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

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。

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.