前列腺癌NF-κB炎症通路与ZFP36负调控糖代谢研究

NF-κB signaling is a key pathway that regulates the reprogramming（Warburg effect）of cancer metabolism. Our previous study have proved that miR-23b promoted glycolysis metabolism in PCa via upregulating the expression of PRDX3 (FEBS Lett. 2012), and have found that the activation of SOCS3-NF-κB pathway in prostate cancer was associated with patient's prognosis (Mol Cell Biochem. 2013). However, the underlying mechanism of NF-κB negative regulatory function during the procedure of glucose metabolism reprogramming in cancer is not fully understood. We further observed that the expression of ZFP36 was increased in the inflammatory model of tumor cells stimulated by LPS（Clin Transl Oncol.2015）. Ristetraprolin, also known as zinc finger protein 36 homolog (ZFP36), is a new mechanism for the degradation of mRNA. In addition, we have discovered the metabolic target genes (ENO2, PDK4, MYC) of ZFP36 through software prediction. Therefore, we hypothesized that “the NF-κB activation in tumor cell will increase the expression of ZFP36, which in turn negatively regulate metabolic genes involved in the metabolism reprogramming”. We intend to use RNA-EMSA and double fluorescence report approaches to verify the downstream target molecules negative regulated by ZFP36. Glucose metabolism test, animal experiments and clinical sample will be used to validate and illustrate the effect of ZFP36 negative regulatory pathway on tumor metabolism. This study could provide the scientific basis for finding new therapy target and prognosis biomarker of prostate cancer.

NF-κB是肿瘤细胞代谢重编程（Warburg效应）的关键通路。我们前期研究发现前列腺癌miR-23b上调PRDX3促进肿瘤的糖酵解代谢，并证实NF-κB/SOCS3的表达与患者的预后密切相关（Mol Cell Biochem. 2013）。但肿瘤细胞NF-κB重编程中负调控的机制和作用是什么？我们进一步在LPS刺激的肿瘤细胞炎症模型中观察到ZFP36的表达升高（Clin Transl Oncol.2015）。ZFP36是mRNA降解的一种新机制，通过软件预测获得ZFP36降解的代谢靶基因(ENO2、PDK4、MYC)。据此，我们提出“ZFP36负调控代谢靶基因，参与NF-κB代谢重编程通路”的假设。拟采用RNA-EMSA和双荧光报告等验证ZFP36负调控的下游代谢靶分子，糖代谢检测、动物实验和临床样本验证以阐释ZFP36负调控通路对肿瘤代谢影响，为前列腺癌的治疗新靶点提供可靠的科学依据。

本项目前期通过Taylor数据库发现ZFP36在前列腺癌无生化复发中具有良好的预后意义，结合大量文献报道及ZFP36的功能：ZFP36对mRNA的稳定性起负调节作用，其能够结合并靶向作用于含有ARE的mRNA，然后快速降解mRNA水平，因此ZFP36在前列腺癌中可能扮演抑制肿瘤生长的作用。我们进一步在Taylor和TCGA数据库中分析发现ZFP36的表达水平与前列腺肿瘤的恶性生物学特性存在负相关。因此我们构建了过表达与敲低的前列腺癌细胞株：DU145和22RV1细胞株，在体外通过细胞功能实验证明了ZFP36能抑制肿瘤的增殖、侵袭和迁移能力，并进一步通过裸鼠移植瘤模型证明ZFP36在体内同样可以抑制肿瘤细胞的生长。而ZFP36如何调控下游基因发挥抑癌作用，我们进行了高通量测序，并对ZFP36调控相关通路的下游基因进行了筛选，我们发现CDK6是ZFP36的下游靶基因，并在细胞株、动物组织上通过Q-PCR进行了mRNA水平上的验证，同时也通过Western blot进行了蛋白水平上的验证，两者均表明ZFP36可以负调控CDK6的表达，此外，通过双荧光素酶实验及RNA免疫共沉淀验证了CDK6是ZFP36的下游靶基因。CDK6是细胞周期信号通路中的重要组分，此外，细胞周期实验结果表明ZFP36是将肿瘤细胞周期阻滞在G1期，从而抑制肿瘤生长，综上所述，我们构建ZFP36过表达和敲低细胞株，采取细胞、动物实验证明ZFP36参与前列腺癌的进展，通过荧光素酶报告、免疫共沉淀等实验揭示ZFP36负调控CDK6参与前列腺癌细胞周期进展的作用网络，新鉴定的ZFP36-CDK6轴部分说明了PCa进展的分子机制，并代表了PCa治疗的新型潜在治疗靶标，为前列腺癌治疗寻找有效的新靶点和新策略。