摘要
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P<0.05);阴性对照组细胞各项观察指标无统计学差异(均P>0.05)。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。
Objective: To investigate the effects of down-regulating the expression of ubiquitin-protein ligase E3 A(UBE3 A) on the biological behaviors in three negative breast cancer(TNBC) cells.Methods: Three constructed UBE3 A shRNA sequences were respectively transfected into the human TNBCMDA-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 UBE3 A 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 UBE3 A shRNA expression were successfully constructed and the UBE3 A shRNA sequence with highest interreference efficiency was picked up(the inhibitory rate was 89.5% for UBE3 A gene and 45.3% for UBE3 A 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 UBE3 A 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 UBE3 A expression can induce cell cycle arrest in TNBC cells and thereby inhibit their proliferation and invasion abilities, and suggests that UBE3 A expression plays an important role in the malignant biological behaviors of TNBC cells.
作者
谢少利
王碧娟
刘家有
李金穗
赵小波
邓世山
侯令密
XIE Shaoli;WANG Bijuan;LIU Jiayou;LI Jinsui;ZHAO Xiaobo;DENG Shishan;HOU Lingmi(Department of Thyroid and Breast Surgery,the Affiliated Hospital of North Sichuan Medical College,Nanchong,Sichuan637000,China;Department of Internal Medicine,the Second Affiliated Hospital of North Sichuan Medical College,Nanchong,Sichuan637000,China;Department of Anatomy,North Sichuan Medical College,Nanchong,Sichuan637000,China)
出处
《中国普通外科杂志》
CAS
CSCD
北大核心
2018年第11期1417-1423,共7页
China Journal of General Surgery
基金
国家自然科学基金资助项目(81172496)
川北医学院课题资助项目(CBY16-A-YB14)
四川省南充市校科技战略合作项目(18SXHZ0555)
关键词
三阴性乳腺癌
泛素蛋白连接酶类
RNA干扰
细胞周期
Triple Negative Breast Neoplasms
Ubiquitin-Protein Ligases
RNA Interference
Cell Cycle
