miR-34a调控下游基因FUT8表达介导乳腺癌多药耐药的实验验证

miR-34a mediates the multidrug resistance of breast cancer by regulating the expression of downstream gene FUT8

目的 探究miR-34a及 FUT8 的异常表达对乳腺癌多药耐药性的调控机制,明确乳腺癌耐药的诊疗靶点。方法 采用Real-time PCR及western blot技术检测MCF-7和MCF-7/ADR中miR-34a和 FUT8 的表达水平;Real-time PCR技术检测乳腺癌细胞系中miR-34a的转染效率;通过生物信息学方法预测并采用双荧光素酶报告实验验证 FUT8 与miR-34a之间的靶向关系;特异性调控miR-34a的表达后,分别通过Real-time PCR、western blot以及免疫荧光染色检测转染细胞系中 FUT8 基因及蛋白质水平变化;采用CCK8实验、免疫荧光实验检测其对MCF-7和MCF-7/ADR细胞增殖、耐药性的调控作用。结果 miR-34a 在MCF-7中的表达水平明显高于MCF-7/ADR( P =0.002 6);FUT8 基因在MCF-7/ADR中的表达水平明显高于MCF-7( P = 0.001 6);FUT8 是miR-34a调控乳腺癌耐药性的靶点( P =0.001 9);特异性上调MCF-7/ADR细胞中miR-34a的水平可明显抑制 FUT8 的表达,并抑制该细胞的多药耐药性及增殖性;而下调MCF-7细胞中miR-34a表达后, FUT8 的表达水平明显升高,同时增强了细胞多药耐药及增殖能力。结论 miR-34a通过调控下游基因 FUT8 的表达介导乳腺癌多药耐药。

Objective To identify the therapeutic target of multidrug resistance of breast cancer by investigating the regulatory mechanism of the abnormal expression of miR-34a and FUT8 on the multidrug resistance of breast cancer. Methods The expression levels of miR-34a and FUT8 in MCF-7 and MCF-7/ADR cells were detected by the real-time PCR and western blot. The transfection efficiency of miR-34a in breast cancer cells was determined by the real-time PCR. The targeting relationship between miR-34a and FUT8 was predicted by the bioinformatics method, and further verified by the dual-luciferase reporter assay. After the expression of miR-34a was specifically regulated, the changes of FUT8 mRNA and protein levels in transfected cells were detected by the real-time PCR, Western blot and immunofluorescence staining, respectively. After the expression of miR-34a was specifically regulated, the proliferation and multidrug resistance of MCF-7 and MCF-7/ADR cells were determined by the CCK8 and immunofluorescence assays. Results The expression levels of miR-34a in MCF-7 cells were significantly higher than that in MCF-7/ADR cells ( P =0.002 6). The expression levels of FUT8 gene in MCF-7/ADR cells were significantly higher than that in MCF-7 cells ( P =0.001 6). FUT8 was the target of miR-34a regulating the drug resistance of breast cancer ( P =0.001 9). The up-regulation of miR-34a in MCF-7/ADR cells could significantly inhibit the expression of FUT8 , and the multidrug resistance and proliferation of MCF-7/ADR cells. While, the down-regulation of miR-34a increased the expression of FUT8 , and enhanced the multidrug resistance and proliferation of MCF-7 cells. Conclusion MiR-34a mediates the multidrug resistance of breast cancer by regulating the expression of downstream gene FUT8 .