利用蛋白质组学技术研究双氢青蒿素抑制卵巢癌细胞上皮-间质转化的分子机制

Ovarian cancer is one of the leading causes of gynecologically malignant cancer death among women. Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of ovarian cancer cells.We have recently found that dihydroartemisinin (DHA), the most potent antitumor artemisinin-like compound and the main metabolites of artemisinin's derivatives, blocks the invasion and metastasis of ovarian cancer cells by inhibiting the progression of EMT. To clarify its molecular mechanisms, firstly, the experiments of proteomics and pull down are employed to check the protein expression profiles of ovarian cancer cells in vitro and in vivo with DHA treatment. Two complementary approaches are used in the present studies, including fluorescent two-dimensional difference gel electrophoresis(2D DIGE) coupled to mass spectrometry(MS) and liquid chromatograph/mass spectrometry labeled by isobaric tags for relative and absolute quantification? (iTRAQ?-LC/MS). The possible target-related proteins on DHA inhibiting the progression of EMT in ovarian cancer cells are identified by MS. Secondly, the possible DHA-targeted proteins are predicted and evaluated by the molecular docking software. The direct binding affinity of DHA towards the possible target-related proteins is confirmed in vitro using surface plasmon resonance (SPR) biosensor analysis. Effects of DHA-targeted proteins treated are assessed by enzyme-linked immunosorbent assay (ELISA), coimmunoprecipitation(CoIP), RNA interference (RNAi), gene overexpression and transfection, et c. Finally, the methods of bioinformatics are used to construct the signal network of the possible protein-protein interaction(PPI), which are from direct DHA-targeted proteins to the proteins related to the signal transduction pathway of EMT treated by DHA. The techniques of molecule biology,such as real-time polymerase chain reaction (PCR) and western blotting(WB),are applied to detect the level of differentially expressed proteins affected by DHA in the signal cascade network. The research will provide the evidence of potential implications for the rational application of DHA as a novel antitumor drug against ovarian cancer in clinic. Understanding of the molecular changes in the signal transduction pathways associated with the invasion and metastasis of ovarian cancer cells could lead to the identification of target proteins for novel preventive and therapeutic interventions.

卵巢癌是危害女性健康常见的妇科肿瘤，上皮-间质转化（EMT）在卵巢癌侵袭和转移中起重要作用；我们发现双氢青蒿素（DHA）通过抑制EMT阻止卵巢癌的侵袭和转移。为了阐明其分子机制，本课题首先采用蛋白质组学技术（双向荧光差异凝胶电泳结合质谱和液相色谱串联质谱）和pull down实验分析DHA对卵巢癌蛋白质表达谱变化的影响，寻找与DHA作用相关的靶点蛋白。其次，运用分子对接软件预测DHA与靶点蛋白的结合，通过表面等离子体共振技术验证；采用ELISA、基因过表达转染、RNAi和CoIP等技术考察DHA对靶点蛋白功能的影响。最后，通过生物信息学手段构建与DHA作用相关的蛋白质相互作用信号网络，运用real-time PCR和western blotting等技术检测DHA对于网络中EMT信号通路蛋白表达的影响。本研究将为DHA今后应用于临床治疗肿瘤奠定基础，同时也将为卵巢癌的防治提供新的靶点蛋白。

卵巢癌是危害女性健康最常见的妇科肿瘤，上皮-间质转化（EMT）在卵巢癌侵袭和转移中起重要的作用；本课题在体外细胞水平和体内动物水平发现双氢青蒿素（DHA）通过抑制EMT阻止卵巢癌细胞的侵袭和转移。为了阐明其分子机制，本课题首先采用双向荧光差异凝胶电泳(DIGE)结合质谱鉴定和基于iTRAQ 标记的液相色谱串联质谱(LC-MS)鉴定的蛋白质组学技术分析DHA对于卵巢癌蛋白质表达谱变化的影响，寻找到与DHA作用相关的21种受体蛋白和13种与细胞侵袭和转移以及EMT相关的蛋白。其次，运用分子反向对接软件预测DHA与通过比较蛋白质组学获得的25种差异表达的蛋白质的结合，通过表面等离子体共振技术验证DHA与受体蛋白PDGFRα的结合。最后，通过生物信息学手段构建了与DHA诱导肿瘤细胞周期阻滞和凋亡，抑制肿瘤细胞增殖、侵袭转移以及肿瘤新生血管生成，逆转肿瘤细胞耐药性以及DHA参与肿瘤细胞氧化应激反应和炎症反应相关的蛋白质相互作用网络。体内与体外实验结果表明DHA通过直接与靶点蛋白PDGFRα结合后引起其泛素化蛋白降解，进而失活下游PI3K/AKT和ERK MAPK信号通路，最终抑制EMT阻止卵巢癌细胞的侵袭和转移。此外，DHA还能够增强卵巢癌细胞对于PDGFRα抑制剂索拉菲尼（Sorafenib）的敏感性。DHA用于肿瘤的临床治疗已有个案报道，本研究为DHA 成为新型抗癌辅助药物提供实验依据，同时也为卵巢瘤的防治提供新的靶点蛋白。本研究为青蒿素类化合物抗肿瘤作用机制研究提供新的内容，同时也为该类化合物进一步的开发和利用提供重要的线索。