沉默中介体19对人乳腺癌MCF-7细胞侵袭及迁移的影响

Influence of silencing Med19 on metastasis and invasion of human breast cancer MCF-7 cells

目的:研究中介体19(Med19)对人乳腺癌MCF-7细胞侵袭及迁移的影响。方法:用慢病毒介导的Med19小分子干扰RNA(siRNA)感染人乳腺癌细胞MCF-7(KD组),并设置空载体转染组(NC组)、空白对照组(CON组)。荧光显微镜下观察细胞感染效率,利用RT-PCR和Western blot检测Med19蛋白表达,用划痕实验和transwell实验检测细胞迁移及侵袭能力。结果:荧光显微镜下观察细胞感染慢病毒效率达90%以上。与NC组及CON组细胞相比,KD组Med19 mRNA表达水平下降了72.3%、72.1%,Med19蛋白表达水平下降了85.4%、85.3%,差异均有统计学意义(P<0.05)。分别在0、6、12、24 h比较迁移的距离,KD组细胞随着时间的延长,较CON组、NC组迁移能力显著下降,差异有统计学意义(P<0.05);KD组、NC组、CON组MCF-7细胞穿过聚碳酸酯膜的细胞数分别为23.8±4.32、43.4±3.65、45.8±5.81,KD组较CON组、NC组细胞的侵袭能力显著降低,差异均有统计学意义(P<0.05)。NC组与CON组相比,细胞迁移和侵袭能力差异无统计学意义(P>0.05)。结论:Med19基因沉默抑制细胞的迁移及侵袭能力,从而抑制乳腺癌的恶性生物学行为。

Objective To investigate the influence of silencing Med19 on metastasis and invasion of human breast cancer MCF-7 cells. Methods lentivirus expression vector delivering small hairpin RNA （shRNA） against Med19 gene was constructed, then transfected of human breast cancer MCF-7 cell line. There were three experimental groups： non-infected （CON） group, Lv-NC-infected （NC） group, and Lv-shMed19-infected （KD） group. To determine the lentiviral infection efficiency, expression of GFP was detected with fluorescence microscopy. The mRNA and protein levels of Med19 in three groups MCF-7 cells were detected by real-time PCR and Western blotting. Scratching and transwell tests were employed to determine the ability of metastasis and invasion of cells. Results The highest infection efficiency was obtained, resulting fluorescent expression identified in more than 90% of MCF-7 cells 120 h after infection. The expression of Med19 mRNA in the KD group was dramatically decreased by 72.3% compared with the NC group, and by 72.1% compared with the CON group, respectively （P 〈 0.05）. The Med19 protein expression in the KD group was significantly lower than that of the NC group and the CON group, with a reduction of 85.4% and 85.3%, respectively （P 〈 0.05）. No statistical significance was detected between the NC groups and the CON groups. Thus, the constructed Lv-shMed19 was demonstrated to be active and specific in inhibiting the expression of Med19. Moreover, the invasive distances of KD group, NC group and CON group MCF-7 cells were compared at 6 hour, 12 hour and 24 hour. The invasive ability of MCF-7 cells decreased significantly with the extension of time （P 〈 0.05）. The cells passed through polycarbonate membrane in KD group, NC group and CON group were 23.8 ±4.32, 43.4 ±3.65 and 45.8 ± 5.81 respectively. The metastatic ability of KD group was significant reduced （P 〈 0.05）. Conclusion Silencing of Med19 gene significantly decreases the ability of metastasis and invasion of human breast cancer MCF-7 cells, and then suppresses the malignant biological behavior of breast cancer.