扶正解毒祛瘀法干扰Beclin1与YAP1相关分子通路逆转肺癌顺铂耐药机制研究

Lung cancer was one of frequent malignant tumors in China. Cisplatin was widely used in the treatment of lung cancer. However,cisplatin resistance in the treatment of lung cancer limited its clinical application. How to overcome cisplatin resistance had became important problems in the treatment of lung cancer. Beclin1 and YAP1 were related to cisplatin resistance, and the YAP1 may affect the expression of Beclin1. Researchers had reported that there are binding sites of Beclin1 in YAP1 protein. We speculated that Beclin1 and YAP1 interacted in the process of cisplatin resistance and promoted drug resistance together. Previous findings also found Fu-zheng Jie-du Qu-yu Therapies could reverse cisplatin resistance and increase the efficacy of cisplatin in lung cancer. And Fu-zheng Jie-du Qu-yu Therapies reduced the expression of Beclin1 and other genes. We also speculated that Fu-zheng Jie-du Qu-yu Therapies could reverse the cisplatin resistance by interfering with Beclin1 and YAP1. In present study, we adopted the vivo and in vitro experiments to illustrate the interaction between Beclin1 and YAP1 during cisplatin resistance and the molecular mechanism of Fu-zheng Jie-du Qu-yu Therapies reversing drug resistance. Through this study, it provides a new idea for the treatment of drug resistance in cancer.

肺癌是我国常见的恶性肿瘤之一，顺铂广泛应用于肺癌的治疗中，然而肺癌治疗过程中顺铂耐药发生率较高，限制了其临床应用。克服耐药成为肿瘤治疗中不可回避的问题。我们前期发现Beclin1和YAP1与顺铂耐药密切相关，YAP1的变化又影响Beclin1的表达。结合文献YAP1蛋白存在有Beclin1结合位点，我们考虑Beclin1与YAP1在顺铂耐药过程中相互作用，共同促进耐药。前期研究我们还发现扶正解毒祛瘀法可逆转顺铂耐药，增加顺铂作用疗效，并且能够降低Beclin1等基因的变化。据此我们推测扶正解毒祛瘀法通过干扰Beclin1和YAP1等基因表达，进而逆转肺癌细胞株对顺铂的耐药。本研究通过体内外实验配合基因组学和蛋白组学实验初步阐明顺铂耐药过程中Beclin1与YAP1相互的作用及扶正解毒祛瘀法干扰Beclin1和YAP1等基因进而逆转顺铂耐药的分子机制，从中医角度为治疗肿瘤耐药供新的思路。

肺癌治疗过程中顺铂耐药发生率较高，限制了其临床应用，克服耐药成为肿瘤治疗中不可回避的问题。本研究通过构建了稳定高表达和低表达Beclin1的细胞株Ad Beclin1 A549/DDP和Si Beclin A549/DDP，我们发现Si Beclin1 A549/DDP细胞株中，Beclin1、P-gp、LRP和HMGB1 mRNA和蛋白的表达较A549/DDP细胞株降低，说明 Beclin 1 与顺铂耐药密切相关。同时构建了稳定高表达和低表达YAP1的细胞株Ad YAP1 A549/DDP和Si YAP1 A549/DDP，通过细胞克隆、Transwell、划痕实验等实验检测Beclin1对顺铂耐药细胞株的影响，结果发现而低表达可以降低肺癌顺铂细胞增殖、侵袭和转移，说明YAP1与肺癌顺铂耐药密切相关。我们同样在Ad YAP1 A549/DDP、Si YAP1 A549/DDP和A549/DDP检测耐药相关基因mRNA和蛋白的相关表达，结果发现Si YAP1 A549/DDP细胞株中、P-gp、LRP，YAP1和HMGB1 mRNA和蛋白的表达较A549/DDP细胞株降低。此结果同样说明 YAP1与顺铂耐药密切相关。细胞实验发现消岩汤作用于A549/DDP后，无论Beclin1和YAP1表达情况如何，消岩汤联合顺铂较单独消岩汤和顺铂抑制细胞增殖、侵袭和转移能力更强，其P-gp和LRP 表达更低。但相比于高表达Beclin1表达，消岩汤联合顺铂抑制低表达Beclin1和正常表达Beclin1细胞增强、侵袭和能力更强，其P-gp和LRP 表达更低，而消岩汤联合顺铂抑制低表达YAP1细胞增强、侵袭和能力更强。动物实验方面，发现消岩汤灌胃降低肿瘤的生长，进而在不同基因表达的移植瘤模型中，发现消岩汤抑制低表达Beclin1作用最强，其次是低YAP1肺癌细胞株，而对高表达YAP1抑制作用与对照组差异无统计学意义。体内外实验说明消岩汤通过抑制Beclin1-YAP1通路进而逆转肺癌细胞顺铂耐药。消岩汤不仅对肺癌细胞有抑制作用，并且可能改善耐药情况，增加DDP作用效果，消岩汤和顺铂在治疗肺癌中起到协同作用。本研究证实了在肺癌耐药过程中Beclin1-YAP1环路发挥重要作用，消岩汤通过干扰Beclin1-YAP1环路进而改善肺癌细胞顺铂的敏感性，从中医角度为治疗肿瘤耐药供新的思路。