title: [Preparation of the recombinant lentiviral expression vector targeting human Wnt5a gene and its inhibitory effect on melanoma cell invasion].
abstract: OBJECTIVE
  To construct a recombinant lentiviral vector expressing small-hairpin RNA (shRNA) targeting human Wnt5a gene and investigate its silencing effect on WM793B human melanoma invasion.
  
  
  METHODS
  Based on the sequence of human Wnt5a gene in GenBank, Wnt5a siRNA was designed and synthesized. The single-stranded primers were annealed to double-stranded oligonucleotide sequences and subcloned into linear pLKO.1 lentiviral plasmid digested by enzyme to produce pLKO.1-shWnt5a lentiviral vector. After being identified by PCR and sequencing, plasmid pLKO.1-shWnt5a was transfected into HEK293T cells to package lentiviral particles. Human malenoma WM793B cells were infected by the lentiviral particles. Expression of shWnt5a in WM793B cells was detected using Western blotting. Then the Transwell(TM); invasion assay was performed to assess its effect on melanoma cell invasion.
  
  
  RESULTS
  Lentivirus expressing shWnt5a was successfully constructed and WM793B(Wnt5a-);, a strain of melanoma cells with low expression of Wnt5a was also established. Transwell(TM); invasion assay revealed that cell migration was inhibited in Wnt5a-inhibited melanoma cells.
  
  
  CONCLUSIONS
  Down-regulated Wnt5a expression exerts a significant inhibitory effect on the invasion of melanoma cells.
authors.affiliation: Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Miao
authors.affiliation: Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Xinyu
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Liya
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Molecular Biology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Lu
authors.affiliation: Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Wenjun
authors.initials: W
authors.lastname: Liao
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009361:Neoplasm Invasiveness; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism; D000071818:Wnt-5a Protein
chemical_list: D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; C085389:WNT5A protein, human; D051153:Wnt Proteins; D000071818:Wnt-5a Protein