基于CAFs来源的外泌体miRNA-20a介导PTEN/PI3K/AKT通路探讨加味龟鹿二仙胶汤干预非小细胞肺癌顺铂耐药机制

The modified Guilu Erxian Jiao Formula can reverse cisplatin chemoresistance in non-small cell lung cancer (NSCLC).Cancer associated fibroblasts (CAFs) have a close relationship with cisplatin chemoresistance in NSCLC. Previous studies have confirmed that A549 cells can activate CAFs and promote the up-regulation of miRNA-20a, and then CAFs-derived exosomes transmit miRNA-20a to promote chemoresistance in NSCLC. Further study found that the modified Guilu Erxian Jiao Formula can inhibit CAFs to release exosomes and regulate PTEN/ PI3K / AKT pathway. Therefore, we speculated that the Formula’s effects of increasing drug sensitivity and reversing drug resistance were related to the regulation of CAFs. The hypothesis is as follows: modified Guilu Erxian Jiao Formula can regulate miRNA-20a-mediated PTEN / PI3K / AKT signaling pathway to sensitize and reverse chemoresistance by inhibiting the release of CAFs derived exosomes. In order to confirm this hypothesis, this project will build a lung cancer mouse model of deficiency of both qi and yin, and demonstrate the above hypothesis by detecting the miRNA-20a-mediated PTEN / PI3K / AKT signaling pathway in CAFs derived exosomes in vivo and in vitro. This project will provide a scientific basis for finding a drug and method to increase NSCLC chemosensitivity and reverse of drug resistance.

加味龟鹿二仙胶汤能够逆转非小细胞肺癌(NSCLC)顺铂化疗耐药，肿瘤相关成纤维细胞（CAFs）与NSCLC顺铂化疗耐药关系密切。前期研究证实A549细胞活化CAFs并促进miRNA-20a表达上调，进而CAFs来源的外泌体传递miRNA-20a促进NSCLC化疗耐药。进一步研究发现，加味龟鹿二仙胶汤能够抑制CAFs释放外泌体，调控PTEN/PI3K/AKT通路。因此，我们推测该方的增敏及逆转耐药作用与调控CAFs有关，提出“加味龟鹿二仙胶汤通过抑制CAFs释放外泌体，调节miRNA-20a介导的PTEN/PI3K/AKT信号通路以增敏及逆转化疗耐药”假说。本课题将构建气阴两虚型肺癌小鼠模型，通过体内和体外实验检测CAFs来源外泌体miRNA-20a介导的PTEN/PI3K/AKT信号通路以论证上述假说，为找到针对NSCLC化疗起到增敏及逆转耐药的药物和治疗方法提供科学依据。

肺癌严重危害人类健康，非小细胞肺癌（NSCLC）是肺癌的主要类型。化疗耐药是影响非小细胞肺癌治疗的重要原因之一。找到抑制或逆转化疗耐药的药物或方法对提高NSCLC化疗疗效迫在眉睫，同时这也是目前肺癌化疗中面临的重要课题。扶正培本法在中医肺癌治疗中占据重要地位，加味龟鹿二仙胶汤属于扶正培本类方剂，本身无直接杀伤肿瘤细胞作用。本课题从肿瘤微环境入手，选择微环境中的主要成分——CAFs及关键miRNA(miR-20a)为主要研究对象，构建气阴两虚型肺癌小鼠模型，通过体内和体外实验，证实miR-20a在CAFs来源的外泌体中表达上调并促进NSCLC细胞增殖、抑制凋亡和促进对顺铂(DDP)耐药。GW4968和Rab27a沉默能够下调NSCLC细胞中的miR-20a，miR-20a在体外诱导NSCLC细胞增殖和DDP耐药。进一步发现，PTEN是miR-20a的靶基因，过表达PTEN抑制NSCLC细胞增殖和化疗耐药，同时促进细胞凋亡。miR-20a通过抑制PTEN表达促进NSCLC细胞进展和化疗耐药。CAFs来源的外泌体将miR-20a传递给肿瘤细胞，通过PTEN/PI3K/AKT通路以增强肺癌细胞增殖DDP耐药。加味龟鹿二仙胶汤含药血清能够调控miR-20a，抑制CAFs来源外泌体细胞增殖、化疗耐药，促进NSCLC细胞凋亡。加味龟鹿二仙胶汤通过抑制CAFs释放外泌体，调节miRNA-20a介导的PTEN/PI3K/AKT信号通路以增敏及逆转化疗耐药。本项目验证了“加味龟鹿二仙胶汤通过抑制CAFs释放外泌体，调节miRNA-20a介导的PTEN/PI3K/AKT信号通路以增敏及逆转化疗耐药”假说。本项目的完成初步阐明了加味龟鹿二仙胶汤增强肺癌化疗药物敏感性的具体作用机制，对于继承扶正抗癌的学术思想，揭示中医肿瘤"扶正培本法”的学术内涵具有重要意义。