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Objective:The aim of the study was to investigate the interference and anti-tumor effects of lentiviral vector of miRNA targeting IGF1R gene regulated by survivin promoter.Methods:The fragment of the survivin promoter was acquired by PCR amplification and inserted into pPRIME to recombinant plasmid sur-pPRIME.The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into the sur-pPRIME vector,named sur-pPRIME-IGF1R-miR30-shRNA.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 sur-pPRIME-IGF1R-miR30-shRNA on IGF1R expression of Hep3B cells was detected by RT-PCR and Western blot.The antitumor potential of sur-pPRIME-IGF1R-miR30-shRNA to Hep3B cells was evaluated by CCK-8 assay.Results: sur-pPRIME-IGF1R-miR30-shRNA was constructed successfully.Functional PFU titers of sur-pPRIME-IGF1R-miR30-shRNA were 4.58×109 PFU/mL.Sur-pPRIME-IGF1R-miR30-shRNA was more effective to inhibit IGF1R expression in mRNA or protein levels and the proliferation of Hep3B cells.Conclusion:sur-pPRIME-IGF1R-miR30-shRNA expressing IGF1R-siRNA can inhibit IGF1R expression and may be used for further investigation of gene therapy of liver cancer.
Objective: The aim of the study was to investigate the interference and anti-tumor effects of lentiviral vector of miRNA targeting IGF1R gene regulated by survivin promoter. Methods: The fragment of the survivin promoter was acquired by PCR amplification and inserted into pPRIME to recombinant plasmid sur-pPRIME. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the sur-pPRIME vector, named sur-pPRIME-IGF1R-miR30-shRNA. 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 sur-pPRIME-IGF1R-miR30-shRNA on IGF1R expression of Hep3B cells was detected by RT-PCR and Western blot The antitumor potential of sur-pPRIME-IGF1R-miR30-shRNA to Hep3B cells was evaluated by CCK-8 assay. Results: sur-pPRIME- IGF1R-miR30- shRNA was constructed successfully. Functional PFU titers of sur-pP RIME-IGF1R-miR30-shRNA were 4.58 × 109 PFU / mL. Sur-pPRIME-IGF1R-miR30-shRNA was more effective to inhibit IGF1R expression in mRNA or protein levels and the proliferation of Hep3B cells. Confluence: sur-pPRIME-IGF1R -miR30-shRNA expressing IGF1R-siRNA can inhibit IGF1R expression and may be used for further investigation of gene therapy of liver cancer.