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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 observe the inhibitory effect of serum and glucocorticoid-regulated protein kinase 1 (SGK1) gene silencing on the growth of human breast cancer cells and provide experimental evidence for the treatment of breast cancer. Methods RNA interference (shRNA) plasmid pGen-3-siSGK1 and short hairpin RNA (shRNA) plasmid pGen-3-control were transfected into human breast cancer cell line MDA-MB- 231 was screened to obtain the stable SGK1 gene silencing breast cancer cell model. The expression of SGK1 in shR-NA interference group, negative control group and untransfected group was detected by real-time quantitative PCR, immunofluorescence and Western blotting In vitro invasion assay and cell migration assay were used to detect the invasion and metastasis of cells in each group. Thiazolyl blue colorimetry was used to detect the cell growth in vitro. Results shRNA interference plasmids targeting SGK1 gene were successfully constructed and stable SGK1 gene silencing breast cancer cell model was established. The expression of SGK1 protein in model cells decreased by 60% (P <0.01) compared with that in control group. The expression of β-catenin and SGK1 The relative intensity of SGK1 mRNA in PGen-3-siSGK1 group was 0.35, which was lower than that of the other two groups (0.96 and 1.00, respectively), decreased by 65.5% compared with pGen-3-control group The difference was statistically significant (P <0.05). The number of transmembrane cells in pGen-3-siSGK1 group was 22 cells / HPF under high magnification microscope, and 66,62 cells / HPF in the other two groups respectively. The expression of pGen-3-siSGK1 The cell migration rate was 17.01% in the other two groups and 72.22% and 68.47% in the other two groups, respectively. The growth of pGen-3-siSGK1 group was inhibited at different concentrations of thrombin, and the growth rate was slow at 0.1,1.0 U / mL , Cell content (absorbance) were 0.10,0.05, much lower than the other two groups, the difference was statistically significant (P <0.05). Conclusion Silencing SGK1 can inhibit the expression of SGK1 gene and decrease the infiltration and migration of human breast cancer cell line MDA-MB-231 and inhibit the growth of breast cancer cells.