Lentiviral vector-mediated doxycycline-inducible USP39 shRNA or cDNA expression in triple-negative breast cancer cells
Lentiviral vector-mediated doxycycline-inducible USP39 shRNA or cDNA expression in triple-negative breast cancer cells
复制标题
慢病毒载体介导的三阴性乳腺癌细胞中多西环素诱导的 USP39 shRNA 或 cDNA 表达
DOI：
10.3892/or.2015.3872
复制
发表时间：
2015-05-01
期刊：
影响因子：
4.2
通讯作者：
Sun, Ming
中科院分区：
文献类型：
--
作者：
Liu, Shihai;Liu, Xiangping;Sun, Ming
三阴性乳腺癌（TNBC）具有独特的生物学和临床病理学特征，由于缺乏有效的治疗靶点，预后较差。我们之前的数据显示，与正常乳腺组织样本相比，TNBC 样本中选择性地存在高水平的 USP39，并且 USP39 在培养的 TNBC 细胞和正常乳腺细胞中也有不同水平的表达。然而，USP39 的潜在细胞和分子机制仍不清楚。在本研究中，我们描述了一种多西环素（DOX）调节的慢病毒载体系统，在TNBC细胞系MDA-MB-231中表达USP39基因的shRNA或cDNA。 USP39 表达被基于 miR-30 的诱导型慢病毒短发夹 RNA (shRNA) 递送系统敲低，或被诱导型 cDNA 系统过度表达。诱导型shRNA介导的USP39表达下调显着降低了MDA-MB-231细胞的增殖和集落形成能力，而诱导系统过度表达USP39并不会促进癌细胞增殖。慢病毒载体介导的Tet-on系统证明了TNBC细胞中USP39的有效和可诱导的敲低或USP39的过表达，促进了基因敲低和过表达实验在体外和体内基因功能研究中的广泛应用：
Triple-negative breast cancer (TNBC), characterized by distinct biological and clinicopathological features, has a poor prognosis due to lack of effective therapeutic targets. Our previous data revealed that high levels of USP39 were selectively present in TNBC samples compared with their normal breast tissue samples and USP39 was also expressed at different levels in cultured TNBC cells and normal breast cells. Yet, the underlying cellular and molecular mechanisms of USP39 remain unclear. In the present study, we describe a doxycycline (DOX)-regulated lentiviral vector system expressing shRNA or cDNA of the USP39 gene in the TNBC cell line MDA-MB-231. USP39 expression was knocked down by the miR-30-based inducible lentiviral short hairpin RNA (shRNA) delivery system or overexpressed by the inducible cDNA system. The inducible shRNA-mediated downregulation of USP39 expression markedly reduced the proliferation and colony-forming ability of MDA-MB-231 cells, while overexpression of USP39 by the inducible system did not promote cancer cell proliferation. The lentiviral vector-mediated Tet-on system demonstrated efficient and inducible knockdown of USP39 or overexpression of USP39 in TNBC cells, facilitating a wide variety of applications for gene knockdown and overexpression experiments in gene functional studies in vitro and in vivo: