[Preparation of the recombinant lentiviral expression vector targeting human Wnt5a gene and its inhibitory effect on melanoma cell invasion].

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.

CONCLUSION

Down-regulated Wnt5a expression exerts a significant inhibitory effect on the invasion of melanoma cells.