慢病毒载体PLKO.1-shp65的构建及在乳腺癌细胞MDA-MB-231中功能的研究

Construction of p65 shRNA knockdown vector and its effect on breast cancer cell MDA-MB-23

目的:构建p65降表达质粒并包装成慢病毒,检测对MDA-MB-231细胞增殖转移能力的影响。方法:将体外合成的p65干扰片段与PLKO.1-puro连接形成重组慢病毒质粒PLKO.1-shp65,在293FT细胞中进行包装,产生重组慢病毒颗粒,感染MDA-MB-231细胞。实时定量聚合酶链反应(PR-QPCR)、Western blot检测细胞中p65表达情况。采用细胞计数的方法检测稳定降表达p65之后对细胞增殖能力的影响,用transwell实验检测细胞迁移侵袭能力的变化。用RT-QPCR、Western blot检测增殖转移相关基因细胞周期蛋白E1(CCNE1)、细胞周期蛋白D1(CCND1)、细胞周期蛋白B1(CCNB1)、周期蛋白依赖性激酶(CDK1)、细胞分裂周期蛋白7(CDC7)、细胞分裂周期相关蛋白7(CDCA7)表达量的变化。结果:用重组慢病毒颗粒PLKO.1-shp65感染MDA-MB-231细胞,p65的表达量显著降低,并且MDA-MB-231细胞的增殖以及迁移侵袭能力降低。同时增殖相关基因CCNE1和CCND1,转移相关基因Snail、Slug的表达量降低。结论:干扰p65之后MDA-MB-231细胞的增殖、转移能力降低。

Objective： To construct p65-silenced breast cancer cells by RNA interference technology and lentiviral vector, and to study the influence on proliferation and metastasis of breast cancer cell MDA-MB-231. Methods： Short hairpin RNA （shRNA） was designed and sub-cloned into PLKO. 1-puro to construct lentiviral vector. MDA-MB-231 cells were infected, and the expression of p65 was detected by Western blot and RT-QPCR. The effect of p65 on metastasis was detected by transwell assay, and the proliferation ability was measured by cell counting experiment. The expression of proliferation and metastasis related genes were detected by RT-QPCR, Western blot. Results： The PLKO.1-shp65 was successfully constructed, and was stably expressed in MDA-MB-231 cells and could interfere with the expression of human p65 effectively, preliminary function study showed that p65 knockdown inhibited MDA-MB-231 cell proliferation and metastasis. The expression of proliferation and metastases related genes like Snail, Slug, CCNE1 and CCND1 were decreased significantly. Conclusion： p65-siIenced may reduce the proliferation and metastasis ability of MD A-MB-231 cells.