[Construction and identification of lentiviral vector containing human ILK-shRNA and mda7 gene]

To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene.

Based on the human ILK gene sequences, RNAi target sequences were designed and cloned into the lentiviral vector pSicoR-eGFP by restriction endonuclease HpaI and XhoI double digestion and T4 DNA ligase ligation. Based on the human mda7 gene sequences, PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro. After the candidate clones were identified by DNA sequencing, the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles. Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector. The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot, respectively. The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively.

ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed. Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector. The transfection efficiency of the collected virus exceeded 90% in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency. The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly. The mda7-pLVX-Puro lentiviral vector increased the expression of mda7 in PC-3 cells, and the ability was maintained for one month. Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells (Ps<0.05).

The lentiviral vectors of ILK knockdown and mda7 over-expression have been successfully constructed and identified. The recombinant lentivirus can efficiently infect human prostate cancer PC-3 cells, in which ILK expression is inhibited and mda7 is over-expressed.