潜在抑癌基因NEK11和癌基因NEK2对卵巢癌耐药的调控机制研究

Ovarian cancer is the leading cause of death among malignancies of the female reproductive system, with a high rate of mortality worldwide. The main obstacle to a successful treatment for ovarian cancer is the development of drug resistance. It has been proven that the mechanisms of drug resistance in ovarian cancer are complicated and involved in many biological and cellular processes. However, regardless of mechanisms, abnormal expression of drug resistance-related genes often plays an important role in drug resistance, and among all these genes, tumor suppressor genes and oncogenes are clearly the key players. NEK11 is a potential tumor suppressor gene, with rarely research on its association with cancer, and NEK2 is an oncogene, with only several studies indicated its relationships with drug resistance in cancers. However, the study on these two genes with drug resistance in ovarian cancer has never been reported. Based on the mRNA expression data of the NEK11 and NEK2, according to the array data retrieved from Oncomine and GEO online databases, we found that the NEK11 and NEK2 was significantly down-regulated and up-regulated in ovarian cancer tissues and drug-resistant ovarian cancer cells, respectively, which were consistent with our results of RT-qPCR and western blot detection. Network and interaction-based comprehensive bioinformatics analyses suggested that the NEK11 and NEK2 might associate with drug resistance in ovarian cancer. Therefore, in this application, a scientific hypothesis is proposed that the potential tumor suppressor NEK11 and the oncogene NEK2 are contributed to the modulation of drug resistance in ovarian cancer. By means of RNA/protein expression analysis, lentiviral systems for overexpression/RNA interference, microRNA transfection and in vivo xenograft growth assay, we aim to illustrate the role of NEK11 and NEK2 in the modulation of drug resistance in ovarian cancer. The impact of this application is to better understand the mechanisms of drug resistance, and to set the stage for molecular-targeted therapies, drug resistance reversion and clinical application in ovarian cancer.

卵巢癌患者的死亡率高居妇科恶性肿瘤之首。耐药是导致卵巢癌治疗失败和死亡率高的主要原因，而抑癌基因和癌基因的异常表达则是卵巢癌耐药调控的重要因素。NEK11与肿瘤相关研究较少，是潜在抑癌基因，而癌基因NEK2仅有少许研究表明其与耐药相关，但这两个基因参与调控卵巢癌耐药的研究未见报道。基于芯片数据挖掘和我们在蛋白/基因水平上的检测,以及综合生物信息学分析，我们发现在卵巢癌组织和耐药细胞中显著下调表达的NEK11和显著上调表达的NEK2可能通过多种方式与卵巢癌耐药相关联。因此我们提出科学假设：潜在抑癌基因NEK11和癌基因NEK2参与调控卵巢癌耐药的发生发展。本课题将通过表达分析、过表达/RNA干扰、microRNA调控及动物模型等手段，深入探讨NEK11和NEK2对卵巢癌耐药的调控机制。该研究成果将有助于加深对肿瘤耐药分子机制的认识，并为卵巢癌分子靶向治疗、耐药逆转及临床应用提供理论依据。

卵巢癌是死亡率最高的女性生殖系统恶性肿瘤，而耐药是卵巢癌治疗失败的主要原因。癌基因和抑癌基因是卵巢癌耐药调控的重要因素。NEK2为癌基因，NEK11为潜在抑癌基因，均与肿瘤进展相关，但与卵巢癌耐药相关研究鲜见。基于细胞、小鼠、临床组织及基因表达调控等多层次研究，本项目旨在系统研究NEK2和NEK11与卵巢癌耐药的关系。我们发现和8例癌旁正常组织相比，NEK2在218例卵巢癌组织中显著上调，NEK11显著下调；和27例卵巢癌敏感组织相比，NEK2在24例耐药组织中显著上调，NEK11显著下调。Kaplan-Meier分析显示，在51例组织中，NEK2高表达和NEK11低表达与不良DFS和OS均显著相关，这在1815例卵巢癌大样本中进一步得到了确证，且联合NEK2和NEK11具有更为显著的OS预后效能，提示这两个基因是潜在预后标记物。基于DNA甲基化、lncRNA及microRNA三个层面的研究，发现lncRNA和microRNA，特别后者是这两个基因在卵巢癌耐药组织/细胞中差异表达的调控机制。miR-150显著负调控NEK2蛋白表达，miR-203和miR-590-5p显著负调控NEK11表达，且双荧光素酶报告系统证明miR-590-5p能靶向结合NEK11。基于细胞和动物模型研究，明确了NEK2上调能提高卵巢癌耐药细胞HeyA8-R和SKOV3-R对紫杉醇的耐药性，NEK2下调和NEK11上调能削弱细胞对紫杉醇的耐药性，说明这两个基因是卵巢癌耐药调控的重要因子。通过分析NEK2和NEK11过表达/干扰表达细胞中互作蛋白的动态变化，筛选出受这两个基因显著调控的AKT1、GSK-3beta、KCNN3、PDIA4、ALDH1A2 及TRPC1，绘制了其协同调控细胞死亡和凋亡响应卵巢癌耐药的分子通路，初步阐明这两个基因调控卵巢癌耐药的分子机制。.综上，在lncRNA和microRNA的调控下，卵巢癌耐药组织/细胞中NEK2上调和NEK11下调可能通过介导细胞死亡和凋亡等途径而显著提高卵巢癌耐药细胞对紫杉醇的耐药性，且NEK2高表达和NEK11低表达与卵巢癌不良预后显著相关。本研究首次比较系统的阐明了NEK2和NEK11与卵巢癌耐药相关，有望作为治疗靶点和预后标记物应用于临床，具有重要的理论和应用价值。.