论文部分内容阅读
目的:探讨乳腺癌细胞株MCF-7和耐药株MCF7/ADR中miR-34a的表达差异及miR-34a对于多柔比星化疗敏感性的影响。方法:采用实时定量PCR技术检测MCF-7细胞及MCF-7/ADR细胞中miR-34a的表达差异。采用转染技术,以脂质体为载体,在MCF-7/ADR细胞株中过表达miR-34a后,检测其对于多柔比星耐药活性的影响,同时采用实时定量PCR技术和蛋白质印迹法技术检测靶基因的表达变化。结果:MCF-7细胞株和耐药株MCF-7/ADR中miR-34a的相对表达量分别为1.01±0.03和0.76±0.04,在耐药细胞株中呈现下调表达,P=0.007。MTT结果显示,MCF-7及其耐药株MCF-7/ADR细胞多柔比星半数抑制浓度IC50分别为(0.22±0.02)和(18.7±0.09)μmol/L。对耐药株MCF-7/ADR过表达miR-34a后,MCF-7/ADR细胞对多柔比星的IC50降低为(10.7±0.11)μmol/L,明显增强此细胞株对于多柔比星的耐药敏感性。实时定量PCR结果显示,与转染阴性对照RNA相比,于耐药株MCF-7/ADR细胞中转染miR-34a后耐药相关靶基因Bcl-2和CCND1的mRNA水平分别降低了0.46±0.02(P=0.002)和0.33±0.02(P=0.008)。蛋白质印迹结果显示,过表达miR-34a后,可明显下调靶基因Bcl-2和CCND1的表达。结论:上调表达miR-34a可以增加耐药细胞株MCF-7/ADR对于多柔比星的耐药敏感性。
Objective: To investigate the difference of miR-34a expression in breast cancer cell line MCF-7 and drug-resistant MCF7 / ADR and the effect of miR-34a on doxorubicin chemosensitivity. Methods: Real-time quantitative PCR was used to detect the expression of miR-34a in MCF-7 cells and MCF-7 / ADR cells. The expression of miR-34a in MCF-7 / ADR cell line was detected by lipofectamine 2000, and its effect on resistance to doxorubicin was detected by real-time quantitative PCR and Western blot Method of detecting the target gene expression changes. Results: The relative expression levels of miR-34a in MCF-7 cells and drug-resistant MCF-7 / ADR cells were 1.01 ± 0.03 and 0.76 ± 0.04, respectively, and were down-regulated in drug-resistant cell lines, P = 0.007. MTT results showed that the IC50 of doxorubicin in MCF-7 and its multi-drug resistant MCF-7 / ADR cells were (0.22 ± 0.02) and (18.7 ± 0.09) μmol / L, respectively. The IC50 of MCF-7 / ADR cells against doxorubicin was (10.7 ± 0.11) μmol / L after MCF-7 / ADR overexpression of miR-34a, significantly enhanced the sensitivity of this cell line to doxorubicin Drug resistance. The results of real-time PCR showed that the mRNA level of Bcl-2 and CCND1 in the drug-resistant MCF-7 / ADR cells transfected with miR-34a decreased by 0.46 ± 0.02 (P = 0.002) and 0.33 ± 0.02 (P = 0.008). Western blotting showed that overexpression of miR-34a significantly down-regulated the expression of Bcl-2 and CCND1. Conclusion: The up-regulation of miR-34a can increase the sensitivity of drug-resistant cell line MCF-7 / ADR to doxorubicin.