基于lncRNA探讨益气小复方逆转三阴性乳腺癌顺铂耐药的作用与机制

Triple-negative breast cancer (TNBC) is known to have poor diagnosis. By now, chemotherapy is still the mainstay of treatment as a systemic therapy. Recently, More and more attention has been paid to platinum containing regimens, but their strong drug-resistance in patients makes the solution clinically hopeless. The excessive activation of PI3K/AKT pathway is one of the major mechanisms underlying the drug-resistance in cisplatin (DDP), where lncRNA is involved in regulating the pathway. .In our previous studies we have found that Yiqi Formula was capable of down -regulating the expression of p-AKT in breast cancer while at the same time up -regulating that of PTEN, to reversal of DDP resistance Is there a key lncRNA ,base on which , Yiqi Formula was capable of down -regulating p-AKT and up regulating of PTEN and reverse DDP resistance in TNBC ?. Based upon these foundations and hypothesis, we further investigate the Function and mechanism of Yiqi Formula on reversing drug resistance in TNC/DDP by 1)locking resistance-related target-lncRNA through microarrays screening incorporated by literatures; 2) using Yiqi Formula in Vitro and Vivo TNC/DDP resistance model to observe its drug resistance; 3) further utilizing modern technologies such as Western blotting, RT-qPCR, RNAi and bioluminescence in microbes determination.The investigation offers an experimental basis for the clinical practice that the Yiqi fu zheng principle is adopted in the treatment of the TNBC resistant to chemotherapy.

三阴性乳腺癌（TNBC）预后较差，化疗是其主要的全身治疗手段，含铂的方案越来越受到重视，然而其耐药现象让临床束手无策。PI3K/AKT通路过度活化是TNBC铂类耐药的重要机制，lncRNA参与调控该通路。.我们在前期研究中发现，益气小复方能下调p-AKT、上调PTEN表达发挥逆转TNBC顺铂（DDP）耐药作用。益气小复方是否通过某些关键lncRNA下调p-AKT、上调PTEN表达，逆转TNBC顺铂耐药？.基于以上基础及假设，通过高通量芯片技术筛选、锁定TNBC/DDP耐药相关目标lncRNA；运用益气小复方作用于体内、外TNBC/DDP耐药模型，观察耐药情况，并进一步综合采用Westernblotting、RT-qPCR、RNAi、生物发光学检测等技术，深入探索益气小复方逆转TNBC/DDP耐药的作用及机制，从而为益气扶正法治疗TNBC化疗耐药的临床实践提供实验依据。

本研究首次发现lncRNA HCP在MDA-MB-231顺铂耐药细胞株中高表达，MTT及流式检测细胞凋亡实验结果显示，益气小复方同顺铂联用能增强MDA-MB-231/DDP细胞株对顺铂的敏感性，MDA-MB-231/DDP细胞株较MDA-MB-231细胞株，PTEN蛋白表达降低，p-Akt蛋白表达增加（p<0.001）；益气小复方加顺铂组对比顺铂组PTEN蛋白表达增加，p-Akt蛋白表达降低（p<0.001）。敲减HCP5基因后，MDA-MB-231/DDP-KD-HCP5细胞株较MDA-MB-231/DDP细胞株对顺铂敏感性增强，PTEN蛋白表达增加，p-Akt蛋白表达降低（p<0.001）；过表达HCP5基因后，MDA-MB-231-OE-HCP5细胞株较MDA-MB-231细胞株对顺铂敏感性降低，PTEN蛋白表达降低，p-Akt蛋白表达增加（p<0.001），体内实验，同样以顺铂干预，MDA-MB-231/DDP组与MDA-MB-231组相比，瘤体体积更大、重量更重（p<0.001）；益气小复方加顺铂组与顺铂组相比，瘤体的体积、重量均有减小（p<0.001），瘤体的lncRNA HCP5、p-Akt蛋白表达降低， PTEN蛋白表达增加（p<0.001），结果提示益气小复方同顺铂联用可增强三阴性乳腺癌顺铂耐药细胞株MDA-MB-231/DDP对顺铂的敏感性，体内实验可增强顺铂对MDA-MB-231/DDP裸鼠移植瘤的增长抑制作用，其机制可能与其下调lncRNA HCP5，提高PTEN蛋白、降低p-Akt蛋白表达有关。lncRNA HCP5可能是临床诊治三阴性乳腺癌的一个潜在靶点值得进一步研究和推广。