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目的探讨有丝分裂期检查点蛋白BubR1在乳腺癌细胞发生紫杉醇耐药中的作用。方法设计并合成针对BubR1基因的特异性干扰RNA(siRNA)片段,连接入穿梭质粒后,包装成腺病毒(Ad-BubR1-siRNA),感染MCF-7细胞,采用实时荧光定量RT-PCR和Western blot法评价BubR1干扰对MCF-7细胞BubR1基因和蛋白表达的影响。分别通过cck8试验、染色体计数分析、细胞集落生成试验、Hochest染色和细胞划痕试验评价干扰BubR1后,MCF-7细胞在紫杉醇作用下增殖、染色体稳定性、凋亡及迁移能力等生物学功能。结果重组腺病毒的滴度为1010 pfu/ml,其能有效抑制BubR1基因在mRNA和蛋白水平的表达。干扰BubR1表达后,MCF-7细胞在紫杉醇作用下表现为增殖和迁移能力增强,染色体不稳定性增加,集落生成能力增强,凋亡能力下降。结论下调BubR1基因可能导致乳腺癌细胞MCF-7对紫杉醇的耐药性增强。
Objective To investigate the role of BubR1, a checkpoint protein of mitosis, in paclitaxel resistance in breast cancer cells. Methods The specific siRNA fragment targeting BubR1 gene was designed and synthesized. After being inserted into the shuttle plasmid, the recombinant plasmid was packaged into Ad-BubR1-siRNA and infected with MCF-7 cells. Real-time fluorescent quantitative RT-PCR and Western blot method to evaluate the effect of BubR1 interference on the expression of BubR1 gene and protein in MCF-7 cells. The biological functions of MCF-7 cells such as proliferation, chromosome stability, apoptosis and migration were evaluated by cck8 assay, chromosome counting assay, colony forming assay, Hochest staining and cell scratch assay respectively after interference with BubR1. Results The titer of the recombinant adenovirus was 1010 pfu / ml, which effectively inhibited the expression of BubR1 gene at mRNA and protein levels. After interfering with the expression of BubR1, MCF-7 cells showed enhanced ability of proliferation and migration, increased chromosome instability, enhanced colony formation ability and decreased apoptosis capacity under the action of paclitaxel. Conclusion Down-regulation of BubR1 gene may result in enhanced resistance to paclitaxel in breast cancer cells MCF-7.