miR-301a在缺氧诱导胰腺癌EMT中的功能机制研究

Our preliminary studies have shown that hypoxia up-regulated miR-301a expression, which targeted Smad4 expression. Given that hypoxia induces HIF-1α expression, we hypothesize that hypoxia induces HIA-1 expression and promotes miR-301 expression, which targets Smad4 and other unknown molecules to regulate TGF-β-mediated Smad dependent and independent signaling, promoting STAT3 aberrant activation and EMT process in pancreatic cancer. In this proposal, we will analyze the potential association of miR-301 and its downstream molecules with pancreatic cancer metastasis in clinical samples, determine the molecular mechanisms underlying the role of miR-301a in hypoxia-induced EMT in pancreatic cancer, and investigate the potential cross-talk between the TGF-β and STAT3 pathway in the absence of Samd4, and finally identify the regulatory effect of HIF-1α on hypoxia-induced miR-301a expression. Our data may demonstrate that miR-301a is valuable biomarker for the prognosis of pancreatic cancer and reveal molecular mechanisms underlying the action of miR-301a in regulating EMT and metastasis of pancreatic cancer. Potentially, our findings may provide a basis for the design of new therapeutic strategy for the intervention of pancreatic cancer in the clinic.

课题组前期的研究发现缺氧微环境上调miR-301a的表达，miR-301a又可能通过其靶基因Smad4影响TGF-β信号通路，并通过串扰效应引起STAT3过活化，诱导胰腺癌EMT。但目前尚不知miR-301a如何调控TGF-β信号相关的STAT3过度活化，也不知缺氧诱导的HIF-1α是否可调控miR-301a的表达。本课题拟分析miR-301a及其下游分子对胰腺癌分期及转移的关系，研究miR-301a在缺氧诱导胰腺癌EMT的功能，分析miR-301a调控TGF-β信号通路与STAT3信号通路串扰关系及相关机制，并初步探讨HIF-1α对miR-301a表达的影响。我们的研究结果将首次揭示miR-301a在缺氧条件下通过TGF-β通路诱导胰腺癌EMT中的功能及机制,可为揭示胰腺癌发病机理提供依据，也为基于microRNAs临床靶向性治疗奠定基础。

前期研究发现缺氧微环境能上调胰腺癌细胞株miR-301a的表达，下调miR-301a表达水平抑制胰腺癌细胞株体外侵袭转移能力。本课题组以此为基础首先应用基因转染技术上调或下调miR-301a在胰腺癌细胞株中的表达，观察其对体外侵袭及转移、克隆形成，体内肿瘤形成和生长的影响，结果显示上调miR-301a可增强胰腺癌侵袭转移能力、克隆形成能力，而下调miR-301a减低胰腺癌侵袭、转移、克隆形成能力。课题组进一步利用慢病毒构建miRNA-301a的稳定过表达细胞株以及利用CRISPR/Cas9技术构建miR-301a敲除细胞株，研究发现稳转miRNA-301a能促进低氧下胰腺癌细胞发生EMT，而敲除miR-301a能够抑制低氧下胰腺癌细胞EMT。我们进一步揭示了miRNA-301a下游调控机制。miR-301a能够结合Smad4、PTEN、TP63等靶基因的3’UTR区，并在转录后水平抑制其靶基因的表达，miR-301a通过抑制Smad4促进胰腺癌细胞增殖和转移；miR-301a通过抑制TP63上调Vimentin、Fibronection等间质表型标记物，同时下调E-cadherin、β-catenin等上皮表型标记物，从而促进胰腺癌细胞EMT；miR-301通过抑制PTEN降低胰腺癌细胞对吉西他滨的敏感性。miR-301在低氧中的调控机制鲜有报道，我们在胰腺癌细胞中转染siRNA分别对HIF-1α、HIF-2α进行干扰，结果发现miR-301a表达下调，其中干扰HIF-2α后miR-301表达下调更显著。进一步将miR-301a上游约4000bp的启动子区构建到PGL4.27荧光素酶报告基因载体中，发现Hif-2α可明显促进miR-301a启动子区活性。证明miR-301a在低氧中表达受Hif-2α调控。课题组利用裸鼠皮下种植瘤模型及裸鼠胰腺癌细胞原位种植模型验证miR-301a可促进胰腺癌细胞体内生长及转移。应用胰腺癌组织芯片检测miR-301a表达与临床病理及患者预后的关系，揭示了miR-301a表达与肿瘤分期、分级、转移及患者预后密切相关。.我们的研究结果首次揭示miR-301a在低氧条件下诱导胰腺癌EMT和转移中的功能及机制，可为揭示胰腺癌发展提供线索，也为基于microRNAs临床靶向性治疗奠定基础。