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。
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-47 D 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-47 D 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-47 D. RESULTS Results of MTT assays showed that miR-489 significantly enhanced the cytotoxicity of carboplatin to T-47 D. Results of bioinformatics, Western blot analysis and luciferase reporter assay showed that XIAP was the target of miR-489 in T-47 D. 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-47 D. 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 Mi R-489 targets XIAP to enhance the anti-tumor effect of carboplatin on breast cancer.
作者
郑璐
胡静
陈雪
许权
ZHENG Lu, HU Jing, CHEN Xue, XU Quan(Department of Radiotherapy, Li Huili Hospital of Ningbo Medical Center, Ningbo 315000, Chin)
出处
《中国现代应用药学》
CAS
CSCD
北大核心
2018年第3期319-324,共6页
Chinese Journal of Modern Applied Pharmacy
关键词
乳腺癌
miR-489
X连锁凋亡抑制蛋白
卡铂
凋亡
breast cancer
miR-489
X-linked inhibitor of apoptosis protein （XIAP）
carboplatin
apoptosis
