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。