Sox2下调表达对MDA-MB-231乳腺癌细胞β-catenin与转录中介因子γ的影响

Effect of targeted Sox2 silencing on expression of β-catenin and TIF1γ in human breast cancer MDA-MB-231 cells

目的下调乳腺癌MDA-MB-231细胞Sox2基因表达,观察对细胞增殖、侵袭和凋亡的影响以及信号因子β-catenin和TIF1γ表达的影响。方法将MDA-MB-231细胞分为重组质粒转染组(转染Sox2-shRNA干扰载体)、阴性对照组(转染p Mafic7.1-shRNA-NC质粒)、空白对照组(未经处理的MDA-MB-231细胞,n=6),并将shRNA-Sox2-1瞬时转入乳腺癌MDA-MB-231细胞,通过Western blot检测乳腺癌MDA-MB-231细胞中Sox2蛋白表达水平,FCM检测细胞凋亡的情况,Transwell检测细胞的侵袭能力,MTS检测细胞活力和增殖能力。另外,采用Western blot和免疫组化检测靶向抑制Sox2对信号因子β-catenin和TIF1γ蛋白的表达与分布。结果成功构建重组载体p Mafic7.1-shRNA-Sox2。与空白对照组(未转染组)和阴性对照组(转染p Mafic7.1-shRNA-NC)相比,p Mafic7.1-shRNA-Sox2转染至MDA-MB-231细胞后,Sox2蛋白的表达水平明显下调(P<0.01),细胞凋亡率明显增加(P<0.05),细胞的侵袭能力、活力和增殖能力均明显下降(P<0.05)。与空白对照组(0.79±0.07)和阴性对照组(0.74±0.10)相比,重组质粒转染组β-catenin(0.15±0.04)蛋白的表达水平明显下调(P<0.05);同样,与空白对照组(0.74±0.05)和阴性对照组(0.64±0.07)相比,重组质粒转染组TIF1γ蛋白(0.11±0.01)的表达水平明显下调(P<0.05)。结论靶向沉默Sox2基因能促进细胞凋亡,抑制细胞的增殖和侵袭,其作用可能与抑制Wnt/β-catenin信号通路与TIF1γ调控有关。

Objective To determine the effect of short hairpin RNA-mediated silencing of Sox2（ sex determination region of Y chromosome-related HMG-box 2） on the invasion,proliferation and apoptosis in human breast cancer cells（ MDA-MB-231） and on the expression of β-catenin and transcriptional intermediary factor 1γ（ TIF1γ）. Methods Constructed shRNA recombinant vector of targeted Sox2 gene was transfected into MDA-MB-231 cells. The cells transfected with plasmid p Mafic7. 1-shRNA-NC and those without served as negative and blank control. The protein expression of Sox2 in the 3 groups of cells was detect by Western blotting. The cell apoptosis was measured by flow cytometry. Transwell assay was used to detect the cellular invasiveness and metastasis. Cell vitality and proliferation were tested by MTS assay. The expression and distribution of β-catenin and TIF1γ protein were detected by Western blotting and immunohistochemical staining, respectively. Results Recombinant vector of p Mafic7. 1-shRNA-Sox2 was constructed successfully. When compared with negative and blank control cells,the transfected cells had obviously reduced level of Sox2 protein（ P〈0. 01）,significantly enhanced apoptotic rate（ P〈0. 05）,and remarkably reduced invasion,vitality and proliferation capacities（ P〈0. 05）. The expression level of β-catenin was significantly decreased in the transfected cells（ 0. 15 ± 0. 04） than the negative（ 0. 74 ± 0. 10） and blank control cells（ 0. 79 ± 0. 07,both P〈0. 05）. So was that of TIF1γ（ 0. 11 ± 0. 01 vs 0. 64 ± 0. 07 and 0. 74 ±0. 05,both P〈0. 05）. Conclusion Targeted silencing of Sox2 enhances the apoptosis,and inhibits the proliferation and invasion in breast cancer cells,which may be related in the inhibition of Wnt / β-catenin signal pathway and TIF1γ.