抗菌肽merecidin通过JNK信号途径抑制非小细胞肺癌的分子机制研究

As the side effects and resistance of chemotherapy drugs, so the development of highly effective drugs to conquer lung cancer is urgently. Through the previous study of our group has found: Antimicrobial peptide merecidin which derived by LL-37 inhibits the growth of non-small cell lung cancer A549 through the apoptotic pathway. Furtherly, we found that the expression of JNK signaling pathway was significantly different when merecidin was acted on A549. So we put forward the hypothesis: Through the JNK signaling pathway, apoptosis and autophagy was caused by antimicrobial peptide merecidin. In order to prove this hypothesis we designed the following experiment: First, Western-Blot and other series of experiments were used to demonstrate that apoptosis and autophagy were mediated by antimicrobial peptides through the JNK signaling pathway. The second step is to observe the changes in the downstream genes and protein expression when overexpressed and knocked out the key genes in the JNK signaling pathway. To achieve this goal, lentivirus systems and CRISP / CAS9 overexpression techniques were used separately. Finally, animal experiments were conducted to study the effect of antimicrobial peptides on tumor growth in nude mice transplanted tumor model. Using molecular, cellular and animal experiments in the above, the mechanisms of apoptosis and autophagy caused by merecidin through the JNK signaling pathway is described in detail.

由于化疗药物存在副作用强和引发耐药性问题,故研发高效抗癌药物是征服肺癌所面临的迫切要求。课题组前期研究发现：LL-37衍生抗菌肽merecidin可能通过细胞凋亡途径抑制非小细胞肺癌A549细胞的生长，且merecidin作用于A549细胞后JNK信号通路的相关基因的表达差异大，故我们提出假说：抗菌肽merecidin通过JNK信号通路介导细胞凋亡并影响细胞自噬，为证明该假说特设计以下实验：首先通过WB等系列实验证明抗菌肽是通过JNK信号通路介导细胞凋亡和自噬，其次分别利用慢病毒系统和CRISP/Cas9技术过表达及敲除JNK信号通路中关键的基因JNK，观察在过表达和敲除之后JNK信号通路下游基因和蛋白表达的变化情况，最后通过动物实验研究抗菌肽抑制移植瘤模型裸鼠的肿瘤生长情况。通过上述分子、细胞、动物实验，详细阐述merecidin通过JNK信号途径在细胞凋亡和自噬过程中的作用机制。

抗菌肽merecidin在体外具有良好的抑制肺腺癌A549细胞的活性，但是具体机制不得而知，课题组首先利用蛋白质组学分析技术检测了抗菌肽作用于A549细胞后细胞内所有蛋白质的磷酸化水平变化来确定抗菌肽对于肺癌A549细胞生物学功能的影响以及可能涉及到的信号通路及作用靶点，磷酸化蛋白组学最终检测出经抗菌肽merecidin处理后，差异磷酸化蛋白共有753个，参与的主要的细胞信号通路有：PI3K-Akt，mTOR，AMPK和Wnt等。选取其中变化水平比较明显的MAPK1分子作为研究对象，利用慢病毒包装系统在人肺癌A549细胞中成功构建MAPK1过表达稳定转染株；利用CRISPR/CAS9系统在人肺腺癌细胞A549中成功构建永久敲除的MAPK1敲除细胞系。利用CCK8实验、划痕实验、Transwell小室实验检测其侵袭能力结果显示过表达MAPK1可以促进肺癌A549细胞的增殖、迁移、侵袭；敲除MAPK1抑制肺癌A549细胞增殖、迁移、侵袭；利用透射电镜及MDC、AO、CYTO-ID染色检测抗菌肽merecidin之后自噬小体的变化情况结果发现：merecidin处理细胞后，处理组A549发现被双层膜包裹细胞器自噬小体和自噬溶酶体。利用Western Blot实验检测MAPK/ERK信号通路中 MAPK1上游、下游蛋白ERK1/2、RSK1、MEK2、ETS-1、 c-jun等蛋白及涉及到自噬相关信号通路的蛋白ATG5、ATG7、LC3II、ULK1、ATG13等表达变化情况，实验结果显示：ATG5、ATG7、LC3II、ULK1、ATG13蛋白表达上调，P62、MAPK1蛋白表达下调。实验结果说明抗菌肽merecidin可能通过靶向MAPK1调控MAPK/ERK信号通路从而对肺癌A549的增殖、迁移、侵袭、自噬生物学功能等功能起到调控作用。