从DC-Treg轴探讨microRNA在氧化苦参碱逆转肺癌顺铂耐药中的作用

We have proved an inverse correlation between the chemotherapeutic efficiency and the increased FOXP3+/CD8+ ratio in patients with non-small cell lung cancer. Correction of immunosuppression could improve chemoresistance of tumor cells. However, the current immunotherapy may cause subsequent imbalance due to the overinhibition of regulatory T cells (Treg). As the active ingredient of traditional Chinese medicine, oxymatrine(OMT)is proved to weaken the chemoresistance of series of cancer cell lines and can modulate bipolarly the proliferation and function of Treg. The results of our preliminary experiment indicated that OMT can down-regulate TRAF6 expression in A549 lung adenocarcinma cell line. And it's demonstrated that TRAF6 is a critical factor in determining the maturity of dendritic cells (DC) and controlling the response and function of Treg. As reported recently, TRAF6 enhancement can upregulate miR-146a expression and the increased miR-146a level, in turn, suppresses TRAF6 and NFkB activity, thus composing a negative feed back loop. Therefore, our hypothesis is the modulation of Treg polarization and function can another mechanism for OMT to ameliorate the chemoresistance of cancer cells. And microRNA(miRNA) could be the key regulator in this process. At first, the current study will screen, validate and confirm the potential miRNAs involving the modulation of OMT-mediated Treg activation. Secondly, selected miRNAs will be further validated both in vitro. and in vivo.to clarify the regulatory mechanisms for specific miRNA and its target genes. Finally, we will observe the impact of miRNA on the effect of OMT combined with cisplatin to treat tumor-bearing mices. By miRNA modulation to maintain the immune balance in tumor host, it can advance the efficient of OMT to overcome chemoresistance of tumor cells and avoid side effects associated with autoimmune disorders. Out results will provide a original version and new targets for cancer immunotherapy.

已证实肺癌局部FOXP3+/CD8+比值增高预示患者化疗疗效差，纠正免疫抑制状态能改善肿瘤耐药。现有免疫治疗去除调节性T细胞(Treg)后往往诱发新的免疫失衡。中药成分氧化苦参碱(OMT)能逆转肿瘤多药耐药性，且双向调控Treg增殖及功能。我们发现OMT能明显下调A549肺腺癌细胞中TRAF6表达；已证实TRAF6是决定树突状细胞(DC)分化及Treg功能的关键基因并与miR-146a形成负反馈环。推测影响Treg极化及功能是OMT逆转肿瘤耐药的重要机制之一，而miRNA可能是OMT免疫功能的调控靶点。本课题首先筛选、验证并确定参与调控OMT介导DC-Treg的目标miRNA。体外及动物实验剖析miRNA及其靶基因的调控机制；证实miRNA靶向调控对OMT逆转荷瘤小鼠顺铂耐药的增效作用。通过miRNA与其靶基因的相互作用，提高OMT疗效并维持免疫平衡稳态，为肿瘤免疫治疗提供新思路和新靶点。

中药成分氧化苦参碱(OMT)能逆转肿瘤多药耐药性，目前国内外文献证实，OMT能刺激树突状细胞(DC)成熟分化，且能双向调控调节性T细胞(Treg)增殖及功能。Treg在肿瘤免疫应答过程中发挥至关重要的作用。本课题的主要研究内容及重要结果包括：1) 发表英文综述一篇，除了介导免疫应答失衡，miRNA引起能量代谢改变从而诱导肿瘤化疗耐药已被证明。本文总结5个miRNA在调节葡萄糖和脂质代谢以及其他能量代谢中的作用。.2)体外细胞学研究证实，OMT能促进体外培养的DC成熟分化；OMT预处理DC促进细胞因子的分泌；OMT预处理DC能诱导单个核细胞向FOXP3+/CD4+Treg亚群分化；OMT预处理后的免疫细胞(DC及DC-T)均能促进顺铂耐药株发生凋亡。结果提示，在非接触性共培养模式中，OMT-DC与OMT-DC-T均能削弱肿瘤细胞对顺铂的抵抗力。.3)动物实验部分，证实OMT和顺铂可协同抑制与外周血单个核细胞共培养的非小细胞肺癌细胞的生长。OMT和顺铂协同抑制非小细胞肺癌异种移植瘤生长。此外，OMT和顺铂能协同增加小鼠脾脏淋巴细胞中CD8+/Treg比例；并增强CD8+T细胞的抗肿瘤免疫效应。分子机制研究结果提示，OMT和顺铂能通过协同上调miR-155并下调SOCS1在脾脏CD8+T细胞中的表达，发挥其免疫调控作用，进而增强顺铂敏感性。.4)延伸研究部分探讨miR-1205在人类喉癌鳞状细胞癌(LSCC)中的作用。结果发现，临床LSCC组织中miR-1205表达水平明显低于邻近正常组织，且与T期、淋巴结转移及临床分期相关。miR-1205高表达预示LSCC患者预后良好。miR-1205过表达抑制了LSCC体外生长、迁移和侵袭。E2F1被认为是miR-1205新的功能靶点。E2F1异位表达部分逆转了miR-1205对LSCC细胞的抑制作用。而且，E2F1可以直接与miR-1205启动子结合，过表达的E2F1可以抑制miR-1205的表达。提示miR-1205/E2F1循环可能是该疾病潜在的诊断和预后生物标志物。