Construction of retroviral vector of p125FAK specific ribozyme genes and its effects on BGC-823 cells

Abstract

AIM: 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.

METHODS: A hammerhead ribozyme DNA targeting p^125FAK mRNA from nt 1010 to nt 1032 was synthesized and recombinated 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.

RESULTS: 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 P¹²⁵ 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.

CONCLUSION: p^125FAK ribozyme decreases p^125FAK gene expression and induces apoptosis of human gastric cancer cells in vitro.

Keywords: Ribozyme, p^125FAK gene, Stomach neoplasm, Apoptosis