title: [Over-expression of human S-phase kinase-associated protein 2 (Skp2) promotes proliferation of MCF-7 breast cancer cells and increases the number of S phase cells].
abstract: OBJECTIVE
  To construct a eukaryotic expression vector for the human gene S-phase kinase-associated protein 2 (Skp2) with a FLAG tag (pcDNA3-FLAG-Skp2) and detect the effect of Skp2 over-expression on the cell growth and cell cycle in MCF-7 breast cancer cells.
  
  
  METHODS
  Skp2 was amplified from MDA-MB-231 breast cancer cells by reverse-transcription polymerase chain reaction (RT-PCR) and then used to construct the eukaryotic expression vector pcDNA3-FLAG-Skp2. Integration of Skp2 into the vector was confirmed via restriction digest and sequencing; The pcDNA3-FLAG-Skp2 was then transfected into MCF-7 breast cancer cells. Expression of Skp2 protein was verified by Western blotting. Cell growth was assessed by Alamar blue proliferation assay, cell cycle analysis was carried out by flow cytometry.
  
  
  RESULTS
  The PCR amplified fragment was matched up with the anticipated result and its sequence was the same as the data published on GenBank, indicating that the recombinant plasmid pcDNA3-FLAG-Skp2 was constructed successfully. Western blotting revealed that the expression of Skp2 protein was markedly up-regulated in the pcDNA3-FLAG-Skp2 transfected MCF-7 cells at 48 hours. Furthermore, cell growth was significantly promoted in Skp2 over-expressed MCF-7 cells, and the cell count in S phase were also raised.
  
  
  CONCLUSIONS
  The recombinant eukaryotic expression vector pcDNA3-FLAG-Skp2 has been constructed and expressed in MCF-7 breast cancer cells successfully. Over-expression of Skp2 resulted in the increased cell growth and number of S phase cells in Skp2 transfected MCF-7 cells.
authors.affiliation: Department of Oncology, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Chengcheng
authors.initials: C
authors.lastname: Yang
authors.affiliation: Department of Oncology, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Kejun
authors.initials: K
authors.lastname: Nan
authors.affiliation: Department of Thoracic Surgery, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an 710061, China.
authors.forename: Sida
authors.initials: S
authors.lastname: Qin
mesh_terms: D001943:Breast Neoplasms / Q000473:pathology*; D049109:Cell Proliferation; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D014162:Transfection
chemical_list: D044786:S-Phase Kinase-Associated Proteins