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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 investigate the effect of down-regulation of Sox2 gene expression in breast cancer MDA-MB-231 cells on the proliferation, invasion and apoptosis as well as the expression of signal factors β-catenin and TIF1γ. Methods MDA-MB-231 cells were divided into three groups: the recombinant plasmid transfected group (transfected with Sox2-shRNA interference vector), the negative control group (transfected with p Mafic7.1-shRNA-NC plasmid), the blank control group MDA-MB-231 cells, n = 6). The shRNA-Sox2-1 cells were transiently transfected into breast cancer MDA-MB-231 cells. The expression of Sox2 protein in breast cancer MDA-MB-231 cells was detected by Western blot. Detection of apoptosis, Transwell assay of cell invasiveness, MTS detection of cell viability and proliferation. In addition, Western blot and immunohistochemistry were used to detect the expression and distribution of Sox2, a signaling inhibitor of beta-catenin, and TIF1γ. Results The recombinant vector p Mafic7.1-shRNA-Sox2 was successfully constructed. Compared with the blank control group (untransfected group) and negative control group (transfected with p Mafic7.1-shRNA-NC), after transfecting p Mafic7.1-shRNA-Sox2 into MDA-MB-231 cells, Sox2 protein (P <0.01). The apoptotic rate was significantly increased (P <0.05), and the invasiveness, viability and proliferation of cells were significantly decreased (P <0.05). Compared with the blank control group (0.79 ± 0.07) and the negative control group (0.74 ± 0.10), the expression level of β-catenin (0.15 ± 0.04) in the recombinant plasmid transfected group was significantly decreased (P <0.05) Compared with the negative control group (0.64 ± 0.07), the expression level of TIF1γ protein (0.11 ± 0.01) in the control group (0.74 ± 0.05) was significantly decreased (P <0.05). Conclusion Targeted silencing of Sox2 gene can promote cell apoptosis and inhibit cell proliferation and invasion, which may be related to the inhibition of Wnt / β-catenin signaling pathway and the regulation of TIF1γ.