Optimization and establishment of RNA interference-mediated knockdown of the progestagen-associated endometrial protein gene in human metastatic melanoma cell lines
Optimization and establishment of RNA interference-mediated knockdown of the progestagen-associated endometrial protein gene in human metastatic melanoma cell lines
复制标题
人转移性黑色素瘤细胞系中孕激素相关子宫内膜蛋白基因的 RNA 干扰介导的敲低的优化和建立。
DOI：
10.3892/mmr.2013.1679
复制
发表时间：
2013-11-01
影响因子：
3.4
通讯作者：
Ren, Suping
中科院分区：
文献类型：
--
作者：
Chai, Lina;Qiao, Zhixin;Ren, Suping
Progestagen-associated endometrial protein (PAEP), also termed glycodelin, is a 28-kDa glycoprotein of the lipocalin superfamily that is expressed in a variety of tumors, including gynecological tumors, lung cancer and melanoma. To determine the biological functions of the PAEP gene in tumor development and progression, the production of transient and stable PAEP knockdown cell models is required. In the present study, RNA interference technology was used to silence PAEP gene expression in melanoma and transfection was screened for and the conditions were optimized using fluorescence microscopy, flow cytometry, qPCR and western blot analysis. The results of the present study showed that the transient transfection of melanoma cells with 100 nmol/l PAEP siRNA or lentiviral PAEP small hairpin RNA (shRNA) [multiplicity of infection (MOI), 100 pfu/cell] efficiently knocked down target gene expression. To establish stable PAEP knockdown cell lines, melanoma cells were infected with a low MOI (10 pfu/cell) of lentiviral particles expressing PAEP shRNA. Following puromycin screening, the PAEP gene knockdown efficiency was demonstrated to be >80% in 624-Mel and 624.38-Mel cell lines, which was validated by western blot analysis. Therefore, melanoma cell lines with stable knockdown of PAEP were successfully established and may be used as effective cell models to study the biological functions of the PAEP gene in melanoma.
