Enhancement of expression of survivin promoter-driven CD/TK double suicide genes by the nuclear matrix attachment region in transgenic gastric cancer cells

Abstract

This work aimed to study a novel transgenic expression system of the CD/TK double suicide genes enhanced by the nuclear matrix attachment region (MAR) for gene therapy. The recombinant vector pMS-CD/TK containing the MAR-survivin promoter-CD/TK cassette was developed and transfected into human gastric cancer SGC-7901 cells. Expression of the CD/TK genes was detected by quantitative real-time PCR (qPCR) and Western blot. Cell viability and apoptosis were measured using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry. When the MAR fragment was inserted into the upstream of the survivin promoter, the qPCR result showed that the expression of the CD/TK genes significantly increased 7.7-fold in the transgenic SGC-7901 cells with plasmid pMS-CD/TK compared with that without MAR. MTT and flow cytometry analyses indicated that treatment with the prodrugs (5-FC+GCV) significantly decreased the cellular survival rate and enhanced the cellular apoptosis in the SGC-7901 cells. The expression of the CD/TK double suicide genes driven by the survivin promoter can be enhanced by the MAR fragment in human gastric cancer cells.

Keywords: 5-FC; 5-fluorocytosine; CD; Ct; DMEM; Dulbecco's modified Eagle's Medium; GCV; Gastric cancer; Gene therapy; MAR; MTT; Matrix attachment region; PCR; PI; Suicide gene; Survivin promoter; TK; cytosine deaminase; ganciclovir; matrix attachment region; methyl thiazolyl tetrazolium; polymerase chain reaction; propidium iodide; qPCR; quantitative real-time PCR; threshold cycle number; thymidine kinase.