接和细胞间的接触抑制作用，增强其在间质组织中的迁移浸润能力，从而助于肿瘤的侵袭转移。许多研究表明PI3K/Akt 信号通路可以通过多种方式影响EMT，进而影响肿瘤细胞的侵袭性。Ephrins 及其受体Ephs 可以传递双向信号来调节人体的多种发育及正常生理过程。目前普遍的研究主要集中在eprhin-A1，而关于ephrin-A3肿瘤中的作用的研究较少涉及。我们前期利用TKO小鼠皮肤鳞癌细胞系发现ephrin-A3表达使鳞癌细胞的形态由原来的间质细胞样变为方形的上皮细胞样，且ephrin-A3 使肿瘤细胞E-Cadherin增高、Vimentin降低。. 本项目在前期研究的基础上，主要进行了①Ephrin-A3影响口腔鳞癌进展的相关研究；②Ephrin-A3调节人口腔鳞癌细胞发生EMT的研究；③Ephrin-A3促进人口腔鳞癌细胞发生EMT的机制研究。最终结果表明，miR-210-3P在口腔癌中负向调控Ephrin-A3表达，且EphrinA3下调通过PI3K/AKT信号通路促进口腔鳞癌发生EMT。此外，在拟定研究内容之外我们也进行了扩展研究，发现口腔鳞癌细胞外泌体通过PI3K/AKT信号通路调控内皮细胞中miR-210-3P靶向EFNA3进而促进口腔鳞癌相关血管生成。. 本项目将miRNA和Ephrins以及口腔鳞癌发生上皮间质转化联系起来，深入探讨了口腔鳞癌发生EMT的分子机制。完成该项目的同时进行了深入的扩展研究，对于进一步阐明口腔鳞癌细胞外泌体促血管生成表型的机制，对于揭示肿瘤血管生成及miRNA调控机制均有重要意义，并为相关研究的临床转化提供研究基础。