槐耳通过上调exosomal miR-203逆转胃癌EMT及化疗耐药的机制研究

Reinforcement (chemotherapy) with elimination (Fuzheng) is wildly used in treatment of gastric cancer, however, the effectiveness of chemotherapy is limited and the chemoresistance is the main reason of chemotherapy failure. Our previous studies showed that Huaier could reverse the chemoresistance of gastric cancer through regulating EMT, but the detail mechanism is not clear. The relationship between exosomal microRNAs and chemotheray has become a current research focus, our preliminary studies also revealed that Huaier could significantly increase the level of exosomal miR-203 in chemoresistance gastric cancer cells, meanwhile, some EMT-related transcription factors including Snail、ZEB1 were important target genes regulated by miR-203. Accordingly, we suppose that Huaier could reverse EMT and chemoresistance in gastric cancer through regulating exosomal miR-203. in This project , we will perform in vivo and in vitro experiments, aiming to study: 1) the control rule of Huaier on exosomal miR-203 expression. 2) The effect of exosomal miR-203 on EMT and chemoresistance of gastric cancer, the direct regulation of exosomal miR-203 on its target genes. Our project will not only do benefit to explore the mechanism by which Huaier reverse chemoresistance, but also provide new thinking and theoretical basis for anti-cancer study of traditional Chinese medicine.

攻（化疗）补（扶正）兼施的疗法在胃癌治疗中广泛应用，但化疗的疗效较差，其中化疗耐药是导致治疗失败的主要原因。我们的前期研究提示槐耳能通过调控EMT进而逆转胃癌化疗耐药，然而其具体机制尚不明确。Exosomal microRNAs与肿瘤耐药的关系是目前研究的热点，本项目组预实验发现槐耳能显著提高胃癌耐药细胞分泌的exosomal miR-203水平，而Snail、ZEB1等EMT相关转录因子是miR-203的重要靶基因。据此我们推测槐耳是通过调控exosomal miR-203的表达进而逆转胃癌EMT及化疗耐药的。本项目拟通过体内、外实验来研究：槐耳调控exosomal miR-203表达的规律及机制；Exosomal miR-203对胃癌化疗耐药、EMT的影响及其对靶基因的直接调控作用。本项目的实施有利于阐明槐耳逆转胃癌化疗耐药的机制，同时为传统中草药的抗肿瘤研究提供新的思路和理论基础。

课题组首先对槐耳提取物的成分进行分析，结果得出槐耳提取物分为5个不同的部位，即石油醚部位、乙酸乙酯部位、正丁醇部位、乙醇部位及水提部，并比较这5个不同部位提取产物的抑癌效果，发现正丁醇部位抑癌效果最好。另外从胃癌细胞培养上清和血清中分离获得外泌体并观察其结构。然后研究exosomal miR-203在胃癌患者血清中的表达及其临床意义，以及exosomal miR-203在胃癌细胞中的作用，结果表明胃癌患者的外泌体miR-203表达水平显著低于健康人，同时研究表明miR-203可以抑制胃癌细胞的侵袭转移能力。进一步建立阿帕替尼耐药的胃癌细胞，鉴定并进行转录组测序，发现在阿帕替尼耐药的胃癌细胞中，mRNA表达差异明显的仅有12个基因，我们对其中的关键基因DUSP1进行研究，发现DUSP1上调通过激活MAPK信号通络诱导胃癌细胞对阿帕替尼的耐药。最后，针对槐耳醇提物调控胃癌细胞EMT的机制进行研究，结果表明槐尔醇提物可以通过c-Myc-Bmi1轴及Twsit多靶点逆转EMT抑制胃癌的侵袭转移。本研究对exosomal miR-203及DUSP1在胃癌发展及耐药的具体机制，及槐尔醇提物逆转EMT抑制胃癌侵袭转移的机制分别进行研究。为胃癌治疗提供新的潜在靶点和全新策略，为中医药治疗胃癌提供新的证据。