整合素介导的蒽酮类抗生素zunyimycin C抗肝癌效应机制研究

The high incidence and mortality of liver cancer are a serious threat to human health. It is a worldwide problem to explore new methods for the treatment of liver cancer. Molecular targeted therapy is an important part of comprehensive treatment for hepatocellular carcinoma. Integrin inhibitors have great potential in molecular targeted therapy of liver cancer, which can provide new options for clinical molecular targeted therapy of liver cancer. The source of the halogenation of type two Streptomyces polyketide small molecule zunyimycin C showed similar to cisplatin antitumor activity, the prediction results of the molecular structure of network pharmacology showed that it may be with the integrin binding of target protein based on gene expression analysis showed that the compound can be caused by changes in the expression of integrin mediated hepatocellular carcinoma cell lines by PI3K/Akt metabolic pathway the number of genes mRNA. This study will block the anti hepatoma effect, integrin targeted protein analysis based interaction zunyimycin C and integrin target proteins in the analysis of the zunyimycin C zunyimycin C, looking for a comprehensive effect of metabolic pathways and molecular mechanisms of anti liver cancer eventually confirmed, analysis of zunyimycin C, the development of liver cancer molecular targeting new drug lead compounds, as the clinical target to liver cancer to provide experimental evidence for clinical treatment.

肝癌严重威胁人类健康，探索治疗肝癌新方法是世界性难题。整合素调控肿瘤生长、侵袭和转移，是肿瘤靶向药物治疗的重要靶点，整合素抑制剂在肿瘤靶向治疗中具有极大潜力，可为临床提供新的选择。专利新化合物zunyimycin C具有较好抗肝癌活性，网络药理学预测、分子对接和体外实验结果均表明其可能具有与整合素LFA-1等靶蛋白结合活性，qPCR分析表明该化合物改变肝癌细胞整合素介导的PI3K/Akt代谢通路下游基因的mRNA表达水平。本研究将对比LFA-1激动剂、拮抗剂和zunyimycin C对肝癌细胞影响，明确LFA-1介导zunyimycin C抗肝癌效应，利用RNA-seq、ITRAQ等全面分析zunyimycin C作用靶蛋白及代谢途径，定点突变、SPROX\CTESA确证zunyimycin C与LFA-1作用分子机制，解析zunyimycin C抗肝癌效应机制，为肝癌靶向治疗提供新药。

项目背景：.基于肝癌对国民健康构成的严重威胁，探索新型小分子靶向药物并确定其作用靶点十分重要。研究新抗生素zunyimycin C抑制肝癌细胞的分子机制，可为临床靶向治疗肝癌新药物的开发提供新的思路。..主要研究内容：.（1）检测zunyimycin C对HepG2等肝癌细胞株和L02肝细胞株体外增殖的影响；（2）检测zunyimycin C联合P38MAPK抑制剂SB203580对肝癌细胞株体外增殖的影响；（3）观察zunyimycin C对HepG2肝癌细胞集落形成的影响；（4）分析zunyimycin C影响肝癌细胞株及正常肝细胞株的差异表达mRNA和蛋白质及分子网络；（5）DARTS技术筛选与zunyimycin C发生作用的潜在靶蛋白；（6）检测zunyimycin C对肝癌细胞株、正常肝细胞株的MAPK、PI3K/Akt等信号通路相关基因mRNA表达水平影响；（7）检测zunyimycin C对肝癌细胞株、正常肝细胞株的MAPK、PI3K/AKT等信号通路相关蛋白表达及磷酸化修饰水平影响；（8）检测zunyimycin C对肝癌细胞株的PIP3合成水平的影响。..重要结果：.（1）Zunyimycin C对不同肝癌细胞株的IC50低于20μM，相比于正常肝L02细胞的IC50的41.97μM具有统计学意义（P＜0.05）；（2） zunyimycin C对肝癌HepG2细胞集落形成的影响有统计学意义（P＜0.001）；（3）表达谱测序共筛选出差异表达mRNA 56507个，定量蛋白质组学分析获得差异表达蛋白5281个；（4）DARTS实验筛选到zunyimycin C作用的靶蛋白为GPI、ENO1、GAPDH、ASS1、PGK1和ALDOA；（5）Zunyimycin C上调MAPK和PI3K/Akt信号通路凋亡抑制基因及肿瘤分裂相关基因的 mRNA表达水平；（6）Zunyimycin C上调JNK、P38表达水平，促进P-Erk、P-JNK、P-P38、P-Akt蛋白的磷酸化，下调Erk、PI3K、Akt、CREB和Ras蛋白表达水平；（7）Zunyimycin C可降低肝癌HepG2细胞中PIP3的合成水平。.关键数据及其科学意义：.（1）Zunyimycin C对肝癌细胞的增殖具有抑制作用；（2）Zunyimycin C可激活或抑制MAPK、PI3