NrP1在非小细胞肺癌辐射抗性中的作用机制及放疗分子标志物的可行性研究

It was demonstrated in previous researches that tumor radiosensitivity might be mainly related to the differences of tumor heterogeneity, individual heredity and tumor gene expression profiling. The effective screening of molecular markers in lung cancer radiotherapy and the identification of their biological function could determine the non-small-cell lung carcinoma (NSCLC) treatment protocols and its prognosis. Our previous studies have demonstrated that the expression of NrP1 could up-regulate not only in immune radioreaction, but also in radioresistant tumor cells. In order to explore the mechanisms of NrP1 in the radioresistant NSCLC and its feasibility as molecular marker in radiotherapy, the work below will be studied in the present research. ① The expression of NrP1+Treg in NSCLC tissue will be observed, the relationship between tumor tissue infiltrated Treg cells and pathological characteristic or prognosis in clinic will be analysed, and the mechanisms of NrP1 in radioresistance or tumor in environmental immunoregulation will be investigated. ② The over-expression plasmid (NrP1high) and interference plasmid (shRNA- NrP1) will be constructed using gene recombination technique. And the NrP1 over-expressed and interfered cell lines will be induced by the plasmids in vitro. The radioresistant cell line (H1299-RR) will be constructed. The immunoregulation effect, molecular mechanism and signal network of Treg cells in lung cancer radiotherapy will be approached using three dimensional cultural method. ③ The specificity of NrP1 in different tumor cells and NSCLC cells with different radiosensitivities will be detected, and it will be determined as the molecular marker in radiotherapy. ④ The specific inhibitor of NrP1 (miR-9) will be determined with bioinformatics and molecular biology methods. The targeted relationship between miR-9 and NrP1 will be confirmed. The effect of proliferation, migration and infestation in constructed H1299 cell lines induced by miR-9 (targeted regulate NrP1) will be observed in vitro, and the mechanisms of NrP1 in radioresistance and signal pathway with miR-9 targeted regulation will be investigated in vivo (tumor-bearing mice model). ⑤ The DNA-methylated epigenetic regulation of miR-9 in H1299RR cell line with miR-9 over-expression in vitro and in tumor-bearing mice model in vivo will be confirmed by two different kinds of demethylation methods (using 5-aza-2'-dc and shRNA-DNMT1 plasmid, respectively). The effect of immuno-regulation in lung cancer microenvironment and its mechanism will be approached at the same time. The NSCLC pathological staging, prognosis demonstration and radiotherapeutic effect of NrP1 will be investigated in the present research. These observations may set the stage for developing individualized treatment protocols and new molecular target-specific inhibitors in lung cancer.

肿瘤的放射敏感性与肿瘤异质性、个体遗传差异、肿瘤基因表达谱等有关。有效的肺癌放疗分子标志物的筛选及其生物学功能的深入鉴定，有利于NSCLC治疗方案的细化和预后。我们的先期研究发现，NrP1在免疫系统的辐射反应中表达增高的同时在辐射抗性肿瘤细胞中表达增强。本项目在以往研究基础上拟开展以下工作：首先分析NSCLC组织的NrP1表达水平与临床病理特征及预后关系及肿瘤微环境调节机制，研究 NrP1表达对NSCLC细胞的增殖、迁移、凋亡、细胞周期，荷瘤生长、转移等的影响以及相应的分子机制和信号网络；其次检测NrP1在不同肿瘤和不同辐射敏感性细胞中的特异性；最后阐明 NrP1 的特异性阻断剂-miR-9增强NSCLC细胞放射敏感性作用及其DNA甲基化机制。本研究阐明 NrP1 在NSCLC 病理分期、预后和放疗疗效中的作用机制，为肺癌个体化治疗方案的制定、进一步研发新型分子靶向特异性阻断剂提供基础。

1. NRP1增强肺癌细胞辐射抗性的影响及其信号通路机制.本研究证实了电离辐射作用后NRP1可以使肺癌细胞的增殖、克隆形成、抗凋亡、侵袭迁移及DNA损伤修复能力增强；进一步利用生物信息学方法筛选并验证，NRP1可以通过激活下游信号通路（VEGF、PI3K-Akt、MARK-ERK、P38、NF-κB及TGF-β）参与NSCLC细胞辐射抗性的形成。.2. 探讨靶向抑制NRP1基因增强肺癌细胞放射敏感性.本研究从体外实验和荷瘤裸鼠模型以及临床病理标本，证实了NRP1在NSCLC细胞产生辐射抗性中的作用及其分子机制。结果提示，①靶向抑制NRP1可以协同辐射明显抑制移植瘤的生长，其抑瘤作用可能与辐射的直接杀伤作用及抑制NRP1后一系列信号通路（PI3K-Akt、MAPK-ERK、NF-κB 及TGF-β）的改变有关。② NRP1的特异性阻断剂（miR-9）可以抑制 A549细胞的增殖、迁移及侵袭能力，并通过上述多条信号传导通路提高 A549 细胞的放射敏感性。在荷瘤裸鼠模型中同样显示，miR-9可以抑制NSCLC产生辐射抗性中NRP1的作用，从而抑制肺转移。③临床NSCLC病理标本的回顾性分析显示，在肺癌组织中miR-9表达与NRP1趋势相反，并且肺癌中miR-9表达水平男性高于女性， NRP1的表达与病理分型（腺癌高于鳞癌）及淋巴结转移有关联。.3. 肺癌炎性微环境和迁移微环境在NRP1诱导的辐射抗性中作用.本研究采用三维细胞培养方式建立共培养体系，探讨肿瘤微环境在NRP1诱导肺癌细胞辐射抗性中的作用机制。发现NRP1可通过对肿瘤微环境中部分炎性因子（TNF、IL-10、IL-8和IL-6）以及相关趋化因子（MCP-1、IP-10、CXCL8和RANTES等）分泌产生影响，进而对肺癌细胞的免疫耐受和迁移能力等方面发挥重要作用。.4. 探讨miR-9靶向调控NRP1增强肺癌放射敏感性的DNA甲基化机制.本研究探讨miR-9增强肺癌细胞放射敏感性的DNA甲基化机制，发现辐射可能通过DNMT1的表达增强肺癌细胞中miR-9的DNA启动子甲基化，进而增强肺癌细胞放射敏感性。.本研究首次证实了靶向抑制NRP1可以提高A549细胞的放射敏感性，为其作为NSCLC放疗增敏药物靶点的研究提供实验依据与理论基础。并且已在学术期刊上发表11篇论文，其中二区SCI文章一篇，三区SCI一篇。