RNA编辑酶ADAR1调控PARP1在胃癌细胞化疗耐药中的作用及机制研究

Gastric cancer is the most common cancer and the main leading cause of cancer mortality in China. Chemotherapy is one of the major treatments for gastric cancer, but drug resistance limits the effectiveness of chemotherapy. Thus, it is urgent to identify and develop new treatment strategies regarding gastric cancer molecular mechanisms involved in chemotherapy resistance. In our previous study, we demonstrated that ADAR1 was frequently overexpressed in gastric cancer and ADAR1 overexpression promoted cell proliferation and migration. Furthermore, we found ADAR1 overexpression enhanced the chemo-resistance of the gastric cancer cells to cisplatin, and vice versa. As well，ADAR1 is overexpressed in cisplatin-resistance cell SGC7901/DDP. Mechanistically, we found ADAR1 may confer chemo-resistance to gastric cancer cells via modulating microRNAs targeting PARP1 mRNA. Accordingly, in this project we will testify the connection between ADAR1 expression and the chemo-resistance of gastric cancer cells by way of cell proliferation experiment, flow cytometry analysis and tumorigenicity assay in nude mice. Subsequently, by using miRNAseq and literature survey, we will find out the miRNA targeting to PARP1 which was regulated or edited by ADAR1. Through dual-luciferase reporter assay and mutagenesis, we will show how the miRNA candidate regulates PARP1 expression. Finally, we will try to demonstrate the relevance between ADAR1/PARP1 and the clinic outcomes, taking it as a valid therapeutic target for the chemo-resistance reversal in patients suffering from gastric cancer.

胃癌发病和死亡在我国均居高位，化疗是其治疗的重要手段，而化疗耐药则严重制约了化疗效果。本课题组前期研究证实RNA编辑酶ADAR1在胃癌组织中高表达并促进瘤体生长，高表达ADAR1诱导胃癌细胞对顺铂的抵抗，沉默ADAR1则降低胃癌细胞对顺铂的抵抗，同时顺铂耐药株SGC7901/DDP中ADAR1呈高表达。进一步研究证实其耐药机制可能是ADAR1通过编辑miRNA介导调控DNA修复酶PARP1的表达来实现。据此，本课题拟通过细胞水平和胃癌PDX裸鼠模型验证ADAR1介导PARP1参与了胃癌细胞耐药；采用miRNAseq技术寻找ADAR1编辑和调控靶向PARP1的miRNAs，并利用双荧光素酶实验和定点突变阐明miRNAs对PARP1的调控；最后采用免疫组化、CTC和RNAscope技术验证ADAR1、PARP1在胃癌组织中的关联及与患者化疗疗效的关系，为逆转化疗耐药找到潜在靶点。

胃癌发病和死亡在我国均居高位，手术是胃癌最关键的治疗方式，然而2/3的病人在被诊断为胃癌时并不能实行手术。所以，以化疗为主的综合治疗是提高胃癌患者生存率的关键因素，而化疗耐药则严重制约了化疗效果。本项目研究证实RNA编辑酶ADAR1具有促进胃癌细胞顺铂抵抗的作用并阐述了其促进顺铂抵抗的分子机制。具体的，CCK-8，流式细胞术和克隆形成实验表明ADAR1高表达促进而敲低表达抑制胃癌细胞对顺铂的耐受，同时顺铂耐药细胞株中ADAR1表达显著升高。通过对凋亡相关分子标记物检测发现，PARP1受到ADAR1调控，接着qPCR和双荧光素酶报告基因实验证实ADAR1在mRNA水平调控PARP1表达。功能互补实验显示PARP1介导ADAR1对顺铂耐药的调控。通过生信分析及分子生物学实验，我们发现miR-302a表达受到ADAR1抑制，而miR-302a靶向抑制PARP1表达，提示miR-302a介导了ADAR1对PARP1的调控。动物模型实验也表明，ADAR1和miR-302a对顺铂抵抗的调控作用。最后在临床样本中，ADAR1表达和PARP1正相关，而miR-302a和ADAR1，PARP1均为明显的负相关，进一步证明了三者之间存在的调控关系。因此，本项目揭示一条全新的信号通路即ADAR1/miR-302a/PARP1信号轴线在顺铂化疗耐药中的作用，为以ADAR1为靶点设计抗癌药物提供了理论基础，具有潜在的应用前景。