盐霉素通过下调miR-221抑制乳腺癌MCF-7细胞增殖的实验观察

Salinomycin suppresses the breast cancer MCF-7 cell proliferation via downregulating miR-221

目的探讨盐霉素抑制乳腺癌细胞和乳腺癌干细胞增殖与microRNA-221(miR-221)及其调控的PTEN/PI3K/Akt信号通路的关系。方法脂质体转染miR-221模拟物至乳腺癌MCF-7细胞,盐霉素(1μmol/L)处理后检测细胞增殖能力和乳腺球形成能力。筛选miR-221稳转MCF-7细胞,构建裸鼠荷瘤模型,给予盐霉素腹腔注射治疗。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测细胞和皮下瘤组织中miR-221和PTEN mRNA水平,Western blot法检测PTEN、p-Akt以及ALDH1蛋白表达。结果 RT-qPCR结果显示盐霉素(1μmol/L)可明显降低MCF-7细胞中内源性miR-221表达水平(P <0. 05)。与miR-221组相比,Sal-miR-221组乳腺球体积明显变小,数量明显减少(117±9. 4 vs 180±15. 6,P <0. 05);RT-qPCR显示miR-221表达水平明显降低(P <0. 05),PTEN mRNA水平则显著升高(P <0. 05);Western blot结果显示PTEN蛋白表达增高,p-Akt和ALDH1蛋白表达降低。Sal-miR-221组裸鼠异体移植瘤体积呈明显减小趋势;瘤组织内miR-221表达明显低于对照组(P <0. 05),而PTEN mRNA表达明显高于对照组(P <0. 05);PTEN蛋白表达亦明显升高,p-Akt和ALDH1蛋白则降低。结论体内外实验结果表明盐霉素抑制乳腺癌细胞和乳腺癌干细胞增殖,可能是通过下调miR-221促进PTEN表达,抑制PI3K/Akt信号通路实现的。

To explore the inhibitory effects of salinomycin on the breast cancer cells and stem cells,and the relationship with microRNA-221(miR-221)and its downstream PTEN/PI3K/Akt signaling pathway.Methods miR-221 mimic was transfected into breast cancer MCF-7 cells.After treatment of 1μmol/L of salinomycin,the CCK-8 and mammosphere formation assays were performed.MCF-7 cells were infected by the lentiviral vector of miR-221 to construct the miR-221 stable transfection cells.The nude mice were subcutaneously injected with the sable transfection cells.The real-time quantitative PCR(RT-qPCR)was used to detect the levels of miR-221 and PTEN mRNA in the cells and xenograft tissues.Western blot was used to analyze the expression of PTEN,p-Akt and ALDH1 proteins.Results RT-qPCR showed that salinomycin(1μmol/L)significantly decreased the expression of endogenous miR-221 in MCF-7 cells(P<0.05).Compared with miR-221 group,the mammosphere volume of salinomycin-treated group(Sal-miR-221)was significantly reduced,and the number was dramatically decreased(117±9.4 vs 180±15.6,P<0.05).RT-qPCR analysis showed that miR-221 in Sal-miR-221 group was significantly decreased(P<0.05),while the level of PTEN mRNA was significantly increased(P<0.05).Meantime,the expression of PTEN protein was increased,and p-Akt and ALDH1 proteins were decreased.The volume of subcutaneous tumor had tendency of reduction in the salinomycin treated group.The miR-221 was decreased dramatically in the subcutaneous tumor tissues compared to the negative control(P<0.05),whereas the level of PTEN mRNA was significantly higher than that in the control group(P<0.05),and the expression of PTEN protein was also increased.The expression of p-Akt and ALDH1 proteins was decreased.Conclusion The in vitro and in vivo results showed that salinomycin inhibits the proliferation of breast cancer cells and stem cells,possibly through downregulating miR-221,subsequently promoting PTEN expression and inhibiting PI3K/Akt signaling pathway.