骨唾液酸蛋白对乳腺癌MDA-MB-231细胞PI3K-AKT信号通路的影响

Effect of bone sialoprotien on PI3K-AKT signaling pathway of breast cancer MDA-MB-231 cells

目的:探讨骨唾液酸蛋白(bone sialoprotein,BSP)对乳腺癌MDA-MB-231细胞PI3K-AKT信号通路的影响。方法:BSP基因沉默的乳腺癌MDA-MB-231细胞(简称231BO-BSP27)经重组人BSP(recombinant human BSP,rhBSP)和PI3K-AKT抑制剂LY294002处理后,Western blotting检测磷酸化AKT水平的变化,实时定量PCR检测caspase-3、cyclin D1 mRNA表达水平,MTT法检测细胞增殖能力。结果:与BSP基因未沉默的对照组231BO-Scrambled细胞相比,BSP基因沉默的231BO-BSP27细胞BSP蛋白表达明显下调(74.32±2.18)%(P<0.01);AKT磷酸化水平明显下降(33.30±2.61)%(P<0.01),而caspase-3和cyclin D1 mRNA表达分别上升和下降(1.000±0.000 vs 1.733±0.039,1.000±0.000 vs 0.370±0.012;均P<0.01);231BO-BSP27细胞增殖能力显著下降(P<0.05)。外源添加rhBSP蛋白分别上调231BO-Scrambled和231BO-BSP27细胞AKT磷酸化水平(17.86±2.27)%和(33.78±1.51)%(均P<0.01),231BO-BSP27细胞caspase-3 mRNA表达降低(1.000±0.039 vs 0.541±0.091,P<0.01)、cyclin D1 mRNA表达升高(1.000±0.000 vs 2.921±0.032,P<0.01),促进231BO-Scrambled和231BO-BSP27细胞的增殖(均P<0.01)。LY294002则能逆转rhBSP对231BO-Scrambled和231BO-BSP27细胞AKT磷酸化激活作用(P<0.05),使231BO-BSP27细胞caspase-3 mRNA表达升高(P<0.01)、cyclin D1 mRNA表达降低(P<0.01),使该两种细胞增殖能力下降(均P<0.01)。结论:BSP通过PI3K-AKT信号通路调控乳腺癌MDA-MB-231细胞caspase-3和cyclin D1的表达,并影响细胞的增殖。

Objective： To investigate the effect of bone sialoprotein（BSP） on the PI3K-AKT signaling pathway in breast cancer MDA-MB-231 cells.Methods： BSP-silenced MDA-MB-231 cells were treated with recombinant human BSP（rhBSP） and the PI3K-AKT specific inhibitor LY294002.Western blotting analysis was used to detect the phosphorylation of AKT,qPCR was conducted to evaluate caspase-3,cyclin D1 mRNA expressions,and the proliferation of cells was analyzed by MTT assay.Results： Compared with the 231BO-Scrambled cells in control group,BSP protein expression in BSP-silenced 231BO-BSP27 cells was significantly lower（74.32±2.18）%（P〈0.01）,and expression level of AKT phosphorylation was also significantly lower（33.30±2.61） %（P〈0.01）,resulting in up-regulation of caspase-3 mRNA level（1.000±0.000 vs 1.733±0.039,P〈0.01）,down-regulation of cyclin D1 mRNA（1.000±0.000 vs 0.370±0.012,P〈0.01）,and the inhibition of 231BO-BSP27 cells growth（P〈0.05）.After treatment with exogenous rhBSP,the phosphorylation of AKT was increased in both 231BO-Scrambled and 231BO-BSP27 cells [（17.86±2.27）%,（33.78±1.51）%,P〈0.01].rhBSP treatment decreased caspase-3 mRNA（1.000±0.039 vs 0.541±0.091,P〈0.01）,and increased cyclin D1 mRNA（1.000±0.000 vs 2.921±0.032,P〈0.01） expression in 231BO-BSP27 cells,and stimulated the proliferation of 231BO-Scrambled and 231BO-BSP27 cells（P〈0.01）.Furthermore,rhBSP-induced activation of AKT was reversed by LY294002 in 231BO-Scrambled and 231BO-BSP27 cells（P〈0.05）,with an increase in caspase-3 mRNA and decrease in cyclin D1 mRNA expression in 231BO-BSP27 cells（all P〈0.01）,causing proliferation inhibition in 231BO-Scrambled and 231BO-BSP27 cells（P〈0.01）.Conclusion： BSP can regulate the mRNA expressions of caspase-3 and cyclin D1,and affect the proliferation of breast cancer MDA-MB-231 cells through the PI3K-AKT signaling pathway.