蛋白激酶SGK3过表达对乳腺癌细胞株MDA-MB-231影响的研究

目的 血清和糖皮质激素诱导的蛋白激酶3 (serum and glucocorticoid-inducible protein kinase 3,SGK3)参与细胞增殖、分化和物质转运等生命活动,在某些肿瘤形成中担任重要角色.本研究设计观察SGK3过表达对乳腺癌细胞株MDA-MB-231细胞增殖和存活的影响,并初步探讨其作用的分子机制.方法 免疫组化方法检测乳腺癌组织中SGK3蛋白的表达;通过GenEscortTMⅠ介导用重组质粒pEGFPN1-SGK3瞬时转染乳腺癌MDA-MB-231细胞,荧光显微镜和蛋白质印迹法鉴定转染细胞中融合蛋白SGK3-GFP的表达;细胞增殖实验观察转染细胞的生长增殖情况;流式细胞术分析转染细胞的细胞周期时相变化;蛋白质印迹方法检测细胞增殖与存活相关基因的表达.结果 SGK3在乳腺癌组织中的表达量明显高于癌旁组织和正常乳腺组织(F=29.672,P＜0.001),与二者组间比较,均P＜0.01.荧光显微镜下可见转染细胞因外源基因的表达而发出绿色荧光,且蛋白质印迹法检测可见融合蛋白SGK3-GFP的表达;过表达SGK3的MDA-MB-231细胞,其细胞生长速度加快,细胞倍增时间缩短(F=19.557,P-0.001),与亲本细胞及转染空载体细胞组间比较均P＜0.01.与亲本细胞和转染空载体组比较,SGK3过表达使细胞凋亡比例明显下降(F=12.156,P=0.008),与前两者组间比较P值分别为0.019和0.030;细胞内源性Bad的表达量降低(F=11.152,P=0.010),与前两者组间比较P值分别为0.008和0.005;而Bcl-xL(F=8.810,P=0.016,与前两者组间比较P值分别为0.014和0.009)以及p-GSK3β(F=42.253,P＜0.001,与前两者组间比较P值分别为＜0.001和0.009)的表达量均升高.结论 乳腺癌组织高表达SGK3,SGK3过表达可促进MDA-MB-231细胞的增殖与存活,影响细胞增殖与存活相关基因的表达可能是其发挥作用的主要分子机制.

Overexpression of protein kinase SGK3 promotes proliferation and survival of breast cancer cell line MDA-MB-231

OBJECTIVE Serum and glucocorticoid induced protein kinase 3 (SGK3) is involved in cell proliferation,differentiation and material transport,and it plays an important role in the formation of some tumors.This study was de signed to investigate the effects of SGK3 overexpression on the proliferation and survival of breast cancer cell line MDAMB-231,and explore the molecular mechanism.METHODS The expression of SGK3 protein in breast cancer tissues was detected by immunohistochemistry.Transient transfection of MDA-MB-231 cells with pEGFPN1-SGK3 plasmid mediated by GenEscortTM Ⅰ.The expression of fusion protein SGK3-GFP in transfected cells was identified by fluorescence micros copy and western blotting.The growth of the transfected cells was observed by cell proliferation assay,and the expression of fusion protein SGK3-GFP was detected by Western blotting.Cell cycle phase was analyzed by flow cytometry.The expression of cell apoptosis related genes was detected by Western blotting.RESULTS The expression of SGK3 in breast cancer tissues was significantly higher than that in adjacent tissues and normal breast tissues(F=29.672,P＜0.001,compared with the control groups P values were less than 0.01).Under the fluorescence microscope,the green fluorescence was observed in the transfected cells,and the secretory granules increased obviously.Compared with the control cells,the cell growth rate was accelerated and the cell doubling time was shortened(F=19.557,P =0.001,compared with parental cells and transfected empty vector cells,P values were less than 0.01).Compared with the parental cells and transfected empty vector group,SGK3 overexpression significantly reduced the proportion of cell apoptosis in MDA-MB-231 cells(F=12.156,P=0.008,compared with the control groups,the P values were 0.019 and 0.030,respectively).Overexpression of SGK3 decreased the expression of Bad(F=11.152,P=0.010,compared with the control groups,the P values were 0.008 and 0.005,respectively)and increased the expression of Bcl-xL(F=8.810,P=0.016,compared with the control groups,the P values were 0.014 and 0.009,respectively)and p-GSK33(F=42.253,P＜0.001,compared with the control groups,P＜0.001 and P=0.009)in MDA-MB-231 cells.CONCLUSIONS SGK3 is highly expressed in breast cancer tissues,and overexpression of SGK3 promotes cell proliferation and survival in MDAMB-231 breast cancer cells.To influence the expression of proliferation and survival related genes may be its mechanism.