肺腺癌中双链RNA 依赖的蛋白激酶介导的能量代谢变化及相关机制研究

It is of great significance for cancer related energy metabolism. We have confirmed that Double-stranded RNA-dependent protein kinase (PKR) is an independent prognostic variable in patients with non–small cell lung cancer (NSCLC),which can inhibit the protein synthesis and promote the apoptosis. On the other hands, We previously found PKR plays an essential role in glycolysis and regulates AMP-activated protein kinase (AMPK) signal pathway. What’s more important, we found that PKR up regulated p-AMPK Thr172 and p-AMPK Ser485, down regulated total AMPK, inducing the appearance of multiple, phosphatase-sensitive, low-mobility forms of the AMPK α-subunit. As a result, we suppose that PKR/AMPK can predict the prognosis of lung adenocarcinoma as a combined metabolism related biomarkers; PKR can activate AMPK by inhibiting ATP/AMP ratio, resulting in inhibiting cellular glucose uptake and lactate level. This is the reason why PKR can inhibit protein synthesis and promote the apoptosis of lung cancer cells. We plan to explore the PKR/AMPK expression and Metabolic enzymes expressions in the paired primary lung adenocarcinoma tissues, tumor-adjacent tissues and normal lung tissues. Then we explore the glucose uptake, lactate and ATP level, ROS expression in adeno-virus transfected PKR in lung adenocarcinoma cells. At last we try to confirm the directly and indirectly mechanism of activation of AMPK by the PKR and the effect of energy metabolism values. We aim to confirm the target value and mechanism of PKR/AMPK pathway in the effect of engery metabolism of lung adenocarcinoma.

能量代谢在肿瘤领域的研究日趋重要。我们首次发现双链RNA依赖的蛋白激酶PKR在肺腺癌细胞糖酵解等代谢方面有显著作用，且可通过AMPK代谢途径调控肺癌细胞生长，证实PKR可促进肺癌细胞凋亡，其高表达的肺癌患者预后较好，但具体机制未明确。我们推测PKR/AMPK可预测肺腺癌患者的预后，PKR通过降低ATP激活AMPK，进而抑制葡萄摄取及糖酵解，最终抑制蛋白质合成、促使细胞凋亡，因而可预测肺腺癌预后。我们将检测配对肺腺癌原发灶、癌旁组织、正常肺组织中PKR/AMPK及能量代谢酶的表达，明确该联合标记物在预测肺腺癌预后的价值;在此基础上采用腺病毒转染PKR建立肺腺癌过表达细胞株，研究PKR作为能量代谢关键分子引起葡萄糖代谢过程中ATP、活性氧簇、代谢酶等变化的规律，以及对AMPK的调节机制和后续能量代谢及细胞增殖变化；探讨PKR在肺腺癌中能量代谢的作用,初步探索PKR/AMPK通路靶向治疗的意义。

我们首次发现双链RNA依赖的蛋白激酶PKR在肺腺癌细胞 糖酵解等代谢方面有显著作用，且可通过AMPK代谢途径调控肺癌细胞生长，证实PKR可促进肺 癌细胞凋亡，其高表达的肺癌患者预后较好。因此，我们提出“PKR/AMPK 为预测肺腺癌预后的联合肿瘤标记物，且PKR通过消耗ATP激活AMPK的能量代谢途径，后续的能量效应为抑制葡萄糖代谢及糖酵解，最终抑制肺腺癌细胞的生长”的假说，旨在阐明 PKR 在肺腺癌相关的肿瘤能量代谢相关性变化，为寻找预测肺腺癌可靠的联合肿瘤标记物并探索相关机理、靶向应用代谢相关靶点药物如 AMPK 激活剂二甲双胍、糖酵解抑制剂的应用提供实验基础。结果显示，在腺癌患者中，根据PKR和pAMPK172不同的表达组合，生存差异明显（P<0.0001）。在肺癌脑转移患者中，PKRhigh/AMPKhigh(37.3%)的总生存明显比PKRhigh/AMPKlow (23%),PKRlow/AMPKlow (13%), 以及PKRlow/AMPKhigh (15%)患者的总生存高 (p=0.039)。2-NBDG葡萄糖摄取实验、乳酸水平测定及ATP测定证实Ad Wt-PKR抑制细胞内葡萄糖、乳酸摄取，降低ATP，从而增加AMP而激活AMPK。与腺病毒转染的Ad mutant PKR，Ad Luciferace，及对照物处理蛋白相比，Ad Wt-PKR能明显上调p-AMPK Thr172及p-AMPK Ser485的表达，下调总AMPK的表达。功能方面，PKR抑制自噬相关蛋白，上调内质网应激的各蛋白。流式细胞仪显示CompounC联合 Ad Wt-PKR处理后，肺癌Sub G1期死亡细胞比例高于二者单独处理组比例之和，两者有协同作用。综上所述，PKR/AMPK组合对NSCLC预后有重要的价值，而且对肺癌脑转移有预测预后的价值。PKR通过能量代谢的改变激活AMPK的表达，使后者在多个位点磷酸化而被激活，从而负反馈抑制PKR介导的细胞死亡。,本研究提示,PKR在肺腺癌的能量代谢中有重要作用，且对于脑转移的患者，有潜在的预测预后的价值，值得进一步深入研究。本项目培养研究生三名,本科生一名；发表SCI文章(IF5分以上)两篇，2分以上文章1篇，中文综述1篇，另有一篇SCI论文正在投稿；专利一项。