基于miR-34a去甲基化激活miR-34a/SIRT1/p53抑癌通路探讨新加良附方抗胃癌机制

Gastric cancer is one of the most common malignancies in China. Alterations in DNA methylation are likely to be key early steps in the process of carcinogenesis. According to the Epigenetic Progenitor Model of Cancer, tumors arise from epigenetic disruption of progenitor or stem cells, and epigenetic changes, especially alterations in DNA methylation, may lead to aberrant inactivation of tumor suppressor genes and activation of oncogenes. .Our previous studies showed that treatment of gastric cancer cells with MLFD leads to a block of proliferation, angiogenesis and induction of apoptosis, which is dependent on the activation of the p53 pathway. It seems that MLFD may potentiate the effects therapeutic, which operate through a p53-mediated apoptosis, thus becoming a potential adjunct in the treatment of gastric cancer patients. miR-34a and displayed tumor suppressive activities, i.e. miR-34a cause induction of apoptosis and senescence, inhibition of cell cycle progression, and a decrease of angiogenesis. Cellular and animal studies have confirmed the clinical link between miR-34a and cancer. Furthermore, the current status of the research on the connection between p53 and miRNAs, as well as alterations in the p53/miRNA pathways found in cancer will be summarized and discussed. SIRT1 is reported to inactivate p53 by deacetylating a critical lysine residue. These effects substantiate the existence of a tumor suppressor network, linking p53 and SIRT1 through miR-34a. miR-34a plays a pivotal role in a positive feedback loop that includes p53, miR-34a and SIRT1. miR-34a induces apoptosis in part through a pathway that involves: miR-34a/SIRT1/p53. Downregulation of p53 would contribute to cell cycle arrest and apoptosis, which is a common result of p53 activation following DNA methylation. .Our pilot studies showed miR-34a is significantly reduced by MLFD in gastric cancer cells, and the change of epigenetic such as methylation of the putative promoter regions, plays an important role in the regulation of miRNAs expression and tumorigenesis. Dioscin is chemical compound obtained from MLFD. We hypothesize that MLFD and dioscin inhibiting gastric cancer’s effect activate a functional loop linking SIRT1 and p53 through the demethylation of miR-34a, and epigenetic regulation of these tumor suppressor genes by MLFD and dioscin may play a role in gastric cancer prevention. In this study we will further explore the expression of miR-34a is regulated by epigenetic mechanisms and p53/miR-34 pathway in gastric cancer. We will investigate for the first time that MLFD can inhibit cancer’s effect through epigenetic events, including alteration of DNA methylation, which may provide a novel scaffold for discovery and development and may steer its future direction for its clinical development as a novel epigenetic agent.

肿瘤相关miRNA启动子CpG岛甲基化异常在胃癌发生发展中起着重要作用，研发去甲基化药物治疗胃癌成为肿瘤基因治疗的热点之一。课题组依据胃癌“寒凝血瘀、气机阻滞”的病机特征，确立“散寒理气活血”治则，以经典名方良附丸为基础，加味的“新加良附方”治疗胃癌显示较好的临床疗效。前期基础研究证实新加良附方通过下调p53下游靶标Bcl-2、Survivin蛋白表达、诱导细胞凋亡、抑制肿瘤新生血管生成与癌基因表达，发挥抗胃癌效应。而新加良附方是否能从去甲基化等表观遗传学方面调控p53抑癌网络而抑制胃癌尚未涉及。本研究通过建立高表达miR-34a胃癌细胞系模型等方法，探明新加良附方与单体成分薯蓣皂苷通过对miR-34a去甲基化从而激活miR-34a/SIRT1/p53抑癌通路达到抑制胃癌生长的作用，从表观遗传学去甲基化角度为胃癌的治疗提供新的靶点和药物。

近年来研究证实miRNA在胃癌发生发展过程中起着重要作用。课题组依据经典名方良附丸加味的“新加良附方”治疗胃癌显示较好的临床疗效，本课题在前期研究基础上，结合体外细胞实验及体内动物实验，采用qPCR、Western-Blot、流式细胞术和慢病毒转染建立高表达miR-34a胃癌细胞系，并构建胃癌荷瘤裸鼠模型等方法，对新加良附方及其主要成分薯蓣皂苷元抑制胃癌细胞功能作用的分子机制展开深入研究，结果显示miR-34a表达异常与胃癌发生密切相关，新加良附方及薯蓣皂苷元可通过调控miR-34a/SIRT1/p53通路，并调控miR-34a凋亡相关靶基因及Caspase凋亡通路发挥抑制胃癌细胞增殖、迁移、诱导凋亡等作用。本研究基本探明中药复方新加良附方及薯蓣皂苷元抗胃癌作用的分子机制，为新加良附方治疗胃癌的提供了科学依据。