胰腺癌发病中Hippo通路YAP活化的分子调节机制研究

Pancreas cancer (PCa) is a common malignant tumor without effective treatment method in the clinical practices. Nowadays, the pathogenesis is still unknown. Several studies have demonstrated that the Hedgehog (HH)-Gli1 is one of the core signaling pathways in PCa tumorigenesis, as well as inflammatory factor and the Hippo-YAP activity also involved. Our previous studies showed that the miR-301a is high aberrantly expressed in PCa cell lines and tissues, and which can regulate Mst1 gene (YAP inhibitory factor) expression in pancreatic cancer cell lines. In the other hand , it was showed that the regulatory effect of inflammatory factor （IL-1 and TNF）, as well as SHH, on the YAP protein activity in our experiments. There exist the binding sites of NF-kB and Gli1 in the Pri-miR-301a promoter. Additionally the low expression of Mst1 protein and the high expression of YAP protein were observed by immunohistochemistry in PCa tissues. These above evidences implied that there maybe exist the key molecular interaction of "miR-301a targeting Mst1" and the tumorigenesis depend on their regulation role of in the progression of PCa, which link the crosstalk action from the inflammatory factor to Hippo/YAP activity, and as well as the HH activity to Hippo/YAP activity. In this research, first we perform gene expression intervention experiments and rescue experiments in PCa cell lines to prove that the key molecular interaction of "miR-301a targeting Mst1" play an important role in PCa tumorigenesis. Second, we observe the pathogenic characteristics of the expression of the miR-301a and Mst1, as well as their cooperative effect in PCa tumorigenesis. Last, after the construction of the transgenic mice (Pdx-miR-301a) we detected the formation of neoplastic lesions and the expression of the core members of Hippo pathway in both PanINs lesions and primary neoplastic cells, and compared with the local block of the miR-301a action, or administration of agonists and inhibitors for YAP. This projection could add new knowledge of the molecular mechanism of pathogenesis in the tumorigenesis of PCa and provide new molecular targets in the prognostics and therapeutics for PCa.

已证实慢性炎症和Hedgehog通路（HH）活化为胰腺癌致病机制，Hippo通路YAP活化也参与胰腺癌发生。我们前期发现，炎性因子刺激和HH活化可促YAP蛋白活化；同时发现miR-301a在胰腺癌中异常高表达，可调控Mst1（YAP抑制因子）表达，Pri-miR-301a启动子区存在NF-kB及HH-Gli1转录结合位点。提示在胰腺癌发生中可能存在"miR-301a靶向调控Mst1" 分子作用环节，且此环节为炎性因子刺激和HH通路活化对Hippo通路串话（crosstalk）调节的关键环节。本研究首先在癌细胞株中采用基因干预和补救等实验证实"miR-301a靶向调控Mst1"的致病作用及调控Hippo/YAP活化的分子作用；进而，在临床癌标本中观察此分子环节的致病特点；最后，构建转基因小鼠（Pdx-miR-301a）in vivo证实此环节具有促癌生成作用。提出胰腺癌发病新的分子调节机制。

前期工作基础上已证实慢性炎症和Hedgehog通路（HH）活化具有胰腺癌致病作用，机制在于Hippo通路YAP去磷酸化入核活化下游基因；并且发现miR-301a-3p启动子区存在NF-kB及HH-Gli1转录结合位点。在此研究中，我们发现HH通路配体SHH、炎性因子IL-1β和TNF-α刺激胰腺癌细胞株可促进miR-301a-3p表达，miR-301a对YAP通路上游的MST1基因表达具有靶向敲低的作用，双荧光素酶报告基因实验证实miR-301a-3p对人STK4和小鼠STK3的3’UTR序列具有直接结合并抑制表达的作用，增加内源性miR-301a-3p表达后，对细胞周期无显著影响，但可显著增加细胞增殖，抑制细胞凋亡，促进细胞的侵袭力。在研究中，我们采用CRISPR-Cas9技术构建了DICER1基因敲除的Panc-1细胞株（Panc-1-KO），但此细胞活力显著降低，难以进行大规模后续实验。在单纯Panc-1细胞中，SHH、TNF-α和IL-1β这三种因子刺激对YAP通路关键分子的作用类似，即可调低上游Mst1和Mst2的表达，导致YAP磷酸水平降低，YAP蛋白入核活化下游基因，同时对比发现在Panc-1-KO细胞中，这三种因子刺激对LATS1和YAP总蛋白无显著调节作用，YAP磷酸化水平无改变，而YAP通路抑制剂verteporfin处理单纯Panc-1细胞，虽然也可降低Mst1表达减少，Mst2表达减少，但下游的LAST1和YAP总蛋白表达几乎缺失，阻断了通路的传导，结果表明，SHH、TNF-α和IL-1β对YAP通路的调节主要通过miR-301a-3p对靶基因Mst1的调控。细胞学实验进一步表明，Shh、IL-1β和TNF-α联合miR-301a inhibitor处理Panc-1细胞可逆转SHH、IL-1β和TNF-α对细胞恶性特征的促进作用，具有显著抑制细胞增殖、促进细胞凋亡的作用。本研究为胰腺癌发病机制提供了新理论。