BACKGROUND Ovarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women. Treatment with PARP inhibitors (PARPi), such as Olaparib, has been recently introduced for OC patients, but resistance may occur and underlying mechanisms are still poorly understood. The aim of this study is to identify target genes within the tumor cells that might cause resistance to Olaparib. We focused on Neuropilin 1 (NRP1), a transmembrane receptor expressed in OC and correlated with poor survival, which has been also proposed as a key molecule in OC multidrug resistance. METHODS Using three OC cell lines (UWB, UWB-BRCA and SKOV3) as model systems, we evaluated the biological and molecular effects of Olaparib on OC cell growth, cell cycle, DNA damage and apoptosis/autophagy induction, through MTT and colony forming assays, flow cytometry, immunofluorescence and Western blot analyses. We evaluated NRP1 expression in OC specimens and cell lines by Western blot and qRT-PCR, and used RNA interference to selectively inhibit NRP1. To identify miR-200c as a regulator of NRP1, we used miRNA target prediction algorithms and Pearsons' correlation analysis in biopsies from OC patients. Then, we used a stable transfection approach to overexpress miR-200c in Olaparib-resistant cells. RESULTS We observed that NRP1 is expressed at high levels in resistant cells (SKOV3) and is upmodulated in partially sensitive cells (UWB-BRCA) upon prolonged Olaparib treatment, leading to poor drug response. Our results show that the selective inhibition of NRP1 is able to overcome Olaparib resistance in SKOV3 cells. Moreover, we demonstrated that miR-200c can target NRP1 in OC cells, causing its downmodulation, and that miR-200c overexpression is a valid approach to restore Olaparib sensitivity in OC resistant cells. CONCLUSIONS These data demonstrate that miR-200c significantly enhanced the anti-cancer efficacy of Olaparib in drug-resistant OC cells. Thus, the combination of Olaparib with miRNA-based therapy may represent a promising treatment for drug resistant OC, and our data may help in designing novel precision medicine trials for optimizing the clinical use of PARPi.

中文翻译：

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MiR-200c通过靶向Neuropilin 1来增强抗奥拉帕利抗性的卵巢癌细胞的敏感性。

背景技术卵巢癌（OC）是最致命的妇科恶性肿瘤，是女性癌症相关死亡的第二大主要原因。最近，已为OC患者采用PARP抑制剂（PARPi）进行治疗，例如Olaparib，但可能会产生耐药性，并且尚不清楚其基本机制。这项研究的目的是在肿瘤细胞中鉴定可能导致对Olaparib耐药的靶基因。我们专注于Neuropilin 1（NRP1），这是一种在OC中表达且与较差的生存率相关的跨膜受体，它也被认为是OC多药耐药性的关键分子。方法使用三种OC细胞系（UWB，UWB-BRCA和SKOV3）作为模型系统，我们评估了Olaparib对OC细胞生长，细胞周期，DNA损伤和凋亡/自噬诱导的生物学和分子作用，通过MTT和集落形成测定，流式细胞术，免疫荧光和蛋白质印迹分析。我们通过Western印迹和qRT-PCR评估了OC标本和细胞系中NRP1的表达，并使用RNA干扰来选择性抑制NRP1。为了鉴定miR-200c作为NRP1的调节剂，我们在OC患者的活检中使用了miRNA目标预测算法和Pearsons相关分析。然后，我们使用稳定的转染方法在Olaparib抗性细胞中过表达miR-200c。结果我们观察到，长时间的Olaparib治疗会导致NRP1在耐药细胞（SKOV3）中高水平表达，并在部分敏感细胞（UWB-BRCA）中上调，从而导致不良的药物反应。我们的结果表明，选择性抑制NRP1能够克服SKOV3细胞中的Olaparib抗性。而且，我们证明了miR-200c可以靶向OC细胞中的NRP1，从而导致其下调，而miR-200c的过表达是恢复OC耐药细胞中Olaparib敏感性的有效方法。结论这些数据表明，miR-200c显着增强了Olaparib在耐药性OC细胞中的抗癌功效。因此，奥拉帕里布与基于miRNA的疗法的组合可能代表了耐药性OC的有前途的治疗方法，我们的数据可能有助于设计新颖的精确医学试验，以优化PARPi的临床应用。结论这些数据表明，miR-200c显着增强了Olaparib在耐药性OC细胞中的抗癌功效。因此，奥拉帕里布与基于miRNA的疗法的组合可能代表了耐药性OC的有前途的治疗方法，我们的数据可能有助于设计新颖的精确医学试验，以优化PARPi的临床应用。结论这些数据表明，miR-200c显着增强了Olaparib在耐药性OC细胞中的抗癌功效。因此，奥拉帕里布与基于miRNA的疗法的组合可能代表了耐药性OC的有前途的治疗方法，我们的数据可能有助于设计新颖的精确医学试验，以优化PARPi的临床应用。