LncRNA-PVT1通过Pygo2调控细胞自噬在胰腺癌吉西他滨耐药中的分子机制研究

The molecular mechanism of acquired gemcitabine resistance for pancreatic cancer is very complicated. It becomes particularly important to clarify, anticipation, and reverse gemcitabine resistance for pancreatic cancer patients. Our previous study showed that exogenous expressed Lnc-PVT1 could up-regulate the level of Pygo2, and then increase the expression of Beclin-1, a protein essential for autophagy initiation, presenting huge possibility for pancreatic cancer cells to acquire gemcitabine resistant through the autophagy pathway. Further study showed Pygo2 was inhibited by the exogenous miR-152 mimic, which contains the binding sites of both Lnc-PVT1 and Pygo2 3' UTR by the bioinformatics analysis. Suggesting the role of Lnc-PVT1 binds to miR-152 and acts as a sponge to competitively inhibit miR-152 from binding to the 3' UTR of Pygo2. Based on our preliminary results, in Aim 1 we propose to clarify the regulation ship between Lnc-PVT1, miR-152 and Pygo2 by using the luciferase reporter assay, RIP, biotin labeled pull-down assay. After that, the role of Lnc-PVT1/miR-152/Pygo2 in autophagy pathway was evaluated using autophagy activation and inhibition agent. In Aim 2 we propose to investigate the regulation ship between Beclin-1 expression and Pygo2. And in Aim 3 we propose to examine the impact of Lnc-PVT1/Pygo2/Beclin-1 signaling pathway to the autophagy activation and gemcitabine resistant for pancreatic cancer in vitro and in vivo by Lnc-PVT1/Pygo2/Beclin-1 over-expression or knockdown. Successful completion of experiments outlined in this proposal will enhance our current understanding of the mechanism and additional pathway for pancreatic cancer to acquired gemcitabine resistant, and may provide novel therapeutic strategies and targets for pancreatic cancer.

胰腺癌对吉西他滨化疗获得性耐药的机制非常复杂。我们发现，吉西他滨处理胰腺癌细胞后，Lnc-PVT1表达升高。Lnc-PVT1能够通过Pygo2蛋白提高自噬基因Beclin-1蛋白的表达，且miR-152同时具有Pygo2和Lnc-PVT1结合位点。据此推测：Lnc-PVT1能够以ceRNA的方式通过miR-152调控Pygo2蛋白的表达，进一步通过Beclin-1促进细胞自噬，而产生较强的吉西他滨耐药性。本课题拟从三个方面进行研究：①Lnc-PVT1通过miR-152调控Pygo2的机制及其对自噬的作用；②Pygo2提高Beclin-1表达并促进自噬的分子机制；③从体内和体外实验2个角度分析Lnc-PVT1/Pygo2/Beclin-1通路激活的细胞自噬对肿瘤耐受的影响。本课题研究成果有助于丰富使用吉西他滨治疗胰腺癌信号调控网络及其耐药理论，更为临床上对胰腺癌的治疗提供全新的思路。

胰腺癌恶性程度很高，5年生存率<5%，预后极差。吉西他滨（2',2'-二氟脱氧胞苷，dFdC）是目前治疗胰腺癌的一线主要化疗药物。然而，吉西他滨在治疗胰腺癌患者的同时，其药物毒副作用及耐药性非常严重。这为我们对胰腺癌的治疗提出了重大挑战。因此，研究胰腺癌吉西他滨耐药性的作用机制，对胰腺癌的治疗及创新药物研发具有重大意义。. 在这项工作中，我们首先在吉西他滨耐药的胰腺癌细胞系中发现了LncRNA-PVT1的表达显著升高，同时在细胞和动物模型中，我们进一步验证了胰腺癌细胞在稳定敲低PVT1后能够改变细胞对吉西他滨的敏感性。根据我们在细胞自噬和Wnt/β-catenin信号通路方面的研究经验，当利用化学试剂羟氯喹CQ和XAV-939抑制细胞自噬或者Wnt/β-catenin信号通路时，PVT1敲低介导的胰腺癌吉西他滨的敏感性受到极大的影响。通过后续一系列的筛选，我们发现，PVT1能够吸附miR-619-5p，使得其在吉西他滨耐药细胞内的水平降低，进而导致miR-619-5p特异性下游靶标Pygo2蛋白和自噬相关蛋白ATG14的水平上调。Pygo2是Wnt/β-catenin信号通路重要的组成成分，该通路的上调能够使耐药基因MDR1的激活，并促进p-糖蛋白表达增加。而ATG14的上调则能够促进自噬信号通路的激活，最终发挥吉西他滨耐药的功能。. 更重要的是，我们还阐明了Wnt/β-catenin和自噬通路在发挥吉西他滨耐药功能时的协同效应。我们发现，在PVT1基因启动子区存在Wnt/β-catenin信号通路典型的TBE结合位点（TCF/LEF binding elements），当吉西他滨处理时，Wnt信号通路的关键组分β-catenin能够通过直接结合到PVT1的启动子区而上调PVT1的水平。 通过后续RIP（RNA immunoprecipitation）及Co-IP实验， 我们确定了细胞质内的PVT1还能够直接与ATG14发生相互作用，促进自噬起始复合体PtdIns3K-C1的组装，并且能够令该起始复合体中VPS34的激酶活性增强。这项研究成果为胰腺癌吉西他滨治疗后的耐药性和创新药物研发提供了扎实的理论基础。