摘要
目的 探索miR-200a对卵巢癌化疗敏感性的影响及其可能的作用机制.方法 首先利用慢病毒表达载体,在卵巢癌细胞株SKOV-3和ES-2中,构建稳定上调miR-200a的细胞模型;接下来采用MTT实验检测miR-200a对紫杉醇及顺铂化疗敏感性的影响;进一步通过荧光定量PCR和免疫印迹实验,检测miR-200a对耐药相关三磷酸腺苷结合盒转运子（ABC）家族表达水平的影响;最后利用双荧光素酶实验验证miR-200a与ABCG2mRNA 3＇末端非编码区（3＇-UTR）的相互作用关系.结果 qRT-PCR验证携带miR-200a转基因的SKOV-3和ES-2卵巢癌细胞较对照细胞中miR-200a的表达水平分别升高了3.15和273.76倍;证实miR-200a过表达增加了SKOV-3和ES-2细胞株对紫杉醇的敏感性,但对顺铂的敏感性没有明显影响;miR-200a不同程度的下调了ABC家族（ABCB3,ABCC1,ABCC2,ABCC3,ABCG2）的表达水平,但与ABCG2的3＇-UTR并不存在直接相互作用关系.结论 miR-200a可能通过调控耐药相关ABC家族基因的表达,增加了卵巢癌细胞对紫杉醇的敏感性,但其下调ABCG2表达的方式并非直接作用其mRNA的3＇-UTR.
Objective To explore the role of miR-200a in chemosensitivity regulation of ovarian cancer.Methods Firstly miR-200a was up-regulated in ovarian cancer cell lines （SKOV-3 and ES-2） by lentiviral vector.Then the effects of miR-200a on cytotoxicity of paclitaxel and cisplatin were investigated by methyl thiazolyl tetrazolium （MTT）.Furthermore miR-200a regulation of chemoresistance associated with ATP-binding cassette （ABC） family genes expression was detected by quantitative real-time polymerase chain reaction （PCR） and Western blot.Finally the interaction between miR-200a and ABCG2 mRNA 3＇ untranslated region （3＇-UTR） was verified by dual-luciferase reporter assay.Results An over-expression of miR-200a were successfully achieved in SKOV-3 and ES-2 cells.MiR-200a enhanced the chemosensitivity of SKOV-3 and ES-2 to paclitaxel,but not to cisplatin.Chemoresistance associated ABC family （ABCB3,ABCC1,ABCC2,ABCC3,ABCG2） were down-regulated by miR-200a at several levels.However,the direct interaction between miR-200a and the 3＇-UTR of ABCG2 mRNA was not found.Conclusion An over-expression of miR-200a may increase chemosensitivity to paclitaxel in ovarian cancer cells through negatively regulated chemoresistance associated ABC family.However,no direct action on 3＇-UTR of ABCG2 was not found after its down-regulation by miR-200a.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2014年第27期2148-2151,共4页
National Medical Journal of China
基金
广东省教育厅科技创新项目（2013KJCX0042）
南方医科大学珠江医院人才引进基金
