下调泛素蛋白连接酶E3A表达对三阴性乳腺癌细胞生物学行为的影响

目的:探讨降低泛素蛋白连接酶E3A(UBE3A)的表达对三阴性乳腺癌细胞生物学行为的影响。方法:采用慢病毒载体分别将构建的3条UBE3A shRNA序列转染人三阴性乳腺癌细胞株MDAMB-231,检测干扰效率后,筛选出干扰效率最高的序列用于实验。分别采用CCK8法、Transwell小室实验、流式细胞术观察MDA-MB-231细胞经所选用的UBE3A shRNA序列转染后侵袭能力、增殖能力与细胞周期的变化,以转染阴性对照组序列和无处理的MDA-MB-231细胞作为阴性对照及空白对照。结果:成功构建UBE3A shRNA慢病毒表达载体并筛选出干扰率最高的UBE3A shRNA序列(UBE3A基因和蛋白表达抑制率分别为89.5%、45.3%)。与空白对照组细胞比较,下调UBE3A的MDAMB-231细胞的增殖、侵袭能力均明显降低,且细胞周期出现明显的S期阻滞(均P0.05);阴性对照组细胞各项观察指标无统计学差异(均P0.05)。结论:下调UBE3A表达能诱导三阴性乳腺癌细胞的细胞周期阻滞,从而抑制其增殖与侵袭能力,提示UBE3A在三阴性乳腺癌细胞的恶性生物学行为中发挥了重要作用。

Effects of down-regulating expression of ubiquitin-protein ligase E3A on biological behaviors in three negative breast cancer cells

Objective: To investigate the effects of down-regulating the expression of ubiquitin-protein ligase E3A (UBE3A) on the biological behaviors in three negative breast cancer (TNBC) cells. Methods: Three constructed UBE3A shRNA sequences were respectively transfected into the human TNBC MDA-MB-231 cells through lentiviral vectors, and the shRNA sequence with highest interference efficiency was selected for experiments after interference efficiency tests. In MDA-MB-231 cells after transfected with the selected UBE3A shRNA sequence, the changes in proliferation, invasion and cell cycle were determined by CCK-8 assay, Transwell invasion assay and flow cytometry respectively, using the MDA-MB-231 cells without any treatment and transfected with a scrambled sequence as blank control and negative control. Results: The lentiviral vectors with UBE3A shRNA expression were successfully constructed and the UBE3A shRNA sequence with highest interreference efficiency was picked up (the inhibitory rate was 89.5% for UBE3A gene and 45.3% for UBE3A protein). Compared with the cells in blank control group, the proliferative and invasion abilities were significantly decreased with significant S-phase cell cycle arrest in MDA-MB-231 cells with UBE3A down-regulation (all P<0.05); all the observed indexes in cells of negative control group showed no significant changes (all P>0.05). Conclusion: Down-regulating UBE3A expression can induce cell cycle arrest in TNBC cells and thereby inhibit their proliferation and invasion abilities, and suggests that UBE3A expression plays an important role in the malignant biological behaviors of TNBC cells.