[Construction of a lentiviral vector carrying short?hairpin RNA targeting PAX6 and its effect on proliferation of glioma U251 cells in vitro].

OBJECTIVE

To construct a lentiviral vector for delivering short hairpin RNA (shRNA) targeting PAX6 and investigate its effect on the proliferation of glioma U251 cells in vitro.

METHODS

Two small interfering RNA sequences targeting PAX6 gene were designed based on the reported sequence of PAX6 and annealed to form a double?stranded chain, which was inserted into a lentiviral vector to construct the recombinant lentiviral vector shRNA?PAX6. The recombinant vector was infected into U251 cells, and the expression of PAX6 mRNA and protein in the cells was detected by real?time PCR and Western blotting, respectively. The changes in the proliferation of U251 cells after the infection was assessed using MTT assay.

RESULTS

Double enzyme digestion of the lentiviral vector pLKD?CMV?G&NR?U6?shRNA yielded an 8208?bp fragment, and colony PCR and sequencing analysis confirmed successful construction of the lentiviral vector shRNA?PAX6. Infection of the cells with shRNA?PAX6 caused a significant reduction of the expressions of PAX6 mRNA and protein (P<0.05) and resulted in obviously increased proliferation of U251 cells (P<0.05).

CONCLUSION

We successfully constructed the recombinant vector shRNA?PAX6 for silencing PAX6 gene. PAX6 gene silencing results in increased proliferation of U251 cells in vitro.