title: [Construction of lentivirus vector of interference of EGFL7 gene and its inhibitive role on the invasion of laryngeal cancer cell].
abstract: OBJECTIVE
  To construct a lentivirus vector of RNA interference (RNAi) of EGFL7 gene and observe its inhibitive role on the invasion of laryngeal cancer cell.
  
  
  METHODS
  The effective sequence of siRNA targeting EGFL7 gene was confirmed. Both sense and antisence Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pLV vector,which contained H1 promotor and green fluorescent protein (GFP). The resulting lentivirus vector containing EGFL7 shRNA was called LV-sh EGFL7,and it is confirmed by PCR and sequencing. After that, EGFL7 shRNA was transfected into Hep-2 cells and Western blot was used to test the expression of EGFL7. At last, boyden chamber was used to observe the invasion of the Hep-2 cells. Colony formation assay using a EGFL7 gene silencing on the Hep-2 cell colony forming ability.
  
  
  RESULTS
  PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of EGFL7 (LV-sh EGFL7) producing EGFL7 shRNA was constructed successfully. The titer of concentrated virus was 5 x 10(8) TU/L. Western blot showed that the expression of EGFL7 was negative in the EGFL7 siRNA Hep-2 cells. And boyden chamber showed the invasive capability of Hep-2 cells transfected EGFL7 siRNA were obviously decreased. EGFL7 gene silencing of cell colony formation rate of cloned Hep-2 cells and compared with empty vector cells, cell cloning and colony formation was significantly reduced.
  
  
  CONCLUSIONS
  The lentivirus RNAi vector of EGFL7 was constructed successfully. And EGFL7 silence can inhibit invasion of laryngeal cancer in vitro. After silence EGFL7, Hep-2 cell colony formation was significantly lower, that is, gene expression can be down EGFL7 some extent laryngeal cancer cells inhibited anchorage independent growth capacity.
authors.affiliation: Department of Otolaryngology, the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, 710061, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Wang
authors.forename: Xiaobao
authors.initials: X
authors.lastname: Yao
authors.forename: Xiansheng
authors.initials: X
authors.lastname: Ji
authors.forename: Jinghua
authors.initials: J
authors.lastname: Chen
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Hongliang
authors.initials: H
authors.lastname: Zhu
mesh_terms: D002135:Calcium-Binding Proteins; D045744:Cell Line, Tumor; D066255:EGF Family of Proteins; D016228:Endothelial Growth Factors / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics / Q000473:pathology*; D016086:Lentivirus / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D014162:Transfection
chemical_list: D002135:Calcium-Binding Proteins; D066255:EGF Family of Proteins; C527877:EGFL7 protein, human; D016228:Endothelial Growth Factors; D034741:RNA, Small Interfering