摘要
目的验证所设计的Mps1小干扰RNA(siRNA)的特异性,为其应用于肿瘤治疗提供研究基础。方法针对Mps1基因的5'端设计合成一个siRNA(简称为siMps1),利用Western印迹检测siMps1的干扰效果,以及干扰后对肿瘤细胞的增殖和有丝分裂的影响。进一步构建稳定表达siMps1的细胞系SW480-YFPMps1siR/shRNA,检测过表达外源Mps1能否恢复其内源基因缺失的表型。结果转染siMps1能够降低细胞中的Mps1蛋白水平,导致有丝分裂指数降低,染色体中期排列异常;进而通过克隆形成实验发现,HeLaS3细胞转染siMps1后大量死亡,出现多核细胞;构建的稳定细胞系SW480-YFPMps1siR/shRNA能够恢复Mps1缺失后的表型。结论该设计得到的siMps1具有很高特异性,有望应用于肿瘤的治疗中。
Objective To validate the specificity of a small interference RNA （siRNA） for Mpsl in order to pave the way for its application in tumor therapy. Methods A siRNA against the 5＇end coding sequence of Mpsl （ siMpsl ） was designed and synthesized before the knockdown efficiency of siMpsl was determined by detecting the Mpsl protein level via Western blotting, and by checking tumor growth inhibition and mitotic defects upon Mpsl depletion. A stable cell line SW480-YFPMpsl siR/shRNA was generated to find out whether the expression of a siMpsl-resistant YFPMpsl sir could res-cue the defects by the Mpsl depletion with a retrovirns-expressed siMpsl. Results The transfection of siMpsl decreased the protein level of Mpsl significantly, causing a reduced mitotic index and chromosomal misalignment. Furthermore, HeLaS3 cells transfected with siMpsl committed extensive cell death, characterized by the increasing percentage of large cells with a muhilobed nucleus. The retrovirus-expressed siMpsl could also deplete endogenous Mpsl but failed to induce any detecta-ble defects in SW480-YFPMpslsiR/shRNA cell line. Conclusion siMpsl can deplete Mpsl with high efficiency and spe-cificity, with potential application in tumor therapy.
出处
《军事医学》
CAS
CSCD
北大核心
2013年第3期201-206,共6页
Military Medical Sciences
基金
国家自然科学基金面上资助项目(30971444)
