基于miR-21信号介导肿瘤微环境的行气化痰法增加食管癌化疗药物敏感性机制研究

Chemoresistance is a major obstacle in the treatment of esophageal carcinoma. The tumor microenvironment mediated by miR-21 signal plays an important role in the tumor drug resistance. Method of promoting qi and removing phlegm is the basic treatment of TCM clinical differentiation and treatment, whether it participating in adjuvant chemotherapy or used alone has curative effects. Classical formula Qige powder can promote the apoptosis of esophageal cancer cells, reduce tumor angiogenesis, intervene multiple signaling pathways, inhibit miR-21 expression, and increase its target molecule. Using different cell co-culture and other cellular and molecular biology techniques, this project aims to study the relationship between the sensitivity of a commonly used chemotherapeutic drug cisplatin for esophageal cancer cells and tumor microenvironment,based on miR-21 signal, in vivo and in vitro. The growth and apoptosis of esophageal carcinoma cells will be observed in hypoxia and different cell co-culture condition, and miR-21 expression, intercellular transfer and its upstream an downstream molecular expression of indicators will be detected, so as to clearly definite the role of tumor microenvironment mediated by miR-21 signal in esophageal cancer resistance, explain the molecular mechanism of drug resistance in esophageal carcinoma and clarify the mechanism of increasing the sensitivity of chemotherapeutics by method of promoting the circulation of Qi and removing phlegm. These studies will provide the experimental basis for clinical application, and further deepen understanding of the pathogenesis of esophageal carcinoma the understanding and method of promoting qi circulation and removing phlegm.

肿瘤耐药为治疗食管癌的主要障碍，miR-21信号介导肿瘤微环境在肿瘤耐药过程中起重要作用。行气化痰法是中医临床辨治食管癌基本治法，辅助化疗或单独使用疗效确切，行气化痰法代表方启膈散可促进食管癌细胞凋亡、减少肿瘤血管生成、干预多个信号通路、抑制miR-21表达、增加miR-21靶分子。本项目围绕miR-21信号，通过体内、外实验，利用细胞共培养、胞外体（exosomes）的分离和标记转染、电泳迁移率变动分析（EMSA）等细胞分子生物学技术，观察不同细胞共培养或缺氧条件下食管癌细胞生长、凋亡情况，检测miR-21表达、细胞间传递及其上下游调控分子表达等指标，研究食管癌细胞对常用化疗药物顺铂敏感性与肿瘤微环境的关系，探索miR-21信号介导肿瘤微环境在食管癌耐药中的作用，阐释食管癌耐药分子机制，阐明行气化痰法增加化疗药物敏感性作用机制，为临床应用提供实验依据，加深对食管癌病机和行气化痰法认识。

肿瘤耐药为治疗食管癌的主要障碍，miR-21信号介导肿瘤微环境在肿瘤耐药过程中起重要作用。行气化痰法是中医临床辨治食管癌基本治法，多用于辅助化疗。为了研究行气化痰法代表方启膈散增加食管癌化疗药物敏感性作用及其机制，本项目通过体内、外实验，利用细胞共培养、转染、胞外体（exosomes）的分离和标记转染等细胞分子生物学技术进行了研究。发现低氧下、或与内皮细胞、成纤维细胞共培养可以导致食管癌细胞EC9706对顺铂敏感性降低，而EC9706细胞miR-21表达升高，miR-21靶分子PDCD4、PTEN mRNA和蛋白表达降低。经过转染高表达miR-21慢病毒的EC9706细胞对顺铂敏感性降低，而转染miR-21-inhibitor慢病毒的EC9706对顺铂敏感性增加。无论是在常氧、低氧，或和内皮细胞、成纤维细胞共培养，启膈散含药血清都可以增加食管癌细胞EC9706对顺铂的敏感性，增加顺铂引起的凋亡率，干预细胞周期，降低miR-21表达，升高miR-21靶分子PDCD4、PTEN mRNA和蛋白表达。提取和鉴定内皮细胞exosomes，发现该外泌体可以降低EC9706细胞对顺铂的敏感性，并且可以被EC9706细胞摄取，外泌体内容包涵miR-21。在与成为纤维细胞共培养的研究中，发现启膈散提取物和顺铂序贯给药比同时加药更能增加细胞对顺铂的敏感性。应用裸鼠移植瘤模型研究发现，启膈散可以减慢移植瘤生长速度、减轻瘤体重量，增加顺铂的移植瘤的生长抑制作用,抑制STAT3蛋白表达、增加PTEN、PDCD4蛋白表达、减少微血管生成。该研究从miR-21信号角度阐释了肿瘤微环境导致食管癌耐药机制，阐明了行气化痰法增加化疗药物敏感性部分作用机机理，为临床应用提供实验依据和借鉴。