基于气虚质外泌体miRNA促进肺癌发病的中医“体病相关”理论研究

Chinese theory on "relationship between physique and disease" considers that biased constitution of TCM can promote the development of disease, but its mechanism remains unclear. Our previous studies have found that qi-asthenia constitution in the biased constitution of TCM exhibits the highest risk of developing lung cancer; People with qi deficiency in the biased constitution whose serum and exosomes can significantly accelerate the growth of lung cancer cells. Preliminary experimental results have showed that exosome miR-328-5p in the serum is up-regulated expression with qi deficiency people, and it can clearly promote the growth of lung cancer cells. Hereby, we speculate that exosome miR-328-5p may promote cell proliferation exceedingly by targeting genes and relative signal pathway, thus mediating lung cancer morbidity in constitution of qi-asthenia. Based on the previous researches, the project puts forward that studies about "qi deficiency in constitution associated with lung cancer " are the ideal starting point to explore theory on "relationship between physique and disease". Using the combined method in vitro and in vivo experiments, techniques and methods such as lentivirus gene transfection, RNA interference and DLRTM, etc are involved to carry out the project around the exosome miR-328-5p in the serum with constitution of qi deficiency, which aiming at systematic discussing the molecular mechanism of the qi deficiency qualitative on promoting of lung cancer, and eventually elucidating key target genes of miR-328-5p and its signal pathways. This not only could provide experimental evidence for the theory on "relationship between physique and disease", also will contribute to the prevention of lung cancer, promoting the implementation of traditional Chinese medicine's "preventive treatment of disease" health project.

中医“体病相关”理论认为：偏颇体质可促进疾病的发生发展，但其机制至今尚未阐明。我们前期研究发现在偏颇体质中气虚质罹患肺癌的危险度最高；气虚质血清及其外泌体均可显著促进肺癌细胞的生长。预实验结果显示：气虚质血清外泌体miR-328-5p高表达，并且其可显著促进肺癌细胞的生长。因此推测外泌体miR-328-5p可能通过调节靶基因及其信号通路，促进细胞过度增殖，从而介导气虚质促进肺癌发病。本项目在前期研究基础上，提出“气虚质与肺癌相关”研究是探索中医“体病相关”理论的理想切入点，采用体内实验结合体外实验，围绕气虚质血清外泌体miR-328-5p展开研究，应用慢病毒基因转染、RNA干扰、双荧光素酶报告基因等技术和方法，系统探讨气虚质促进肺癌发病的分子机制，最终阐明miR-328-5p的关键靶基因及其信号通路。这将有利于肺癌的防治，丰富中医“体病相关”理论的科学内涵，促进实施中医“治未病”健康工程。

肺癌是我国发病率与死亡率最高的恶性肿瘤，已经成为我国严重的公共卫生问题。中医“体病相关”理论认为：偏颇体质可促进疾病的发生发展，但其机制至今尚未阐明。本项目提出“气虚质与肺癌相关”研究是探索中医“体病相关”理论的理想切入点。首先通过检索PubMed、CNKI、Wan fang、VIP等数据库进行文献搜集，系统评价分析中医体质与肺癌相关性，发现在偏颇体质中气虚质罹患肺癌的危险度最高，且与肺癌病理分期类型密切相关。其次，采用体内外结合实验方法，以人肺癌细胞细胞株A549和SPC-A1为研究对象，利用慢病毒分别构建miR-328-5p过表达/敲低稳转肺癌细胞株，采用CCK8、平板克隆实验检测细胞增殖能力以及Transwell、细胞划痕实验检测细胞的迁移能力，结果显示过表达miR-328-5p显著促进肺癌细胞的增值和迁移能力。利用miR-328-5p过表达/敲低稳转肺癌细胞株建立皮下移植瘤小鼠模型，采用小动物活体成像仪观察移植瘤的大小，结果表明过表达miR-328-5p显著促进促进肺癌细胞在裸鼠体内成瘤能力。最后，运用转录组高通量测序及靶基因预测网站综合预测miR-328-5p的靶基因，应用双荧光素酶实验进行验证其靶基因LOXL4、NDRG1，进一步采用Western blot法检测miR-328-5p在肺癌细胞中对LOXL4、NDRG1表达的影响，最终阐明气虚质外泌体miR-328-5p促进肺癌发病的分子机制。这将有利于肺癌的防治，丰富中医“体病相关”理论的科学内涵，促进实施“治未病”健康工程升级。