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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 verify the specificity of designed Mps1 small interfering RNA (siRNA) and provide the basis for its application in the treatment of cancer. Methods A siRNA (abbreviated as siMps1) was designed and synthesized according to the 5 ’end of Mps1 gene. The interference effect of siMps1 was detected by Western blot and the effect on the proliferation and mitosis of tumor cells after interference. The cell line SW480-YFPMps1siR / shRNA stably expressing siMps1 was further constructed to detect whether overexpression of exogenous Mps1 restored the phenotype of its endogenous gene deletion. Results Transfection of siMps1 could decrease the level of Mps1 in the cells and lead to the decrease of mitotic index and the abnormal chromosome arrangement in the metaphase. After cloning, HeLaS3 cells died of multinuclear cells after transfected with siMps1. The stable cell line SW480- YFPMps1siR / shRNA can restore the phenotype after Mps1 deletion. Conclusion The designed siMps1 has high specificity and is expected to be used in the treatment of tumors.