MAPK/JNK信号通路介导NEIL1影响放射性肺炎发生的机制研究

Human DNA glycosylase NEIL1 (Nei-like protein 1) plays a critical role in base excision repair pathway. We have demonstrated that NEIL1 rs4462560 single nucleotide polymorphism has significant association with risk of acute radiation-induced esophageal toxicity and radiation pneumonitis. Previous studies showed that NEIL1 was correlated with the JNKI kinase, and MAPK/JNK signal pathway was a critical member enrolled in development of inflammation and fibrosis. We reason that NEIL1 may predict the risk of radiation-induced toxicities via MAPK/JNK signal pathway, which may contain a potential target. Base on these evidences, we are to determine the molecular mechanism of NEIL1 rs4462560 effecting the incidence of radiation pneumonitis from the level of expression of mRNA and proteins, apoptosis, DNA fracture, and epithelial-mesenchymal transition, using both cell culture and mouse models. We propose to establish the basic research for individual radiotherapy and clinical drug intervention for patients with high risk of radiation pneumonitis.

NEIL1是参与碱基切除修复通路的重要成员。申请人前期研究结果表明NEIL1单核苷酸多态性rs4462560与食管癌放疗中出现的急性放射性食管炎、肺炎存在明显相关。通过对国内外文献分析，发现JNK1与NEIL1相关，而MAPK/JNK信号通路是参与炎症及纤维化发生的重要成员，推测NEIL1 rs4462560通过MAPK/JNK信号通路介导影响放射性肺炎的发生，JNK1可能为NEIL1发挥功能的靶点。在此基础上，本课题拟从mRNA、蛋白表达、细胞凋亡、DNA损伤，肺泡上皮细胞间质转化等不同水平，从体内体外不同层面，全面阐明NEIL1 rs4462560通过MAPK/JNK信号通路影响放射性肺炎发生的作用及其分子机制。本项目的完成将为今后更深入探索临床药物干预，定制放射损伤易感患者个体化放疗方案提供研究基础。

放射性肺损伤为胸部肿瘤放射治疗会NEIL1是参与碱基切除修复通路的重要成员。前期临床研究结果表明NEIL1单核苷酸多态性rs4462560与食管癌放疗中出现的急性放射性肺炎存在明显相关。本项目通过CRISPR/Cas9技术成功构建了A549细胞NEIL1 rs4462560突变表型（NC_000015.10: g.75355623C>G）。通过对A549细胞NEIL1 rs4462560突变表型及野生表型的蛋白表达水平检测发现该突变为功能性突变，两组细胞蛋白表达水平明显差异。通过细胞凋亡，DNA损伤，克隆形成等一系列试验证实10Gy X放射线照射下不同NEIL1 rs4462560表型的A549细胞存在显著的放射敏感性差异，NEIL1 rs4462560突变表型放射性损伤较野生型显著增加。进一步通过检测磷酸化MAPK及磷酸化JNK，JNK抑制剂SP600125干预下蛋白表达、细胞凋亡及DNA损伤的变化等，从不同角度阐明NEIL1 rs4462560通过MAPK/JNK信号通路影响放射性肺炎发生，达到预期细胞实验结果。此外，成功建立放射性肺损伤小鼠的动物模型，获取放射性肺损伤小鼠肺泡灌洗液，进一步阐明JNK抑制剂SP600125可影响放射性肺损伤发生。同时，收集了本单位临床研究中放疗患者的外周血，设置放射性肺炎组及对照组，并进行GWAS检测，进一步验证NEIL1 rs4462560为功能性放射性肺损伤关键位点。