PRAS40通过NF-κB转录调控头颈鳞癌上皮间质转化及侵袭转移的分子机制研究

Epithelial-mesenchymal transition (EMT) and metastasis have been research emphasis and highlight in cancer cells. Currently, there is no report about PRAS40 in squamous cell carcinoma of the head and neck(SCCHN) and EMT. However, we previously found that PRAS40 was confirmed to promote the metastasis and EMT of SCCHN. P65 has been discovered to be capable of associating with PRAS40 in our high through input protein protein interaction screening. In addition, we also identified a small peptide which could inhibit this protein protein interaction. Considerng that NF-κB could induce EMT in cancer cell through targeting the transcription level of EMT-related DNA. And, P65 is the most important component of the NF-κB transcriptional complex. Therefore, we come up with this hypothesis: PRAS40 regulates the EMT and metastasis of SCCHN via NF-κB activity in vivo and vitro. In order to confirm this hypothesis, we would employ the cell and nude mice model to study the role of PRAS40 in SCCHN metastasis and EMT process. Co-Immunoprecipitation, dual luciferase reporter assay and Chromatin immunoprecipitation and sequencing experiments would be performed to validate whether PRAS40 regulates the EMT and metastasis through NF-κB transcription activity. The downstream gene related to EMT and targeted by NF-κB would be discovered in this part. Finally, we could test the expression and clinical significance of these molecules like PRAS40, E-cadherin, Vimentin, P65 and the genes targeted by NF-κB in the SCCHN patient samples. If this project has been approved, it would broaden the oncogenetic role of PRAS40 in cancer cells, provide some new content for the molecular mechanism of regulating EMT and possible hints for SCCHN drug therapy.

上皮间质转化（EMT）与肿瘤侵袭转移一直是国内外研究的重点，但其机制尚不明确。目前PRAS40在头颈鳞癌及EMT中的作用均无报道。申请人前期发现：PRAS40既可促进头颈鳞癌细胞EMT及侵袭转移，又能结合P65蛋白；并鉴定出可抑制它们结合的小分子多肽。考虑到NF-κB可诱导肿瘤细胞EMT改变，而P65恰好是NF-κB经典转录活动中的核心蛋白。因此，我们提出假设：PRAS40可通过结合P65调控NF-κB转录，进而影响头颈鳞癌EMT及侵袭转移。为此，本项目将通过体内外实验探索：①PRAS40在头颈鳞癌EMT及侵袭转移的作用；②PRAS40是否通过结合P65调控NF-κB转录活性；③NF-κB是否参与PRAS40调控头颈鳞癌细胞EMT及侵袭转移；④组织中上述蛋白的表达与临床意义。本项目的成功实施将拓展PRAS40在肿瘤细胞的作用；可能为EMT的分子调控机制和头颈鳞癌的药物治疗提供新的理论基础。

上皮间质转化（EMT）与肿瘤侵袭转移一直是国内外研究的重点，但其机制尚不明确。目前PRAS40在头颈鳞癌及EMT中的作用均无报道。我们提出假设：PRAS40可通过结合P65调控NF-κB转录，进而影响头颈鳞癌EMT及侵袭转移。为此，本项目已完成以下内容：1）通过CRISPR/Cas9调控PRAS40的表达探索其在头颈鳞癌细胞生长侵袭转移中的作用；2）通过免疫荧光、免疫共沉淀及GST pull down技术验证PRAS40与P65蛋白的结合作用，并借助荧光素酶报告系统验证PRAS40调控NF-kB转录；.3）继续深入缩小PRAS40与P65蛋白结合的大致区域至短小肽段区域；4）此外，本项目还探索了mTOR信号通路及代谢相关产物在PRAS40调控头颈鳞癌细胞恶性行为的改变及作用。