Livin基因增强骨肉瘤转化和顺铂耐药性的体外实验研究  

Livin Enhances Oncogenic Transformation and Cisplatin Resistance in Osteosarcoma Cells

作　　者：李旭[1] 李岩 程世孝[1] 李雷明[1] 孟凡贺[1] 王立国[1] 王军[1] 范广宇[1] 

机构地区：[1]中国医科大学附属第一医院骨科,沈阳110001

出　　处：《中国医科大学学报》2013年第8期673-677,696,共6页Journal of China Medical University

基　　金：国家自然科学基金(30973021;81272946)

摘　　要：目的探讨凋亡抑制蛋白(IAP)家族新成员Livin基因在骨肉瘤形成以及多药耐药过程中的作用。方法应用亚克隆技术构建Livin基因短发夹RNA(shRNA)表达载体,稳定转染并筛选沉默Livin表达的克隆。细胞克隆形成法测定阳性克隆的非贴附性生长状态。流式细胞计数法测量Livin表达短期沉默后细胞周期和顺铂化疗敏感性的变化,以及长期沉默后的Hoechst33258核染色情况。Western blot检测Cyclin D1、Bcl-2、Bax、活化型Caspase-3和多聚ADP核糖多聚酶的表达变化。结果在骨肉瘤U2-OS细胞中,shRNA介导了Livin mRNA和蛋白水平的下调,细胞克隆形成能力明显下降。在Livin表达沉默早期,处于G0/G1期细胞增加,Cyclin D1的表达减弱,对顺铂药物敏感性增强;长期沉默后凋亡小体形成,Bcl-2蛋白的表达下调,Bax、活化型Caspase-3和PARP的表达上调。结论凋亡抑制蛋白Livin不但促进骨肉瘤的发生而且促进顺铂化疗耐药性的形成。Objective To investigate the roles of Livin,a novel member of the inhibitor of apoptosis protein（IAP）family,in the tumorigenesis and multidrug resistance of osteosarcoma.Methods After the construction of the vector expressing short hairpin RNA（shRNA）specific for Livin,the stable transfectants for silencing Livin expression in U2-OS cells was isolated.The ability of anchorage-independent growth was determined by colony formation assay.Cell cycle distribution,chemosensitivity of cisplatin and formation of apoptosis body were determined by flow cytometry and Hoechst 33258 staining at both early and late stage.The expression of Cyclin D1,Bcl-2,Bax,cleaved Caspase3 and PARP was detected by Western blot.Results shRNA-mediated silence of Livin expression was identified in stable transfectants of U2-OS.Short-term inhibition of Livin resulted in G0/G1 arrest with a reduction of Cyclin D1 expression,as well as enhanced chemosensitivity to cisplatin.Moreover,the long-term knockdown of Livin provoked an increase in the apoptotic fraction,reduced the capacity of oncogenic transformation together with downregulation of Bcl-2 and activation of Bax,Caspase-3 and PARP.Conclusion Livin can not only promote the tumorigenesis but also enhance cisplatin resistance in osteosarcoma in vitro.

关 键 词：LIVIN 骨肉瘤 肿瘤发生 耐药性 

分 类 号：R738.1[医药卫生—肿瘤]