食药用菇类中不同蛋白质抑制肺癌细胞增殖的作用途径和关键靶点解析的研究

Recently, cancer is the most deadly disease in China. Its morbidity and mortality keep increasing. However, currently available radiotherapeutic and chemotherapeutic medication only has limited effect but significantly toxic side effects, limiting their application in clinical treatment. Therefore, it is important to develop high-effective, low-toxic, natural anti-tumor proteins and apply them in medication. As China has rich mushroom resources, this research is promising. However, there are numerous categories of anti-tumor proteins from mushrooms with different structures and functioning mechanisms. This research will comprehensively analyze the different mechanisms of inhibiting the proliferation of lung-tumor cells by yet-to-be-reported anti-tumor proteins from 2 to 3 edible mushrooms that will be screened in this project. It will also use gene microarray to systematically investigate the mechanism and key targets for various anti-tumor proteins to inhibit the proliferation of lung-cancer cells. Then, we will verify the functions of those key targets with methods like RNAi and protein inhibitors. Then, we will also use mouse lung-cancer-cells model to prove the consistency of the anti-tumor proteins’ functions in vivo and in vitro. It will lay the theoretical foundation for the clinical use of anti-tumor proteins from mushrooms and the design of new medicine.

癌症已成为中国疾病死因之首，发病率和死亡率呈上升趋势。然而现有放、化疗药物因疗效有限、毒副作用、耐药性等问题在临床应用上受到限制。因此，利用我国菇类资源丰富优势，从中分离高效、低毒抗肿瘤蛋白质有很好的研究意义和应用前景。然而食药用菇类中抗肿瘤蛋白质种类繁多、结构与作用途径各异，缺乏对抗肿瘤蛋白质作用肿瘤细胞关键靶点解析和功效间的比较。本申请项目以肺癌为研究对象，首先对拟筛选出的2-3种未见报道的食药用菇类抗肿瘤蛋白质抑制肺癌细胞增殖作用途径的异同进行全面探讨，然后通过基因芯片技术，深入系统的解析不同种类抗肿瘤蛋白质之间抑制肺癌细胞增殖的作用机制和关键靶点。在此基础上，通过RNA干扰技术、蛋白质抑制剂等方法验证靶点的重要功效。同时通过肺癌造模，验证不同种类抗肿瘤蛋白质体内外作用的一致性。为菇类抗肿瘤蛋白质的临床应用和新药设计奠定坚实的理论基础。

我国肺癌的发病率和死亡率在所有癌症中占比最高。因此从菇类中分离高效低毒的抗肺癌蛋白质有很好的研究意义。该项目以人源非小细胞肺腺癌A549细胞为研究对象，通过对菇类的筛选，发现葡萄状枝瑚菌(Ramaria botrytis，RBUP)、红葱菌（Boletus bicolor,D1）、阿魏菇（Pleurotus ferulae lenzi，XD1）和美味牛肝菌（Boletus edulis，BEAP）具有高效、低度的抗A549细胞的蛋白成分，它们分子量分别为18.0kDa(RBLP), 39.0kDa(D1), 17.5kDa(XD1)和16.7kDa (BEAP)。在体外实验中，RBUP能够特异性抑制A549细胞增殖，其作用机制是通过抑制肿瘤细胞迁移，阻遏其细胞周期在S期，综合调控死亡受体途径、线粒体信号通路和MAPK途径来诱导细胞凋亡等方式抑制肿瘤；D1通过阻滞细胞周期在G1期和诱导内部线粒体凋亡两个途径发挥作用；XD1通过线粒体凋亡通路和死亡受体途径诱导A549细胞凋亡；BEAP能通过激活旁凋亡、凋亡途径和阻滞细胞周期在G1期抑制A549细胞增殖，同时能激活自噬途径促进其增殖，使用自噬抑制剂预处理后，BEAP诱导A549细胞发生凋亡和细胞周期阻滞的程度显著增强，提示BEAP与自噬抑制剂联合应用可能是一种提高BEAP抗肿瘤活性的有效策略。在体内实验中，BEAP同样显示出较强的抗肿瘤效果，作用机制与体外实验具有一致性。.在靶点研究中，首先通过在A549细胞中转染miR-34a模拟物（mimics）序列，探索miR-34a在A549细胞恶性增殖中的关键作用。通过把miR-34a mimics和miR-34a inhibitor（抑制剂）序列分别瞬时转染于A549细胞，构建miR-34a-A549过表达细胞和miR-34a inhibitor-A549细胞，结果显示，BEAP和D1处理A549细胞后，通过上调miR-34a而抑制细胞增殖；而BEAP和D1处理miR-34a-A549过表达细胞后，细胞的抑制率显著增加。由于miR-34a inhibitor序列可使miR-34a的功能缺失，当BEAP和D1处理miR-34a inhibitor-A549细胞后，细胞的抑制率降低。由此推测miR-34a为BEAP和D1抑制A549细胞增殖的关键靶点之一。

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