复方斑蝥通过miR-520d/Beclin1信号轴逆转三阴乳腺癌化疗耐药的研究

Triple-negative breast cancer (TNBC) is a kind of breast cancer with poor prognosis and high mortality rate. Chemotherapy resistance is an important factor abating the therapeutic efficacy. Excessive autophagy is one of the mechanisms of drug resistance. Compound cantharis is commonly used as auxiliary drug against TNBC. Our previous studies suggested that compound cantharis could down-regulate the expression of autophagy-related protein Beclin1, reduce autophagy levels, and enhance TNBC apoptosis. Microarray resultsshowed that the level of miR-520d decreased in drug-resistant TNBC, however, increased significantly after compound cantharis treatment. Bioinformatics showed that Beclin1 might be the target molecules of miR-520d. This project aims to investigate whether compound cantharis could inhibit TNBC autophagy through upregulating miR-520d and increase the anti-tumor efficacy of chemotherapy. We will establish drug resistant TNBC cell and animal models, observe the efficacy of compound cantharis on TNBC autophagy and chemotherapy resistance, and study the effect of compound cantharis on miR-520d/Beclin1 signal axis; Gene transfection and siRNA interference will be used to down-regulate miR-520d, elucidating the mechanism of compound cantharis reversing chemotherapy resistance of TNBC via the signal axis. Therefore, The study might provide a new therapeutic strategy for the clinical treatment of drug-resistanceof TNBC.

三阴乳腺癌（TNBC）是一类难治性、病死率高的乳腺癌，化疗耐药是影响疗效的重要原因。过度自噬是肿瘤耐药发生的机制之一。复方斑蝥是TNBC常用化疗辅助药物。我们前期发现，复方斑蝥可减少自噬蛋白Beclin1表达，降低自噬水平，增强TNBC凋亡；芯片结果示，乳腺癌细胞耐药株miR-520d表达下降，而复方斑蝥能逆转miR-520d水平；生物信息学发现Beclin1可能是miR-520d靶分子。我们推测复方斑蝥通过上调miR-520d抑制TNBC自噬，增强化疗的抗肿瘤疗效。本项目拟通过构建TNBC耐药细胞及动物模型，观察复方斑蝥对TNBC自噬及化疗耐药的影响，探讨复方斑蝥对miR-520d/Beclin1信号轴的调节；并运用基因转染/干扰技术增强或阻断miR-520d信号，明确复方斑蝥通过该信号轴逆转TNBC化疗耐药的可能机制。该项目研究成果为临床治疗耐药性TNBC提供理论依据和新的策略。

目的：明确 miR-520d/Beclin-1 信号轴与三阴性乳腺癌细胞自噬和耐药间的关系；阐明复方斑蝥注射液如何通过 miR-520d/Beclin-1 信号轴逆转化疗耐药，为耐药性三阴性乳腺癌患者的临床治疗提供实验依据和用药参考。.方法：1. miR-520d/Beclin-1 信号轴对三阴性乳腺癌细胞自噬和耐药的影响：构建三阴性乳腺癌细胞MDA-MB-231、MDA-MB-468的耐药株MDA-MB-231/Doc和MDA-MB-468/Doc，并用MTT法和流式凋亡实验验证耐药模型。采用多西他赛作用于三阴性乳腺癌细胞的亲本株和耐药株，给予Con组（模型组），Beclin-1 OE（Beclin-1稳转系组）组，miR-520d mimics组（过表达miR-520d组），Beclin-1 OE + miR-520d mimics组（联合用药组）干预，与对照组比较观察多西他赛作用下各组细胞的自噬及凋亡情况。2. 复方斑蝥注射液逆转三阴性乳腺癌化疗耐药的可能机制：设立Con组（对照组）、Doc组（多西他赛组）、CCI组（复方斑蝥注射液组）、CCI+Doc组（多西他赛联合复方斑蝥组）以及Con组（模型组），CCI组（复方斑蝥组），CCI+ miR-520d inhibitor（复方斑蝥联合miR-520d抑制剂组）组，检测各用药组对乳腺癌耐药株的影响。3. 动物模型验证：建立三阴性乳腺癌细胞MDA-MB-231/Doc耐药动物模型，设立多西他赛组（Doc组）、复方斑蝥注射液组（CCI组）、多西他赛联合复方斑蝥组（CCI+Doc组），并与对照组（Con组）比较，研究各用药组对于肿瘤的影响。.结果：1. miR-520d/Beclin-1 信号轴与三阴性乳腺癌细胞自噬和耐药的关系： Beclin-1是miR-520d的可能靶分子。2. 复方斑蝥逆转三阴性乳腺癌化疗耐药的可能机制。3. 动物模型结果与细胞实验相同。.结论：复方斑蝥注射液通过调控三阴性乳腺癌动物耐药模型的miR-520d/Beclin-1信号轴逆转三阴性乳腺癌化疗耐药。