抑制胞内产生的ROS作用是肿瘤化疗耐药的重要机制。APE1是具有DNA损伤修复功能的核功能蛋白，可核内调控ROS。新近研究发现APE1亦可定位于线粒体，然线粒体APE1是否调节ROS鲜有报道。前期我们发现化疗耐药骨肉瘤中胞浆APE1表达增高；氧化应激后APE1转位至线粒体可抑制细胞凋亡；APE1在线粒体中可调控线粒体电子转运链（ETC）基因抑制凋亡，且ETC是ROS的主要来源。化疗后骨肉瘤中胞浆APE1高表达和ETC调控作用提示核外APE1与骨肉瘤化疗耐药密切相关，而线粒体调控ROS可能是重要作用点。由此我们提出假说：线粒体APE1高表达可通过下调ROS水平介导骨肉瘤化疗耐药。本项目通过建立骨肉瘤耐药细胞株和裸鼠移植瘤模型，检测耐药和亲本细胞表型及线粒体APE1表达，结合干扰表达、WB、ROS检测等方法，研究体内外线粒体APE1对ROS调控及其机制，为临床解决骨肉瘤化疗耐药问题提供新思路。

The inhibition effect of intracellular reactive oxygen species (ROS) generation is an important mechanism of cancer chemoresistance. Apurinic/ apyrimidinic endonuclease 1 (APE1) is a nuclear protein with DNA damage repair activity which regulates nuclear ROS. The recent research indicated that APE1 could also be localized in mitochondria; however, it was unknown whether mitochondrial APE1 can regulate ROS. In previous studies, we found that the cytoplasmic APE1 expression increased with the resistance to chemotherapy in osteosarcoma; after the oxidative stress, the translocation of APE1 to mitochondria could inhibit cell apoptosis; APE1 in mitochondria could regulate mitochondrial electron transport chain (ETC) and further suppress the apoptosis; furthermore, ETC was the main source of ROS. After the chemotherapy, the high cytoplasmic APE1 expression in osteosarcoma and regulatory effects of ETC suggested that cytoplasmic APE1 and osteosarcoma chemotherapy resistance were closely related. Thus, the regulation of mitochondrial ROS may play an essential role. Therefore, we raised a hypothesis that the high expression of mitochondrial APE1 downregulates the ROS level further promotes drug resistance in osteosarcoma chemotherapy. Through the establishment of xenograft model of osteosarcoma multidrug resistance, this project will detect the resistant and parental cell phenotype and the expression of mitochondrial APE1, combined with research methods, including RNA inference, Western blot,ROS detection etc, and will carry out studies in the regulation of mitochondrial APE1 on ROS in vivo and in vitro and the molecular mechanism, in order to provide new strategy to overcome the clinical drug resistance in osteosarcoma chemotherapy.

本项目按照计划对线粒体APE1调控ROS产量并参与顺铂耐药的分子机制进行了研究。项目首先基于对现有的骨肉瘤细胞株对顺铂敏感性的分析，通过剂量递增诱导的方法构建了耐顺铂的骨肉瘤细胞株。同时，我们构建了APE1敲低和线粒体过表达的骨肉瘤细胞，并进行了顺铂敏感性的检测。在这些细胞模型构建的基础上，我们利用Western blot和荧光染色等方法对APE1通过Rac1磷酸化影响ROS产量的机制进行了分析。现有的研究结果提示，在顺铂抵抗的骨肉瘤细胞株中，APE1表达及线粒体APE1分布均呈显著上调，并且敲低APE1可以恢复抵抗细胞对顺铂的反应性。在线粒体特异性过表达的APE1细胞株中，可以显著提高对顺铂的耐受性，并且可以显著增加Rac1的磷酸化水平并且降低细胞内ROS的产生。本项目证实了骨肉瘤化疗增加了APE1的线粒体转运，并通过抑制顺铂作用后产生的ROS而增加骨肉瘤细胞对顺铂的耐受性。本研究亦进行了一些探索性的研究，针对耐顺铂细胞株模型进行了转录组测序，并分析了其mRNA，miRNA和lncRNA的表达特征，通过生物信息学分析对影响顺铂耐药的信号通路和代谢过程进行了探索，以期更为全面的阐明骨肉瘤细胞对顺铂抵抗的分子机制。