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目的建立稳定抑制血清和糖皮质激素调节蛋白激酶1(SGK1)表达的人乳腺癌细胞系MDA-MB-231,观察SGK1基因沉默对人乳腺癌细胞生物学特性的影响。方法构建针对SGK1的shRNA干扰质粒pGen-3-siSGK1和阴性对照质粒pGen-3-control。转染人乳腺癌细胞系MDA-MB-231,经G418筛选得到稳定抑制SGK1表达的乳腺癌细胞模型;实时定量PCR、免疫荧光以及Western blotting检测shRNA干扰组、阴性对照组及未转染组细胞中SGK1的表达;四甲基偶氮唑盐(MTT)比色法检测细胞体外生长能力;体外浸润实验、细胞迁移实验检测各组癌细胞的侵袭和转移。结果成功构建针对SGK1基因的shRNA干扰质粒,建立了稳定抑制SGK1表达的乳腺癌细胞模型;与阴性对照组和未转染组相比,肿瘤细胞中SGK1的表达水平显著降低(P<0.01);SGK1基因沉默后,人乳腺癌细胞的生长能力、浸润和迁移能力均明显降低(P<0.05)。实验中还发现抑制SGK1基因表达之后,β-catenin的含量也出现明显下降。结论抑制SGK1基因的表达可以显著抑制人乳腺癌细胞MDA-MB-231的生长,并降低其侵袭转移的能力,其中β-catenin参与了SGK1基因的抑制功能。
Objective To establish a human breast cancer cell line MDA-MB-231 stably inhibiting the expression of serum and glucocorticoid-regulated protein kinase 1 (SGK1) and observe the effect of SGK1 gene silencing on the biological characteristics of human breast cancer cells. Methods shRNA interference plasmid pGen-3-siSGK1 against SGK1 and negative control plasmid pGen-3-control were constructed. The transfected human breast cancer cell line MDA-MB-231 was screened by G418 to obtain the stable SGK1-expressing breast cancer cell model. The real-time quantitative PCR, immunofluorescence and Western blotting were used to detect the shRNA interference group, negative control group and untransfected cells The SGK1 expression was detected by MTT assay. The in vitro cell invasion assay and cell migration assay were used to detect the invasion and metastasis of cancer cells in each group. Results shRNA interference plasmids targeting SGK1 gene were successfully constructed and a model of breast cancer cells stably inhibiting the expression of SGK1 was established. Compared with the negative control group and the untransfected group, the expression of SGK1 was significantly decreased (P <0.01). SGK1 gene silencing, human breast cancer cell growth ability, infiltration and migration were significantly reduced (P <0.05). Experiments also found that inhibition of SGK1 gene expression, the content of β-catenin also decreased significantly. Conclusion Inhibition of SGK1 gene expression can significantly inhibit the growth of human breast cancer cell line MDA-MB-231 and reduce its invasion and metastasis, in which β-catenin is involved in the inhibitory function of SGK1 gene.