RNA结合蛋白PUM2参与调控miR-221和miR-1291诱导EMT促进胰腺癌细胞化疗耐药的功能及机制研究

Whether to predict the therapeutic effect of the chemotherapy drugs to specific patient and to reverse the resistance of pancreatic cancer cells are critical for chemotherapy of pancreatic cancer. microRNAs (miRNAs) have been reported to play important roles in the genesis of drug-resistance of various cancer types. There are also many advantages of miRNAs in diagnosis and therapy of disease as their small molecules. We identified differently expressed miRNAs in drug resistance of pancreatic cancer cells and found that miR-221 and miR-1291 were significantly up-regulated in the 5-FU resistant cells. The overexpression of miR-221 or miR-1291 in pancreatic cancer cells strongly promoted cell survival when treated with 5-FU, along with Epithelial Mesenchymal Transition(EMT). ZEB1, as an important positive regulator of EMT, can increase the expression of miR-221 directly, which in turn promoted the progression of pancreatic cancer drug-resistance. In addition, we found RNA binding protein PUM2, as a targeting gene of miR-221, has significant roles in carcinogenesis,development and chemo-resistance in pancreatic cancer. Furthermore,the primary investigation of miR-221 expression in pancreatic specimens suggested that high expression of miR-221 was correlated with worse outcome in PDAC patients treated with 5-FU. We will further study the function and mechanism of miR-221 and miR-1291 in modulating drug resistance of pancreatic cancer cells, and investigate the expression of various miRNAs in the pancreatic specimens to develop a method predicting the drug-resistance of specific patient according to the expression of these miRNAs. Meanwhile to detect targeting RNA moleculers and interactive protein compound of RNA binding protein PUM2. After all, miR-221 and miR-1291 are potentially to be developed as new targets for pancreatic cancer therapy to overcome drug-resistance. These works are useful for understanding the essence of drug-resistance of pancreatic cancer cells and to improve the poor therapeutic effect.

能否预测化疗药物有效性、逆转化疗耐药是胰腺癌化疗研究的核心问题，miRNA广泛参与各种肿瘤细胞的耐药发生。从鉴定胰腺癌耐药相关miRNA出发，研究发现miR-221和miR-1291在胰腺癌细胞5-Fu耐药细胞株中显著上调，两者的过表达能够促进胰腺癌细胞5-Fu耐药，并伴随EMT转化的发生，在EMT中具有促进作用的ZEB1能够活化miR-221的表达，使细胞耐药的进一步发展；miR-221靶基因RNA结合蛋白PUM2在胰腺癌发生、发展及化疗耐药具有重要调控作用；miR-221表达水平与患者术后生存期呈负相关。本课题将深入研究miR-221和miR-1291在胰腺癌5-FU耐药中的作用及分子机制，检测PUM2调控的靶RNA分子和相互作用的蛋白质复合物。在组织及血清中联合检测多个miRNA表达水平预测并指导临床化疗药物的选择。研发以miRNA为靶点的靶向治疗方法，改善并提高胰腺癌化疗耐药现状。

胰腺癌恶性程度高，预后差。以吉西他滨为主的化疗是治疗中的重要部分，但吉西他滨耐药一直是导致胰腺癌复发及患者死亡的关键。因此，探究胰腺癌吉西他滨耐药机制对于提高胰腺癌患者生存率意义重大。. RNA结合蛋白（RBP）是一类可以调节RNA转运、剪接、稳定性及翻译的重要蛋白质，多种RBP在胰腺癌中异常表达且与不良预后相关，因此我们筛选出与胰腺癌吉西他滨耐药最相关的RBP PUM2。构建PUM2稳定敲低及过表达的胰腺癌细胞系，体外及体内实验表明PUM2可促进胰腺癌细胞的增殖、迁移及对吉西他滨的抵抗。. 进一步地，联合RNA-seq和RIP-seq探究PUM2发挥促进胰腺癌吉西他滨耐药过程中所调控的下游RNA，我们发现黏着斑（FAK）通路中的数个基因（ITGA3、ADAM17、ASAP1等）在敲低PUM2后表达显著下降，且RIP-seq中存在上述基因3'UTR区的Peak，并通过qRT-PCR验证了PUM2对FAK基因集中数个基因表达量的影响，进而利用RIP-qPCR验证PUM2可以结合上述基因RNA的3'UTR区，最后我们利用放线菌素D进行RNA稳定性实验，发现PUM2可以上调基因mRNA的稳定性。. ITGA3是上述结果中受PUM2调控最显著的下游基因，因此我们进一步利用双荧光素酶报告实验和MS2 RNA沉淀实验验证了PUM2蛋白与ITGA3 mRNA 3'UTR区的结合，拯救实验证实了ITGA3是PUM2发挥调控胰腺癌吉西他滨耐药功能的下游分子。. 最后，我们探究了调控PUM2表达的上游转录因子，通过对PUM2 RIP-seq中的转录因子、敲低PUM2后表达下调的转录因子、预测的PUM2上游转录因子取交集，发现EGR1既是PUM2的上游转录因子、PUM2也可以调控EGR1 mRNA的稳定性，我们利用qRT-PCR、Western、RIP-qPCR、ChIP-qPCR对EGR1与PUM2的相互调控作用进行了验证。. 综上所述，本课题筛选出与胰腺癌吉西他滨耐药密切相关的RNA结合蛋白PUM2并探究了其影响胰腺癌吉西他滨耐药的具体机制，对未来胰腺癌患者化疗方案的筛选、探索逆转吉西他滨耐药联合用药方案提供了扎实的理论基础。