基于PI3K-Akt-mTOR介导的糖代谢重编程，探讨培土生金法干预NSCLC顺铂耐药的分子机制

Compared to the normal cells that depend on oxidative phosphorylation metabolic pathways responsible for generating ATP, cancer cells increase glycolysis for their reprogramming of metabolic networks. The carbohydrate metabolic reprogramming of cancer cells is not only the central of tumorigenesis, but also an important reason of tumor drug resistance. And PI3K-Akt-mTOR signaling pathway is the focal point which affects carbohydrate metabolic reprogramming and tumor drug resistance. Traditional Chinese Medicine (TCM) holds that “insufficiency of healthy-Qi” is the principal reason of cisplatin resistance of lung cancer. Early research has shown that the method of strengthening the lung by way of reinforcing the spleen can effectively intervene in cisplatin resistance of non-small cell lung cancer (NSCLC) through inducing apoptosis and autophage. Regarding the PI3K-Akt-mTOR signaling pathway as the entry point, with the model of A549/DDP cells higher in cisplatin resistance and carbohydrate metabolic reprogramming as the study object, by techniques of animal PET-CT, RNAi, real-time PCR and immunoblotting, the project study that the method of strengthening the lung by way of reinforcing the spleen effects intervening cisplatin-resistant cells of NSCLC on inner relation and the feedback mechanism of gene and protein expression of signal related proteins and substrate. So the subject explores the mechanism of the method of strengthening the lung by way of reinforcing the spleen intervening cisplatin resistance of NSCLC by reversal carbohydrate metabolic reprogramming on PI3K-Akt-mTOR signaling intervening, which further proves PI3K-Akt-mTOR singal transduction regulation is feasible as a new target which improves cisplatin resistance of NSCLC. And it will provide the referable experimental method and theoretical basis for filtering Chinese medicine to improve cisplatin resistance.

与正常细胞相比，癌细胞的代谢网络发生了重编程——更多地依赖有氧酵解途径获取能量，这既是癌变的中心环节，又是耐药的重要原因。PI3K-Akt-mTOR信号通路是肿瘤细胞糖代谢重编程与耐药调控的汇聚点。中医认为正气虚损是肺癌顺铂耐药的主要原因，前期研究发现培土生金法可通过干预PI3K-Akt-mTOR信号通路诱导细胞凋亡与自噬性死亡有效逆转肺癌顺铂耐药。本项目继续以PI3K-Akt-mTOR为切入点，以顺铂耐药、有氧酵解程度高的A549/DDP细胞为模式细胞，运用小动物PET-CT、RNAi、real-time PCR、免疫印迹等技术，利用体内、外实验观察培土生金法对肿瘤耐药及糖代谢重编程信号通路中相关蛋白及其底物的调控，阐明培土生金法干预PI3K-Akt-mTOR信号通路逆转肿瘤细胞糖代谢重编程增加NSCLC顺铂敏感性的机制，为肺癌的治疗提供干预靶点、为丰富肺癌耐药病机与治法理论提供新思路。

肺癌属“肺积”、“痞癖”、“咳嗽”、“咯血”等范畴，为本虚标实之证，多运用培土生金法治疗。.一、比较A549细胞与顺铂耐药A549/DDP细胞基因差异表达谱.通过Illumina HiSeq 4000测序平台，对亲本A549和顺铂耐药A549/DDP细胞的LncRNA和mRNA表达差异进行检测。测序结果中包含11631条LncRNA信息，同时包含34247条mRNA信息。利用real-time PCR及Western blot方法对上述差异表达基因进行验证，证明了RNA-seq分析结果的准确性。.其中，上调的差异表达基因约有2200个，下调的差异表达基因约有4300个。通过 GO分析和KEGG通路分类条目分析，发现差异表达基因多存在于细胞周期通路、DNA复制通路及细胞代谢通路等相关基因的表达出现显著上调。选取排名前6位的糖代谢基因（ALDOA，ENO1，ENO2，HK1，PKM2，PGAM1，LDHA）进行了ceRNA网络构建，并结合糖酵解通路调控关键酶，最终选择miRNA-21-PKM2作为后续研究对象。.二、miRNA-21通过PI3K/Akt/mTOR信号通路抑制A549/DDP细胞糖酵解增加NSCLC顺铂敏感性的研究.与亲本A549细胞相比，miRNA-21在顺铂耐药A549/DDP细胞中表达显著升高。利用miRNA-21sponge 联合顺铂共同干预A549/DDP细胞，可显著降低细胞葡萄糖消耗、丙酮酸及乳酸生成；同时抑制A549/DDP细胞PKM2和LDHA蛋白的表达。进一步研究发现，miRNA-21sponge联合顺铂共同干预A549/DDP细胞，可通过调控PI3K/Akt/mTOR/HIF-1α信号通路，干预肿瘤细胞糖酵解，诱导细胞凋亡及自噬性死亡。.三、培土生金中药调节 PI3K/Akt/mTOR信号通路抑制A549/DDP细胞糖酵解增加NSCLC顺铂敏感性. 以参麦注射液作为培土生金中药代表方干预顺铂耐药A549/DDP细胞，可明显降低顺铂的IC50值。明显降低A549/DDP细胞葡萄糖消耗和乳酸生成，下调细胞中糖酵解相关蛋白（HK2、PKM1/2、PKM2、GLUT1和PDH）的表达水平。进一步研究发现，培土生金方药联合顺铂可通过抑制AKT-mTOR-c-Myc途径，干预肿瘤细胞糖酵解，抑制A549/DDP细胞增殖、诱导细胞凋亡。