基于miR-133a/IGF-1R/PI3K环路甲基化研究行气化痰法治疗食管癌的机制

Phlegm-qi stagnation syndrome is the most common symptom of esophageal carcinoma. The method of promoting qi and removing phlegm and its classical prescription Qige Powder are effective methods and prescriptions for the treatment or adjunct treatment of esophageal carcinoma. Our team found that Qige Powder could reduce the expression of miR-133a target gene IGF-1R, and inhibit the downstream PI3K signaling pathway. DNMTs can promote the methylation of miR-133a promoter, therefore cause low expression. While PI3K/Akt signaling pathway can positively regulate the expression of DNMTs protein, which suggested that the methylation of miR-133a/IGF-1R/PI3K loop may play an important role in the pathological process of esophageal carcinoma. In this study, the methylation of miR-133a/IGF-1R/PI3K signal loop related molecules, and the effects of Qige Powder on the growth, apoptosis and cycle of esophageal carcinoma cells were observed in vivo and in vitro, using inhibitors and gene transfection techniques. We aim to reveal the molecular mechanism of Qige Powder on the treatment of esophageal cancer, deepen the understanding of the pathogenesis and treatment of esophageal carcinoma with traditional Chinese medicine, and provide new ideas for the treatment of esophageal carcinoma and the development of drugs.

痰气郁结证是食管癌最常见的证候，行气化痰法及其经典方启膈散是治疗或辅助治疗食管癌的有效方法和方药。本团队研究发现，启膈散可以降低食管癌差异基因miR-133a靶基因IGF-1R的表达，并对其下游PI3K信号通路有明确抑制作用。DNMTs可促使miR-133a启动子甲基化而低表达，PI3K/Akt信号通路可正调控DNMTs蛋白表达，提示miR-133a/IGF-1R/PI3K环路甲基化可能在食管癌病理过程中起重要作用。本课题围绕miR-133a/IGF-1R/PI3K环路甲基化，通过体内外实验，应用抑制剂、基因转染等技术，观察启膈散对该环路甲基化相关分子及食管癌细胞生长、凋亡、周期等的影响，揭示启膈散治疗食管癌的分子机制，加深中医药对食管癌病机及治疗的认识，为食管癌治疗和药物研发提供新思路。

启膈散是治疗或辅助治疗食管癌的有效方法和方药。本团队前期研究发现，启膈散可以降低食管癌差异基因miR-133a靶基因IGF-1R的表达，并可以抑制其下游PI3K信号通路。DNMTs可促使miR-133a启动子甲基化而低表达，PI3K/Akt信号通路可正调控DNMTs蛋白表达，提示miR-133a/IGF-1R/PI3K环路甲基化可能在食管癌病理过程中起重要作用。本课题围绕miR-133a/IGF-1R/PI3K信号通路及该信号通路不同环节对miR-133a甲基化的影响，采用AKT抑制剂、转染miR-133a促表达、DNMT1抑制剂，观察启膈散对该信号通路在食管癌细胞生长、增殖、凋亡等的作用，并制作食管癌细胞致瘤动物模型，观察启膈散对裸鼠肿瘤生长、裸鼠肿瘤组织形态变化及肿瘤组织miR-133a/IGF-1R/PI3K信号通路相关分子的影响，探讨启膈散治疗食管癌的分子机制。研究结果发现，启膈散能够抑制食管癌细胞EC9706细胞和裸鼠肿瘤细胞增殖，促进肿瘤细胞凋亡。启膈散能够上调miR-133a表达，降低Akt、mTOR蛋白的表达。与AKT抑制剂联合应用，对miR-133a表达的上调作用及Akt、mTOR蛋白表达的下调作用呈竞争抑制效应。提示其作用与调控miR-133a与其下游的PI3K/Akt/mTOR信号通路有关。采用Akt抑制剂、miR-133a促表达、DNMT1抑制剂,分别干预miR-133a/IGF-1R/PI3K信号通路，发现启膈散可以抑制食管癌EC9706细胞DNA甲基转移酶（DNMT1)蛋白的表达，说明启膈散可能通过影响miR-133a/IGF-1R/PI3K信号环路甲基化对肿瘤的生长、增殖、凋亡起到干预作用。本实验预测了miR-133a甲基化的位点，选择CG碱基较集中的区域，对所选位点的甲基化程度进行检测，启膈散对降低该区域位点miR-133a甲基化程度作用不明显。说明启膈散可能影响miR-133a其他位点甲基化或者影响该通路其他分子的甲基化，后续我们将进一步研究。本研究加深了中医药对食管癌病机及治疗的认识，为食管癌治疗和药物研发提供新思路，为临床应用提供实验依据。