shRNA抑制SGK1基因表达对人乳腺癌细胞系生物学特性的影响

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。

Effect of SGK1 gene inhibition by shRNA on biologic characteristics of the human breast cancer cell

Objective To study the effect of shRNA mediated gene silencing of serum and glucocorticoid induced protein kinase 1(SGK1) on biologic characteristics of human breast cancer cells,MDA-MB-231,and to provide experiment and theoretical evidences for genetic therapy of human breast cancer. Methods Hairpin RNA sequence was synthesized and inserted into pGenesil-3 vector with human U6 promoter.Verified constructs were transfected into MDA-MB-231cells,and then selected by G418.Expression of SGK1 was detected by both real time PCR and Western blotting,and β-catenin expression was detected by Western blotting.MTT growth and matrigel invasion assays were used to study the effect of SGK1 gene silencing.Results Both the mRNA and protein level of SGK1 remarkably decreased after transfection of pGen-3-siSGK1.After gene silencing of SGK1,MDA-MB-231 cells shown strong inhibition of cell growth.The invasive and migratory abilities were inhibited after gene silencing of SGK1.In addition,the change of protein level of β-catenin was consistent with that of SGK1. Conclusion SGK1 inhibition suppresses the growth,invasiveness and migratory abilities of breast cancer cell