miR-489协同卡铂通过抑制XIAP的表达发挥抗乳腺癌作用

MiR-489 Targets X-linked Inhibitor of Apoptosis Protein to Enhance the Anti-tumor Effect of Carboplatin on Breast Cancer

关键词:  乳腺癌  miR-489  X连锁凋亡抑制蛋白  卡铂  凋亡

Key words:  breast cancer  miR-489  X-linked inhibitor of apoptosis protein (XIAP)  carboplatin  apoptosis

摘要:

目的 研究miR-489对卡铂的协同抗乳腺癌效应及机制。方法 MTT细胞活力试验检测卡铂和miR-489对T-47D细胞的杀伤活性。生物信息学、western blot试验及荧光素酶报告基因试验验证X连锁凋亡抑制蛋白（X-linked inhibitor of apoptosis protein，XIAP）是否为miR-489的靶点。分离去除T-47D细胞中的线粒体，western blot试验检测miR-489、卡铂及XIAP质粒处理后T-47D细胞Smac/DIABLO的释放和caspase-9及caspase-3的活化。免疫共沉淀试验检测XIAP与Smac/DIABLO的相互作用。流式细胞术检测T-47D细胞的凋亡情况。结果 miR-489处理能显著增强卡铂对T-47D细胞的杀伤活性。XIAP是miR-489的靶点。miR-489不影响卡铂依赖的线粒体中Smac/DIABLO的释放，但能通过下调XIAP的表达减弱XIAP与Smac/DIABLO的相互作用。另外，转染XIAP质粒能显著抑制miR-489对卡铂的协同抗乳腺癌效应，XIAP质粒能显著抑制miR-489联合卡铂对T-47D细胞凋亡的诱导。XIAP质粒能显著抑制miR-489联合卡铂对T-47D细胞caspase-9及caspase-3的活化。结论 miR-489抑制XIAP的表达发挥对卡铂的协同抗乳腺癌作用。

Abstract:

OBJECTIVE To investigate the effect and mechanism of miR-489 on enhancing the anti-tumor effect of carboplatin on breast cancer. METHODS Cell viability of T-47D which were treated with miR-489 and carboplatin was measured by using MTT assay. Bioinformatics, western blot analysis and luciferase reporter assay were performed to confirm whether the XIAP was the target of miR-489. After removal of mitochondria, western blot analysis was conducted to detect the release of Smac/DIABLO and activation of caspase-9 and caspase-3 in T-47D cells treated with carboplatin and miR-489. Co-immunoprecipitation assay was performed to evaluate the interaction with XIAP and Smac/DIABLO. Flow cytometry analysis was used to measure the cell apoptosis of T-47D. RESULTS Results of MTT assays showed that miR-489 significantly enhanced the cytotoxicity of carboplatin to T-47D. Results of bioinformatics, western blot analysis and luciferase reporter assay showed that XIAP was the target of miR-489 in T-47D. Overexpression of miR-489 couldn't influence the carboplatin-induced release of Smac/DIABLO. However, miR-489 was able to inhibit the interaction of Smac/DIABLO and XIAP through down-regulating the expression of XIAP in T-47D. In addition, transfection with XIAP plasmid significantly abolished the synergistic effect of miR-489 on carboplatin-induced cytotoxicity to breast cancer. Results of flow cytometry showed that transfection with XIAP plasmid significantly abolished the miR-489-promoed apoptosis induced by carboplatin. Results of Western blot analysis showed that transfection with XIAP plasmid significantly abolished the miR-489-promoed activation of caspase-9 and caspase-3 induced by carboplatin. CONCLUSION MiR-489 targets XIAP to enhance the anti-tumor effect of carboplatin on breast cancer.