血清和糖皮质激素诱导激酶3表达水平对ER;乳腺癌细胞增殖与凋亡的影响

Serum-and glucocorticoid-regulated kinase 3 promotes proliferation and inhibits apoptosis of ER;breast cancer cells

目的探讨血清和糖皮质激素诱导激酶3(serum-and glucocorticoid-regulated kinase 3,SGK3)对ER^(+)乳腺癌细胞系MCF-7细胞及T47D细胞增殖和凋亡的影响。方法利用基因表达谱交互分析(gene expression profile interactive analysis,GEPIA)生物信息学工具,分析癌症公共数据库癌症基因组图谱计划(the cancer genome atlas program,TCGA)中乳腺癌组织(breast cancer tissue,BRCA)及对照样本中SGK3蛋白的表达水平,基于TCGA数据库中的BRCA患者样本进行生存分析;选取SGK3可诱导表达乳腺癌细胞系MCF-7-Tet-On-SGK3及T47D-Tet-On-SGK3为研究对象,在诱导SGK3过表达后,用MTT法检测过表达SGK3对细胞增殖的影响;流式细胞仪及Annexin V-FITC凋亡检测试剂盒检测过表达SGK3对细胞凋亡的影响;免疫印迹方法(Western blot)检测凋亡相关蛋白分子的变化。利用SGK3特异性小干扰RNA(small interference RNA,siRNA)敲低SGK3后,MTT法检测敲低SGK3对细胞增殖的影响。流式细胞术检测细胞凋亡情况,Western blot实验检测凋亡相关蛋白分子变化。结果 TCGA数据库共有1 084例BRCA样本及492例乳腺对照样本,与对照组乳腺组织相比,SGK3在BRCA中显著高表达。GEPIA的生存曲线分析表明,SGK3高表达患者的总生存期(overall survival,OS)及无病生存期(disease free survival,DFS)较低表达患者缩短。在MCF-7-Tet-On-SGK3和T47D-Tet-On-SGK3细胞中,诱导SGK3过表达能够促进细胞增殖和减少细胞凋亡水平(P<0.05)。在分子水平,过表达SGK3能够下调凋亡蛋白Bax和Cleaved-PARP表达,上调抗凋亡蛋白Bcl-2水平(P<0.05)。在MCF-7和T47D细胞敲低SGK3能够抑制细胞增殖活力和增加细胞凋亡(P<0.05)。在分子水平,敲低SGK3能够增加Bax蛋白水平,降低Bcl-2表达和上调Cleaved-PARP水平(P<0.05)。结论在ER^(+)乳腺癌细胞中,SGK3促进细胞增殖、减少细胞凋亡,其高表达可能与乳腺癌患者预后不良有关。

Objective To investigate the effects of serum-and glucocorticoid-regulated kinase 3(SGK3) on the proliferation and apoptosis of ER positive breast cancer cell lines MCF-7 and T47D.Methods Gene expression profile interactive analysis(GEPIA) was used to analyze SGK3 expression levels in BRCA(breast cancer tissue) and normal tissue in the cancer public database,the Cancer Genome Atlas(TCGA) database,and survival analysis was performed based on SGK3 levels in the BRCA patients in the TCGA database.After inducement of SGK3 overexpression in SGK3-inducible breast cancer cell lines MCF-7-Tet-On-SGK3 and T47 D-Tet-On-SGK3,MTT assay was used to detect the effect of SGK3 overexpression on cell proliferation.Flow cytometry and Annexin V-FITC apoptosis detection kit were employed to detect the effect of SGK3 overexpression on cell apoptosis.Western blotting was applied to measure the changes of apoptosis-related protein molecules.After SGK3 was knocked down with SGK3 specific small interference RNA,MTT assay was adopted to detect the effect of SGK3 knockdown on the proliferation of MCF-7 cells,flow cytometry for cell apoptosis,and Western blotting for the molecular changes of apoptosis-related proteins.Results The TCGA database had a total of 1 084 BRCA samples and 492 breast control samples.Compared with the control breast tissue,the expression of SGK3 was significantly higher in BRCA.The GEPIA survival curve analysis showed that the expression level of SGK3 was negatively correlated with the overall survival(OS) and disease-free survival(DFS) of BRCA patients.In MCF-7-Tet-On-SGK3 and T47D-Tet-On-SGK3 cells,inducing SGK3 expression promoted cell proliferation and reduced the apoptosis.At molecular level,SGK3 overexpression down-regulated the expression of apoptotic proteins Bax and cleaved-PARP,and up-regulated the level of antiapoptotic protein Bcl-2.SGK3 knockdown in MCF-7 and T47D cells inhibited cell proliferation and increased cell apoptosis.At the molecular level,knocking SGK3 down increased Bax protein level,reduced Bcl-2 level and up-regulated cleaved-PARP.Conclusion In ER^(+)breast cancer cells,SGK3 promotes cell proliferation and reduces apoptosis.Its high expression may be associated with poor prognosis in breast cancer patients.