敲除Pim-3基因表达逆转人胰腺癌细胞化疗耐药的作用和机制研究

Pancreatic cancer due to its characteristics of the disease, the cure rate of surgical resection of pancreatic cancer is less than 10%. In a variety of conservative treatment of pancreatic cancer, Gemcitabine (GEM)-based combination chemotherapy has improved the cure effect of the pancreatic cancer, but the results are not so satisfied. Tumor chemo-resistance remains is one of the biggest obstacles to cancer therapy. Thus, a better understanding of the origins of chemo-resistance is critical to the development of superior combination therapies or the replacement of gemcitabine as the gold standard in pancreatic cancer. Our previous observations suggested that Pim-3 plays an important role in gemcitabine resistance in human pancreatic cancer cells. However, the direct functional mechanism is not fully characterized. Therefore, we propose such a hypothesis that Pim-3 may lead to pancreatic cancer chemo-resistance occur through the STAT3 signaling pathway. To characterize this hypothesis, we will establish gemcitabine-resistant, or stably expressing of Pim-3, or stable knockout of Pim-3 expression cell lines of human pancreatic cancer. We will investigate the roles of Pim-3 in chemo-resistance in human pancreatic cancer from the views of molecular, cells, tissues and animals level experiments, by the in vitro and in vivo experiments with the conditions of normoxia and hypoxia. We will identify the mechanism that Pim-3 through the activation of the STAT3 signaling pathway, mediated expression of anti-apoptotic and resistance-related genes involved in pancreatic cancer chemo-resistance. This project will establish the theoretical basis for developing Pim-3 molecule target therapy, and it is necessary to develop combination therapy to improve the treatment of pancreatic cancer.

胰腺癌由于其发病特点，手术切除率不足10％。在胰腺癌的多种保守治疗方法中，以吉西他滨（GEM）为基础的联合化疗方案效果最佳。但是，胰腺癌耐受GEM治疗仍旧是长期困扰医患的最大课题，亟待早日解决。发现并且利用有效调控胰腺癌耐受GEM的关键分子可以克服胰腺癌耐药性，对于胰腺癌治疗及预后评估具有重要的转化应用价值。本课题组前期实验结果显示，具有丝氨酸/苏氨酸激酶活性的原癌基因，Pim-3，在人胰腺癌细胞耐受GEM作用过程中起着重要作用，并且假说其作用机制为：Pim-3通过STAT3信号通路导致胰腺癌耐药的发生。本申请项目将通过构建人胰腺癌耐药细胞株，Pim-3稳定表达和Pim-3稳定敲出的人胰腺癌细胞株，利用体外和体内实验，分别在常氧和乏氧条件下，从分子、细胞、组织以及动物整体水平等多方面验证上述假说，为开发Pim-3分子靶向治疗药物奠定理论基础，为提高胰腺癌治疗效果提供新的思路。

胰腺癌由于其发病特点，手术切除率不足10％。在胰腺癌的多种保守治疗方法中，以吉西他滨（GEM）为基础的联合化疗方案效果最佳。但是，胰腺癌耐受GEM治疗仍旧是长期困扰医患的最大课题，发现并且利用有效调控胰腺癌耐受GEM的关键分子可以克服胰腺癌耐药性，对于胰腺癌治疗及预后评估具有重要的转化应用价值。本项目在前期研究的基础上，首先明确了吉西他滨可以诱导胰腺癌细胞Pim-3 的mRNA 水平和蛋白水平表达上调，提示Pim-3 可能参与了吉西他滨耐药。体内外研究结果显示，过表达Pim-3 可以拮抗吉西他滨介导的肿瘤生长抑制和细胞凋亡；相反地，胰腺癌细胞敲除Pim-3 的表达，对吉西他滨的细胞毒作用起到增敏效果。间歇浓度递增法诱导建立的胰腺癌吉西他滨耐药细胞株中， Pim-3 蛋白表达上调的同时，多药耐药基因编码的蛋白P-gp、抗凋亡蛋白分子Bcl-XL、细胞周期相关分子蛋白Cdc25A、Cdc25C 表达也增加。其次，我们研究发现胰腺癌患者的血浆中miR-33a 的表达水平低于健康对照人群血浆中水平。并且，明确了胰腺癌患者血浆中的miR-33a 的水平，与组织内的表达水平，以及与胰腺癌患者总体生存时间存在着一定的正相关关系。过表达miR-33a 在体内、外都可以有效抑制细胞增殖，促进细胞凋亡及增强胰腺癌细胞对吉西他滨的敏感性。综上所述，我们阐明了Pim-3 通过调节细胞增殖，细胞周期和凋亡，参与胰腺癌吉西他滨的耐药。进一步明确了miR-33a 通过Pim-3 介导，抑制AKT/Gsk-3β/β-catenin 的信号通路，从而抑制胰腺癌细胞生长及逆转吉西他滨耐药。并且提示，检测miR-33a在血浆中的水平有望成为一个有价值的生物学监测指标，并对胰腺癌预后有着重要的指导作用，也为难治性胰腺癌吉西他滨耐受患者提高疗效提供了新思路。