microRNA-FoxM1调控网络在大蒜素抗骨肉瘤干细胞中的作用及机制研究

Osteosarcoma cancer stem cells (CSCs) are the main reason for drug resistance and recurrence in osteosarcoma. How to eradicate CSCs is the key point in the treatment of osteosarcoma. Recent studies show that there is a cross-talk between microRNA (miRNA) and the FoxM1 transcriptional factor in cancer initiation and progression, which are essential for the maintenance of CSCs self-renewal. In our previous studies, we found that diallyl trisulfide could lower the ratio of CSCs and enhance the chemo-sensitivity of osteosarcoma drug-resistant cells. Moreover, miR-370 and the FoxM1 transcriptional factor may be involved in the anti-osteosarcoma effects of diallyl trisulfide. Therefore, we speculated that diallyl trisulfide could eradicate CSCs via regulation of miRNA-FoxM1 signaling pathways, resulting in the reversion of drug resistance. In the following study, we intend to isolate CSCs from osteosarcoma drug-resistant cells and investigate the effects of diallyl trisulfide on the self-renewal and drug resistance of osteosarcoma CSCs; clarify that diallyl trisulfide can regulate the miR-370-FoxM1 signaling pathway in osteosarcoma CSCs; screen and identify the novel miRNA-FoxM1 signaling pathways and further demonstrate the regulatory mechanisms of diallyl trisulfide-induced eradication of osteosarcoma stem cells; determine the effects of diallyl trisulfide on the eradication of osteosarcoma CSCs in the mouse models engrafted with osteosarcoma CSCs cells. Our study is of great importance in overcoming drug resistance in osteosarcoma.

骨肉瘤肿瘤干细胞（CSCs）是骨肉瘤耐药复发的根源，有效清除CSCs是根治骨肉瘤的关键。新近研究证实，microRNA（miRNA）与转录因子FoxM1可相互调控，对维持CSCs自我更新至关重要。我们研究发现，大蒜素能减少骨肉瘤CSCs数量并增强耐药细胞的化疗敏感性，其机制可能与miR-370-FoxM1通路有关。因此，我们提出假说：大蒜素可能通过调控miRNA-FoxM1环路抑制CSCs，从而逆转骨肉瘤耐药。本课题拟在前期工作基础上，分离纯化骨肉瘤原代/耐药细胞中CSCs，研究大蒜素对骨肉瘤CSCs自我更新及耐药性的影响；明确大蒜素对miR-370-FoxM1的调控作用，阐明其在骨肉瘤CSCs中的地位；利用miRNA芯片寻找并鉴定出新的miRNA-FoxM1环路，揭示大蒜素抗骨肉瘤CSCs的新机制；利用荷瘤小鼠模型，探讨大蒜素清除骨肉瘤CSCs的可行性；为克服骨肉瘤耐药奠定理论基础。

骨肉瘤肿瘤干细胞（CSCs）是骨肉瘤耐药复发的根源，有效清除CSCs是根治骨肉瘤的关键。新近研究证实，microRNA（miRNA）与转录因子FoxM1可相互调控，对维持CSCs自我更新至关重要。前期研究发现，大蒜素能减少骨肉瘤CSCs数量并增强耐药细胞的化疗敏感性，并伴有miR-370上调和FoxM1下调。然而，大蒜素能否通过调控miRNA-FoxM1环路抑制CSCs及逆转骨肉瘤耐药目前尚不明确。本课题成功分选出骨肉瘤CSCs，阐明该群细胞的干细胞特性。首次报道，大蒜素可减弱骨肉瘤CSCs的生存和自我更新能力，促进细胞凋亡，并能够增强骨肉瘤CSCs对多种化疗药物的敏感性。同时证实，骨肉瘤CSCs细胞中miR-370对FoxM1的靶向调节作用，发现上调miR-370表达能够抑制骨肉瘤CSCs自我更新、逆转耐药；下调miR-370表达能产生相反的效果。并且，进一步明确miR-370可负向调控FoxM1通路，阻断FoxM1可减弱骨肉瘤CSCs的生存、自我更新能力，并能增强化疗药物的敏感性；上调FoxM1表达可促进骨肉瘤CSCs自我更新及耐药，阐明miR-370通过调控FoxM1维持骨肉瘤CSCs的存活及耐药。此外，我们采用生物信息学方法寻找并鉴定出新的靶向FoxM1的miR-320分子。阐明miR-320过表达可抑制骨肉瘤CSCs增殖，增强细胞凋亡及药物敏感性，明确FoxM1是miR-320分子的下游靶基因，证实miR-320分子在骨肉瘤CSCs中的重要地位，揭示大蒜素抗骨肉瘤CSCs的新机制。此原创性研究阐明大蒜素可通过调控miR-320/370-FoxM1环路抑制骨肉瘤CSCs、逆转耐药，揭示miR-320/370-FoxM1环路有望成为逆转骨肉瘤耐药治疗的新靶点，为克服骨肉瘤耐药提供靶向治疗的新思路。