MiR-186通过PIK3R3和APAF-1双向调节卵巢癌细胞顺铂敏感性的作用及机制研究

Ovarian cancer is a highly lethal gynaecological malignancy. Cisplatin is the basal chemotherapeutic agent used for ovarian cancer, but resistance to cisplatin leads to chemotherapy failure. The mostly accepted proposal to reverse drug resistance is combined chemotherapy with agents which could up-regulated cisplatin sensitivity. Epigenetic therapy with decitabine or TSA is effective in cisplatin resistant ovarian cancer. However, it is limited by their strong side effects. MicroRNAs, a class of short non-coding RNAs of 19-25nt in length, play roles in multiple physiological and pathological functions by binding to 3’UTR of target mRNAs, inhibiting translation and/or promoting mRNAs decades. Recently，data shows that microRNAs also are related to cisplatin sensitivity. We found miR-186 was significantly down-regulated in cisplatin resistant ovarian cancer tissues and cells SKOV3/DDP and A2780/DDP compared with the sensitivity tissues and cells. Interestingly, ectopic overexpression of miR-186 by low concentration of mimics in A2780/DDP cells, increased cisplatin sensitivity of the cells compared to the control, while ectopic overexpression of miR-186 by high concentration of mimics in A2780/DDP cells, decreased cisplatin sensitivity of the cells compared to the control. It is vital to investigate the relationship of miRNAs concentration and function so as to apply miRNAs in clinic. We supposed that the high level miR-186 targets more low level mRNAs which has the opposite function, may contribute to the opposite results. Bioinformatics analyses predicted that PIK3R3 and APAF-1 are potential targets of miR-186. PIK3R3 is antiapoptotic by activating the PI3K/AKT signal pathway. APAF-1 is proapoptotic by activation of caspase 9. We confirmed that both of the levels of mRNAs and proteins of PIK3R3 and APAF-1 were down-regulated by ectopic overexpression of miR-186. We proposed that miR-186 plays a dule role by targeting PIK3R3 and APAF-1. Continuously, We focus on (1) to investigate the dual role of miR-186 in regulating cisplatin sensitivity of ovarian cancer cells in vitro and in vivo. (2)To certify the targets related to cisplatin sensitivity: miR-186 at low concentration mainly target the more abandent PIK3R3 mRNA, increasing cisplatin sensitivity, while miR-186 at high concentration also target the less abandent APAF-1 mRNA, decreasing cisplatin sensitivity. We are aimed at revealing the dual role of miR-186 in regulating ovarian cancer cisplatin sensitivity through its multi-targets, so as to apply to reversing cisplatin resistance in clinic.

顺铂耐药是导致卵巢癌治疗失败的关键因素。经典逆转耐药的地西他滨等毒副作用大，亟待温和的逆转分子。近来，以转录后调节小分子miRNAs逆转耐药成为研究热点。项目组前期通过芯片及qRT-PCR筛选鉴定了在卵巢癌耐药组织和细胞中miR-186低表达，发现不同浓度miR-186呈现不同的功能：低浓度增加细胞顺铂敏感性，高浓度降低。解析上述miRNAs浓度影响其功能的机制对miRNAs的应用意义重大。功能相反的PIK3R3和APAF-1同为miR-186的预测靶，经证实二者皆受miR-186下调。基于前期工作，项目组提出“不同浓度miR-186通过PIK3R3和APAF-1双向调节卵巢癌细胞顺铂敏感性”假说，拟在细胞和动物水平进一步验证miR-186的双向调节作用，明晰不同浓度miR-186与靶的关系。本项目旨在揭示miR-186的双向调节作用及机制，为临床上逆转卵巢癌顺铂耐药提供新的理论依据。

卵巢癌是女性生殖系统恶性肿瘤，致死率高。以顺铂为代表的铂类药物是治疗包括卵巢癌在内的多种恶性肿瘤的一线化疗药物，但药物耐药长导致化疗失败。MicroRNAs是一类短链非编码RNA，转录后水平调控基因的表达，在肿瘤发生、侵袭、转移、凋亡和耐药性中发挥重要作用。大多数研究证明miR-186是一种肿瘤抑制miRNA，而亦有报道证实miR-186是一种促癌的miRNA（oncomir）。近期研究称miR-186在顺铂耐药的卵巢细胞系中表达下调，过表达miR-186可增加顺铂的敏感性。然而，我们首次发现miR-186对顺铂敏感性的双向调控作用：低浓度过表达miR-186可提高卵巢癌细胞A2780/DDP对顺铂的敏感性，而高浓度miR-186则降低顺铂敏感性。其他类型癌细胞中验证了miR-186对顺铂敏感性的调节作用依赖于剂量和细胞类型。miR-186抑制PTEN和PIK3R3（AKT通路的两个调节分子，功能相反）的蛋白表达水平，呈剂量依赖性。MiR-186还上调促凋亡基因APAF1的蛋白表达水平，亦呈剂量依赖性。利用数据库ENCORI分析肿瘤样本中miR-186与PIK3R3、PTEN、APAF1表达的相关性，进一步佐证在肿瘤组织中miR-186对这些基因可能存在调节作用。我们得出结论，miR-186通过调节这些功能相反的基因的表达水平，双向调控卵巢癌细胞对顺铂的敏感性：当miR-186低浓度时，miR-186通过抑制PIK3R3、促进APAF1表达，使PTEN和APAF1的最终效应大于PIK3R3，从而增加卵巢癌细胞对顺铂的敏感性；而当miR-186高浓度时，miR-186亦可通过抑制PTEN，使PIK3R3的最终效应大于PTEN和APAF1，从而降低顺铂的敏感性。我们的研究结果为基于miRNAs逆转卵巢癌顺铂耐药的策略提供科学的思路和实验依据。

内容获取失败，请点击重试