基于microRNA调控网络的消癌解毒方抗肿瘤作用机制研究

Jiedu Xiaoai Prescription is an effective anti-cancer prescription that has been tested by long-term clinical practice under the guidance of Professor Zhou Zhongying's creative Cancerous Toxin Theory. Previous studies have demonstrated that cancerous toxin exists objectively in tumor clinical syndrome and the prescription has remarkable curative effect. Besides, it has also been confirmed that the antitumor function of Jiedu Xiaoai Prescription results from multi-level and multi-target comprehensive adjustments, and it Is a very good development prospect of antitumor drug, in need of deeply systematic research. Based on previous studies, based on Cancerous Toxin Theory as the leading thought, the project follows traditional Chinese medicine's (TCM's) own characteristics,from molecular miRNA of gene upstream regulation, takes tumor tissue, cell and peripheral blood as objects, takes gene chip technology, overall researchs related miRNA expression spectrum in the intervention of Cancer toxin and Jiedu Xiaoai Prescription; To expression differences apparent function miRNA, We adopt RT - PCR methods and bioinformatics analysis; In order to verify the key target genes and signaling pathways, we take interference of miRNA, RT-PCR and Western blot; Through the research of miRNA expression spectrum and focusing research of particular genes, the overall characterization of Gene chip and accurate analysis of key miRNA, we obtain the function gene network , biomarker of Cancer toxin and targets of TCM closely related Cancer toxin and Jiedu Xiaoai Prescription, explore the antitumor mechanism of this Prescription from the perspective of miRNA control network in the whole and molecular level, expound scientific connotation of Cancerous Toxin Theory, and provide important new ideas for research of TCMs anti-tumor.

消癌解毒方是国医大师周仲瑛教授癌毒理论指导下经长期临床的有效抗癌方。前期研究证实癌毒在临床中客观存在及该方抗肿瘤疗效，表明该方具多环节、多靶点抗肿瘤作用特点，是一种发展前景极好的抗肿瘤药，急需对其进行深入系统研究。 项目以前期研究为基础，以癌毒理论为主导思想，遵循中医学自身特点，从基因上游调控分子miRNA入手，以肿瘤组织、细胞及外周血为对象，基因芯片获得与癌毒及消癌解毒方相关的miRNA表达谱；差异miRNA进行RT-PCR验证及生物信息学分析；miRNA转染、RT-PCR及Western blot对关键靶基因和信号通路进行验证；miRNA表达谱与特定基因聚焦研究结合，基因芯片整体表征和重点miRNA精确分析，获得与癌毒及消癌解毒方相关的miRNA功能基因调控网络、癌毒标志物及中药靶标，在整体及分子水平从miRNA调控网络揭示该方抗肿瘤机制，诠释癌毒理论内涵，为中医药抗肿瘤研究提供新思路

采用H22 荷瘤小鼠，随机分组；取外周血和瘤组织；光镜下检测各组小鼠瘤组织；miＲNA芯片技术检测各组小鼠瘤组织及外周血差异表达 miＲNAs，找出与消癌解毒方作用机制相关的miＲNAs，PCR验证；基因过表达质粒、细胞转染、靶点预测、双荧光素酶验证、检测细胞功能及增殖能力，功能分析miR200b-3p；开展该方对人肝癌SMMC-7721细胞增殖及miR-25-3p，miR-29a-5p，miR-122-3p，miR-124-3p，miR-182-5p、hsa-miR-21-5p、hsa-miR-148a-3p表达的影响。研究显示该方抗肿瘤作用与调控H22 荷瘤小鼠外周血和瘤组织内多种miRNA表达相关，尤其与影响miRNA200b-3p表达、降低Notch1基因表达密切相关。回顾性研究肝癌带瘤生存患者临床症状、肿癌标志物及瘤灶，探讨癌毒理论及该方在肝癌治疗中的有效性；开展癌毒理论在乳腺癌治疗中的应用等其它临床及理论研究。.H22瘤荷小鼠组织 miRNAs表达谱研究.芯片结果，该方上调小鼠瘤组织内miＲ-1298 -5 p、miＲ-874 -3 p、miＲ-721 、miＲ-298 - 5 p、miＲ-551 b-5 p、miＲ-346 -5 p、miＲ-105 表达; 下调miＲ-24 -3 p、miＲ-3963 、miＲ-127 -3 p、miＲ-434 -5 p、 miＲ-1187 、miＲ-468 -3 p、miＲ-221 -5 p、miＲ-6695 -5 p 表达。.H22瘤荷小鼠外周血miRNAs表达谱研究.芯片结果，小鼠外周血差异表达miRNAs233个，上调75个，下调158个；qRT-PCR验证10个；该方与miR200b-3p、miR-669k-5p、miR-206-3p、miR-453、miR-463-3p、miR-135a-1-3p、miR-1960表达密切相关。.miR200b-3p研究.构建miR200b-3p基因过表达质粒，转染肝癌细胞，检测细胞增殖、迁移能力；构建过表达稳定转染细胞株；miR200b-3P作用靶点预测；双荧光素酶报告验证；检测Notch1蛋白表达；构建Notch1基因过表达质粒，共转染肝癌细胞株，检测细胞功能及增殖能力。miRNA200b-3p表达与Notch1基因表达降低有关。