[Construction of a lentiviral vector for RNA interference of PRL-3 gene and its stable expression in SW480 cells].

OBJECTIVE

To construct a lentiviral vector for RNA interference (RNAi) of PRL-3 gene and establish a human colon carcinoma cell line with PRL-3 gene knock-down.

METHODS

The plasmids were constructed expressing two different short hairpin RNAs (shRNA) targeting PRL-3 gene under control by the U6 promoter by lentiviral vector. An entry clone was generated in the pENTR/U6 vector. After identification with sequencing, a recombinant lentiviral vector was obtained using the pENTR/U6 construct and pLenti6/BLOCK-iT TM-DEST vector. The recombinant lentivirus was harvested from 293FT cells co-transfected with optimized ViraPower Packaging Mix and the pLenti6/BLOCK-iT -DEST expression construct. SW480 cells were infected with the recombinant lentivirus and the cells with stable PRL-3 gene knock-down were screened by blasticidin selection. PRL-3 expression was detected using real-time reverse transcription-polymerase chain reaction and Western blotting.

RESULTS

A recombinant lentiviral vector expressing shRNAs targeting PRL-3 gene was successfully established and confirmed by DNA sequencing. The recombinant lentivirus were harvested from 293FT cells with a viral titer of 6 x 10(5) pfu/L. Twelve clones of SW480 cells infected with the recombinant lentivirus were selected, and the Clone 1 exhibited significant knock-down of PRL-3 protein expression (72.9%).

CONCLUSION

The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.