Objective:To study the effects of lentiviral vector of microRNA targeting IGF1R gene on growth of liver cancer cells.
Methods :The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pPRIME vector, named pPRIME-IGFIR-miR30-shRNA. The viruses were propagated on 293T cells. Viruses were purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The effect of pPRIME-IGFIR-miR30-shRNA on IGFIR expression of Hep3B、SMMC7721 cells was detected by RT-PCR and Western blot. The antitumor potential of PRIME-IGFIR-miR30-shRNA to Hep3B、SMMC7721 cells was evaluated by CCK-8 assay and Tunel.
Results:PRIME-IGFIR-miR30-shRNA was constructed successfully. Functional PFU titers of pPRIME-IGFIR-miR30-shRNA were 4.58×109PFU/ml. PRIME-IGFIR-miR30-shRNA inhibited IGFIR expression and the proliferation of Hep3B、SMMC7721 cells.
Conclusions:PRIME-IGFIR-miR30-shRNA expressing IGFIR-siRNA can inhibit IGFIR expression and may be used for further investigation of gene therapy of liver cancer.