SGK1基因沉默对人乳腺癌细胞生长抑制作用  

Inhibitive effect of SGK1 gene silencing on proliferation of human breast cancer MDA-MB-231 cells

作　　者：覃红斌[1] 陈显兵[1] 甘华文[2,3] 张洁[2] 张玲[2] 

机构地区：[1]湖北民族学院医学院,湖北恩施445000 [2]武汉大学基础医学院 [3]武汉大学人民医院

出　　处：《中国公共卫生》2013年第3期343-347,共5页Chinese Journal of Public Health

基　　金：湖北省自然科学基金(2008CDB222)

摘　　要：目的观察血清和糖皮质激素调节蛋白激酶1(SGK1)基因沉默对人乳腺癌细胞生长抑制作用,为探讨乳腺癌治疗靶点提供实验依据。方法采用RNA干扰技术,以pGenesil 3为载体,建立针对SGK1的短发卡RNAC(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,经筛选得到稳定SGK1基因沉默的乳腺癌细胞模型;采用实时定量PCR法、免疫荧光法以及蛋白免疫印迹法,检测shR-NA干扰组、阴性对照组及未转染组细胞中SGK1的表达;采用体外浸润实验、细胞迁移实验检测各组细胞的侵袭和转移能力,噻唑蓝比色法检测细胞体外生长能力。结果成功构建针对SGK1基因的shRNA干扰质粒,建立稳定的SGK1基因沉默乳腺癌细胞模型;模型细胞中SGK1蛋白表达量与对照组相比下降了60%(P<0.01);β-catenin与SGK1表达条带亮度均明显下降;PGen-3-siSGK1组SGK1 mRNA相对含量(RQ)为0.35,低于其他2组(分别为0.96、1.00),与pGen-3-control组相比下降了65.5%,差异有统计学意义(P<0.05),高倍显微镜视野下,pGen-3-siSGK1组的穿膜细胞数为22个/HPF,其他2组分别为66、62个/HPF;pGen-3-siSGK1组的细胞迁移率为17.01%,其他2组分别为72.22%、68.47%;凝血酶不同浓度下pGen-3-siSGK1组细胞生长均受到抑制,生长速度缓慢,其中0.1、1.0 U/mL浓度下,细胞含量(吸光度)分别为0.10、0.05,远低于其他2组,差异均有统计学意义(均P<0.05)。结论 SGK1基因沉默可抑制SGK1基因表达,可使人乳腺癌细胞MDA-MB-231株的浸润能力、迁移能力下降,抑制乳腺癌细胞生长。Objective To examine inhibitive effect of serum and glucocorticoid-sensitive kinase 1 （ SGK1 ） gene si- lencing on porliferation of human breast cancer MDA-MB-231 cells and to provide evidence for genetic therapy for hu- man 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-231 cells and then selected by G418. Expression of SGK1 was detected by real time PCR and western blot, and f3-catenin expression was detected by western blot as well as 3 E 4,5- dimethyl thiazol-2-yl ]-2,5-diphenyl tetrazolium bromide （MTT） assay. Matrigel invasion assays were used to study the effect of SGK1 gene silencing. Results The mRNA and protein of SGK1 remarkably decreased by 60. 0% and 65.5%, respectively, after the transfection of pGen-3-siSGK1. After gene silencing of SGK1, MDA-MB-231 cells showed a strong inhibition of cell growth. The invasive and migratory abilitiy behaviors were inhibited after gene silencing of SGK1. In addition,the change of protein level of 13-catenin was consistent with that of SGK1. Conclusion SGK1 inhibtion sup- presses the growth,invasiveness, and migratory abilities of breast cancer cells.

关 键 词：血清和糖皮质激素调节蛋白激酶1(SGK1) RNA干扰 短发卡RNA(shRNA) 基因沉默 乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]