miRNA-1246通过NF-κB介导乳腺癌细胞紫杉醇耐药的机制研究

Study on the mechanism of miRNA-1246 mediating breast cancer cell resistance to paclitaxel through NF-κB

目的探讨微小RNA-1246(miR-1246)在乳腺癌细胞紫杉醇(PTX)耐药中的作用及相关机制。方法以SK-BR-3细胞构建PTX耐药乳腺癌细胞SK-BR-3/PTX作为SK-BR-3/PTX组,以正常SK-BR-3细胞作为SK-BR-3组;CCK-8实验检测两组细胞活力并计算凋亡指数;RTFQ-PCR检测miR-1246表达水平;免疫荧光实验和Western blot法检测P-gp蛋白表达。SK-BR-3/PTX细胞转染miR-1246 inhibitor和阴性对照(NC)inhibitor,分别设为miR-1246 inhibitor组和NC inhibitor组;Western blot法检测p-NF-κB p65、IκBα和P-gp蛋白表达。PTX处理miR-1246 inhibitor组和NC inhibitor组,流式细胞术检测细胞凋亡。结果成功构建PTX耐药乳腺癌细胞SK-BR-3/PTX,耐药指数为51.3。与SK-BR-3细胞相比,SK-BR-3/PTX细胞miR-1246表达水平显著升高(P<0.01),P-gp和p-NF-κB p65蛋白显著上调,IκBα蛋白明显下调(P<0.01),细胞核中NF-κB p65蛋白显著上调(P<0.01)。转染miR-1246 inhibitor可逆转SK-BR-3/PTX细胞上述蛋白表达变化(P<0.01)。miR-1246 inhibitor还可显著增强PTX诱导的SK-BR-3/PTX细胞凋亡(P<0.01)。结论miR-1246可能通过NF-κB信号通路诱导P-gp蛋白表达上调,从而诱导乳腺癌SK-BR-3细胞PTX耐药。

Objective To explore the role and related mechanisms of microRNA-1246(miR-1246)in paclitaxel(PTX)resistance of breast cancer cells.Methods PTX-resistant cells SK-BR-3/PTX were constructed as SK-BR-3/PTX group,and normal SK-BR-3 cells were used as SK-BR-3 group.CCK-8 assay was performed to detect cell viability and calculate apoptotic index in both groups.miR-1246 expression level was detected by RTFQ-PCR.Immunofluo-rescence assay and western blot assay were performed to detect P-gp protein expression.SK-BR-3/PTX cells were transfected with miR-1246 inhibitor and negative control(NC)inhibitor,which were set up as miR-1246 inhibitor group and NC inhibitor group,respectively.Western blot assay was performed to detect p-NF-κB p65,IκBαand P-gp protein expression.PTX was used to treat miR-1246 inhibitor group and NC inhibitor group,and apoptosis was detected by flow cytometry.Results PTX-resistant breast cancer cell SK-BR-3/PTX was successfully constructed with a resis-tance index of 51.3.Compared with SK-BR-3 cells,the expression level of miR-1246 in SK-BR-3/PTX cells was significantly increased(P<0.01).Compared with SK-BR-3 cells,P-gp and p-NF-κB p65 proteins were significantly up-regulated,and IκBαprotein was significantly down-regulated(P<0.01).Compared with SK-BR-3 cells,SK-BR-3/PTX nucleoprotein-NF-κB p65 protein was significantly up-regulated(P<0.01).Transfection of miR-1246 inhibitor could reverse the above-mentioned protein expression changes in SK-BR-3/PTX cells(P<0.01).miR-1246 inhibitor could also significantly enhance the apoptosis of SK-BR-3/PTX cells induced by PTX(P<0.01).Conclusion miR-1246 may induce the up-regulation of P-gp protein expression through the NF-κB signaling pathway,thereby inducing PTX resistance in breast cancer SK-BR-3 cells.