胰腺癌特异性Hedgehog活化效应miRNAs的鉴定及作用机制研究

Pancreas cancer (PCa) is a kind of aggressive, common malignant tumor, with an unknown pathogenesis. Several studies have demonstrated that the Hedgehog (HH)-Glis (Gli1/2) is one of the core signaling pathways in PCa tumorigenesis, however up to now, no studies were involved in the identification of target miRNAs of HH pathway activity and their molecular mechanism of pathogenesis. In our previous studies, we found that the aberrant expression levels of some miRNAs were positively correlated with the Gli1 expression, suggesting a significance of the research on identification of HH-Glis target miRNAs and their actions in PCa tumorigenesis. In this projection, after sorting and grouping PCa cancer stem cells and cell lines respectively, the HH-Glis target miRNAs are to be selected and identified out through a series of experiments, including miRNAs microarray and electrophoretic mobility shift assay（EMSA）, etc. Then, the effect of HH-Glis target miRNAs in PCa tumorigenesis and the potential application value of targeting the miRNAs for prognostics and therapeutics are to be evaluated through a series of tests, such as the detection of the miRNAs expression in PCa and PanINs tissues, the transgene experiments in PCa cells and in vivo gene therapy, etc. Meanwhile, the target genes of the miRNAs are caught by labeled miRNA pull-down assay（LAMP）and the role of the miRNAs and their targets genes as a feedback control factor in the HH pathway regulatory networks will be explored and validated by time-effect relation of transgenic intervention. This projection could find a novel molecular mechanism of pathogenesis in the tumorigenesis of PCa and provide new molecular biomarkers in the prognostics and therapeutics for PCa.

胰腺癌恶性程度极高，发病机制尚不清楚。Hedgehog(HH)-Glis活化为核心致病机制，但目前尚未见Glis效应miRNAs在胰腺癌发病中的作用研究报道。我们前期发现，胰腺癌中一些miRNAs与Glis表达呈正相关，提示开展胰腺癌特异性Glis效应miRNAs致病机制研究，凸显必要。首先，我们从胰腺癌细胞株和癌干细胞（由临床新鲜标本获得）中应用miRNAs芯片和EMSA等实验鉴定出Glis效应miRNAs，而后，一方面通过在胰腺癌组织中异常表达检测、对胰腺癌细胞（细胞株和干性细胞）转基因干预、及in vivo动物实验等观察Glis效应miRNAs的致病作用特点和诊疗价值；另一方面，应用LAMP方法特异性捕获效应miRNAs调控的靶基因，设计"反馈环路验证实验"，探索和验证这些效应miRNAs可作为HH通路反馈调控因子，并参与调控网络构成的作用。为胰腺癌的防治提供新的理论基础和分子靶标。

胰腺癌恶性程度极高，发病机制尚不清楚。Hedgehog(HH)-Glis活化为核心致病机制，但目前尚未见Glis效应miRNAs在胰腺癌发病中的作用研究报道。本项目在前期工作中发现，胰腺癌中一些miRNAs与Glis表达呈正相关，提示开展胰腺癌特异性Glis效应miRNAs致病机制研究，凸显必要。本研究通过转染HH通路正向活化基因Gli1cDNA和反向调控基因SUFUcDNA入胰腺癌细胞株SW1990和Panc-1中，进过miRNA芯片和mRNA芯片筛查差异表达的miRNA和mRNA，并通过两者之间的相关性分析、生物信息学miRNA靶基因分析、Gli1转录因子结合位点序列分析等，采用CHIP-qPCR实验方法等确定出Gli1核因子的效应miRNA靶基因3个（miR-301a，miR-1228和miR-29b）。进一步实验发现，miRNA-301a对MST1、SHH和HHIP的mRNA和蛋白表达均具有靶向敲低作用；并且发现miR-301a对细胞周期未见显著影响，但可显著促进细胞的增殖及侵袭能力；同时，在胰腺癌组织中发现miR-301a存在显著高表达。本研究的主要科学意义在于发现了在胰腺发病中，Hedgehog（HH）信号通路活化新的靶基因miR-301a，miR-1228和miR-29b。其中miR-301a具有靶向调控YAP通路STK4和自身HH通路HHIP和SHH表达的作用。由此可构成较为复杂的对Hippo通路的串话（crosstalk）调节和对自身HH通路的正反馈（由SHH构成）和负反馈（由HHIP构成）调控网络。这些研究结果为进一步揭示胰腺癌肿瘤发生的分子机制提供了新的理论，为进一步研发新的胰腺癌诊疗技术提供了基础。