抑癌基因CMTM7调控非小细胞肺癌自噬影响其放疗敏感性的作用及机制研究

Radiotherapy is one of the main means of the radical treatments for non-small cell lung cancer (NSCLC), but the local control and the prognosis of NSCLC patients remains poor. It is important to understand the mechanisms of radiotherapy resistance for patients with localized advanced NSCLC. Recent researches have demonstrated that EGFR-AKT signaling pathway negatively regulates autophagy in NSCLC, leading to decreased radiosensitivity. CMTM7, a tumor suppressor gene, has been shown to be down-regulated in lung cancer, exerting a tumor suppressive function by inhibiting EGFR-AKT signaling pathway. Preliminary studies have indicated that overexpression of CMTM7 promotes the autophagy in HeLa cells, while silencing of CMTM7 depresses the autophagy in A549 cells. The autophagy of MEF cells derived from Cmtm7 knockout mice is also inhibited. Further, radiotherapy increases CMTM7 expression in NSCLC A549 cells, and CMTM7 knockdown can decrease the radiation-induced LC3B-II accumulation. Therefore, CMTM7 might probably influence the radiosensitivity of NSCLC by regulating the autophagy. The present project will employ multiple methods of virus expression vectors, CRISPR-Cas9 and Cmtm7 knockout mice to investigate the function of CMTM7 in the radiation-induced autophagy in NSCLC cells and the resulting impact on the radiosensitivity in vitro and in vivo. The role of EGFR-AKT signaling pathway in the process will also be examined. In summary, this project is original and feasible with solid preliminary work, which may provide a new target for the prediction or even improvement of the radiosensitivity of NSCLC.

放射治疗是非小细胞肺癌（Non-small cell lung cancer, NSCLC）根治性治疗的主要手段之一，但局部控制和预后未显著提高，因此亟待探索NSCLC放疗抵抗机制。最近研究发现EGFR-AKT通路负调控NSCLC自噬从而抑制其放疗敏感性。抑癌基因CMTM7在肺癌中表达下调，通过抑制EGFR-AKT发挥抑癌功能；初步结果提示超表达/敲减CMTM7能够促进/抑制细胞自噬。放疗后NSCLC细胞中CMTM7表达量增加，敲减CMTM7可降低放疗诱导的LC3B-II水平。因此，CMTM7极有可能通过调控NSCLC细胞自噬影响其放疗敏感性。本项目将利用病毒载体、CRISPR-Cas9、基因敲除小鼠等方法干预CMTM7表达，通过体内外实验研究其通过EGFR-AKT通路调控自噬在NSCLC放疗敏感性中作用。本项目基础扎实，延续性好，可能为预测甚至提高NSCLC放疗敏感性提供新的靶标。

放射治疗是非小细胞肺癌（Non-small cell lung cancer, NSCLC）根治性治疗的主要手段之一，但局部控制和预后未显著提高，因此亟待探索NSCLC放疗抵抗机制。我们前期结果提示CMTM7极有可能通过调控NSCLC细胞自噬影响其放疗敏感性。本项目中，我们首先证明CMTM7促进了自噬体形成的启动，从而促进了自噬流的后续多阶段过程，即从自噬体组装到自体溶酶体形成和降解。共聚焦和共免疫沉淀显示，CMTM7与Rab5，VPS34，Beclin1和ATG14L相互作用，但与ULK1，UVRAG和LC3B不相互作用。重要的是，CMTM7增加了ATG14L-VPS34复合物的活性及其与Rab5的结合力。体外和体内实验均表明，敲低CMTM7可通过抑制自噬从而促进肿瘤生长。以上结果表明CMTM7在自噬和致瘤性中起重要作用，揭示了CMTM7是Rab5和ATG14L-Beclin1-VPS34复合物相互作用新调控分子。进一步，利用CRISPR-Cas9技术在高表达CMTM7的非小细胞肺癌A549中敲除CMTM7，利用CMTM7敲除的A549细胞，体内体外证明了CMTM7通过EGFR-AKT信号通路调节的自噬增加非小细胞肺癌放射敏感性。发表课题相关论文2篇，选派2人参加2018-2020年召开的Astro放疗大会，并做口头发言及壁报展示。本项目的研究结果为非小细胞肺癌放疗疗效研究提供新的线索，进一步阐明自噬与放疗敏感性的关系，并为预测甚至提高 NSCLC 放疗敏感性提供新的靶标。

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