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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GenEscort TMⅠ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。
Objective Serum and glucocorticoid-inducible protein kinase 3 (SGK3) are involved in the life activities of cell proliferation, differentiation and material transport, and play an important role in some tumorigenesis. This study was designed to observe the effect of SGK3 overexpression on the proliferation and survival of breast cancer cell line MDA-MB-231 cells and to explore its molecular mechanism. Methods The expression of SGK3 protein in breast cancer tissues was detected by immunohistochemistry. The breast cancer cells MDA-MB-231 cells were transiently transfected with recombinant plasmid pEGFPN1-SGK3 by GenEscort ™ I, and the fusion of transfected cells was identified by fluorescence microscopy and Western blotting The expression of SGK3-GFP was detected by cell proliferation assay. The proliferation and proliferation of transfected cells were observed by flow cytometry. The changes of cell cycle were analyzed by flow cytometry. The expressions of survivin and cell proliferation were detected by Western blotting. Results The expression level of SGK3 in breast cancer tissues was significantly higher than that in paracancerous tissues and normal breast tissues (F = 29.672, P <0.001), both P <0.01. Under the fluorescence microscope, the transfected cells showed green fluorescence due to the expression of the foreign gene, and the expression of the fusion protein SGK3-GFP was detected by Western blotting. The MDA-MB-231 cells overexpressing SGK3 accelerated the growth of cells, Doubling time shortened (F = 19.557, P = 0.001), P <0.01 compared with parental cells and empty vector transfected cells. SGK3 overexpression significantly decreased the percentage of apoptotic cells (F = 12.156, P = 0.008) compared with the parental cells and empty vector transfected cells. The P values of the two groups were 0.019 and 0.030, respectively. The expression of Bad decreased (F = 11.152, P = 0.010), compared with the former two groups P values were 0.008 and 0.005, respectively; while Bcl-xL (F = 8.810, P = 0.016, P values were 0.014 and 0.009 respectively) and p-GSK3β (F = 42.253, P <0.001, P <0.001 and 0.009 respectively compared with the former two groups). Conclusion The overexpression of SGK3 and SGK3 in breast cancer tissue can promote the proliferation and survival of MDA-MB-231 cells, which may be one of the main molecular mechanisms that affect the proliferation and survival-related gene expression.