强心苷药物Oleandrin上调铜转运蛋白CTR1增加人骨肉瘤细胞摄取顺铂能力的机制研究

Cisplatin resistance is common in osteosarcoma, which is closely related to the reduction of cisplatin uptake in cells. Copper transporter 1 (CTR1) is the key protein that actively absorbs cisplatin. We have reported that Oleandrin, a kind of cardiac glycoside, shows anti-tumor ability in osteosarcoma cells. Our recent studies have found that Oleandrin can upregulate the expression of CTR1 and increase the uptake of cisplatin. However, the molecular mechanisms regulating the expression of CTR1 in osteosarcoma have not been reported. Cathepsin L, which is highly expressed in osteosarcoma cells resistant to cisplatin, has been reported to effectively hydrolyze and inactive CTR1 in other cells. We hypothesized that this may be an important cause of cisplatin tolerance in osteosarcoma cells. The microRNA microarray of osteosarcoma cells showed that the abundance of mir-214-3p, which had a direct binding sequence with CTR1 mRNA, decreased after interfered by Oleandrin. This suggests that mir-214-3p may also effect the expression of CTR1. In this study, we aim to confirm that Oleandrin may raise the expression of CTR1 by inhibiting the function of cathepsin L and mir-214-3p. The expected results may provide new targets and new ideas for reversing cisplatin resistance in osteosarcoma.

骨肉瘤对顺铂耐受普遍，这与细胞摄取顺铂能力降低关系密切。铜转运蛋白1（CTR1）是主动摄取顺铂的关键蛋白。本课题组曾报道强心苷物质Oleandrin在骨肉瘤细胞中有显著抗肿瘤活性，近期研究发现Oleandrin能上调CTR1的表达，增加细胞对顺铂的摄取。然而，骨肉瘤中调控CTR1表达的分子机制尚无报道。组织蛋白酶L能高效水解CTR1使其失活。耐受顺铂的骨肉瘤细胞中组织蛋白酶L表达水平高，这可能是骨肉瘤细胞产生顺铂耐受的重要原因。microRNA芯片显示被Oleandrin干预过的骨肉瘤细胞中与CTR1 mRNA具有直接结合序列的mir-214-3p丰度降低，提示其可能也影响CTR1的表达。基于此，我们拟证实Oleandrin可能通过抑制组织蛋白酶L和mir-214-3p来上调CTR1的假说，以期为逆转骨肉瘤耐受顺铂提供新靶点和新思路。

骨肉瘤（Osteosarcoma，OS）对顺铂耐受普遍且严重影响患者预后，这与细胞摄取顺铂能力降低密切相关。铜转运蛋白1（Copper transporter 1，CTR1）被认为是肿瘤细胞主动摄取顺铂的关键蛋白，本课题组前期研究发现欧夹竹桃苷（Oleandrin）有显著抗OS作用，本项目旨在研究Oleandrin对OS细胞化疗增敏作用及其介导CTR1转运参与化疗增敏的机制，研究内容主要包括：1）转录组测序观察Oleandrin对OS细胞基因表达的影响，通过GO和KEGG分析观察药物治疗前后差异基因表达和信号通路富集情况，寻找Oleandrin的潜在化疗增敏作用及涉及的分子信号机制；2）明确Oleandrin与顺铂联合应用对OS细胞命运的影响，体内验证两种药物联合作用效果；3）进一步探索Oleandrin在体外增强OS细胞对顺铂化疗敏感性的内在调节机制，尤其是对Oleandrin促进OS细胞摄取顺铂的机制探索。相关结果显示，Oleandrin能够影响OS细胞中多种基因表达，其中涉及多种与顺铂耐受相关的基因与通路，提示其是一种潜在的顺铂增敏剂。此外，Oleandrin可与顺铂联用能够发挥协同效应杀伤OS细胞，且细胞毒性作用具有选择性。这种协同效应与Oleandrin介导CTR1成熟体表达上调进而促进细胞摄取顺铂有关，此过程是通过抑制蛋白酶体对CTR1成熟体的降解来实现。通过本项研究，为Oleandrin在OS化疗增敏方面的潜在应用价值提供了新的理论依据，并明确了CTR1可能为Oleandrin增强OS细胞对顺铂敏感性的关键桥梁分子，为OS的化疗增敏研究领域提供了新的重要潜在靶点。

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