靶向Survivin反义寡核苷酸逆转MCF-7乳腺癌细胞三苯氧胺耐药的研究

Study of converting tamoxifen-resistance of breast cancer MCF-7 cells by Survivin mRNA-targeted antisense oligonucleotide

目的探讨Survivin基因在乳腺癌细胞获得性三苯氧胺(TAM)耐药性形成中的作用以及靶向Survivin mRNA逆转乳腺癌细胞TAM耐药性的可行性。方法建立TAM耐药细胞株(TAM-R),用靶向Survivin mRNA的反义寡核苷酸(ASODN)联合10^(-5)M TAM分别作用于MCF-7细胞和MCF-7/TAM-R细胞72 h后,采用MTT法、平板克隆形成实验、流式细胞仪、原位杂交法检测细胞活性、克隆形成率、凋亡率及Survivin mRNA表达的变化。结果 MCF-7/TAM-R组的Survivin mRNA表达明显高于MCF-7组;ASODN联合应用TAM能够逆转MCF-7/TAM-R细胞对TAM的耐药性,其细胞活性、克隆形成率以及凋亡抑制程度明显强于TAM组(P<0.05);靶向Survivin的反义寡核苷酸能够抑制Survivin mRNA的表达。结论 MCF-7细胞可能通过上调Survivin基因来对抗长期TAM刺激所诱导的细胞凋亡,从而促进TAM耐药性的形成,靶向Survivin mRNA的反义寡核苷酸能够逆转MCF-7细胞对TAM的耐药。

Objective To explore the role of Survivin gene in formation of acquired tamoxifen（ TAM）- resistance in breast cancer cells and the feasibility of converting tamoxifen-resistance of breast cancer MCF-7 cells by Survivin mRNA. Methods Tamoxifen-resistant breast cancer cell line（ TAM-R） was established. After treated by Survivin mRNA-targeted antisense oligonucleotide（ ASODN） combined with 10^-5M TAM respectively in MCF-7 and MCF-7 / TAM-R cell lines for 72 hours,the cell viability,colony formation capability,apoptotic rate,and change of Survivin mRNA expression were measured by MTT assay,clone formation assay,flow cytometry,and hybridization in situ. Results The expression of Survivin mRNA in MCF-7 / TAM-R cells was significantly higher than that in MCF-7 cells; ASODN combined with TAM converted TAM resistance in MCF-7 / TAM-R cells,the cell viability,the clone formation rate,and the degree of apoptotic inhibition in MCF-7 / TAM-R cells treated by ASODN and TAM were statistically significantly higher than those in TAM group（ P〈0. 05）,Survivin mRNA-targeted antisense oligonucleotide inhibited the expression of Survivin mRNA. Conclusion MCF-7 cells may resist to apoptosis induced by long-time TAM stimulation through up-regulating Survivin expression,thus the formation of TAM-resistance is promoted,and resistance of MCF-7 cells to TAM can be converted by Survivin mRNA-targeted antisense oligonucleotide.