title: Optimization and establishment of RNA interference-mediated knockdown of the progestagen-associated endometrial protein gene in human metastatic melanoma cell lines.
abstract: 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.
authors.affiliation: Beijing Institute of Transfusion Medicine, Beijing 100850, P.R. China.
authors.forename: Lina
authors.initials: L
authors.lastname: Chai
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Qiao
authors.forename: Jiexi
authors.initials: J
authors.lastname: Wang
authors.forename: Minxia
authors.initials: M
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Xuanlin
authors.initials: X
authors.lastname: Wang
authors.forename: Min
authors.initials: M
authors.lastname: He
authors.forename: Weijing
authors.initials: W
authors.lastname: Li
authors.forename: Qun
authors.initials: Q
authors.lastname: Yu
authors.forename: Ying
authors.initials: Y
authors.lastname: Han
authors.forename: Suping
authors.initials: S
authors.lastname: Ren
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D005434:Flow Cytometry; D000071816:Glycodelin; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D016086:Lentivirus / Q000235:genetics; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000556:secondary; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
chemical_list: D000071816:Glycodelin; D006023:Glycoproteins; C045246:PAEP protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering