Construction of retroviral vector of p125FAKspecific ribozyme genes and its effects on BGC-823 cells

Abstract

To construct the retroviral vector of p(125FAK) specific ribozyme genes and to explore the feasibility of ribozyme in BGC-823 gene therapy in vitro. A hammerhead ribozyme DNA targeting p(125FAK) mRNA from nt 1010 to nt 1032 was synthesized and recombined into the retroviral vector pLXSN forming pLRZXSN recon. Using the lipofectin-mediated DNA transfection technique, pLRZXSN was introduced into BGC-823 cells. The effects of ribozyme on the growth of BGC-823 cells and apoptosis were studied by cell colony assay, flow cytometry (FCM), reverse transcriptase-polymerase chain reaction (RT-PCR), detection of DNA fragmentation and electron microscopy. The number of BGC-823 cell colonies was inhibited by 56% after the cells were treated for 48 h. The cell proliferation was inhibited effectively by p(125FAK) ribozyme and the inhibitory effect depended on the concentration and the time of incubation. The expression of p(125FAK) mRNA and protein P(125FAK) decreased sharply in BGC-823 cells treated with p(125FAK) ribozyme. The characteristics of apoptosis, namely sub-G1 peak, DNA fragmentation and morphological changes, were revealed in BGC-823 cells treated with p(125FAK) ribozyme. p(125FAK) ribozyme decreases p(125FAK) gene expression and induces apoptosis of human gastric cancer cells in vitro.