The Effect Of Gastric Carcinoma Cells Growth And Apoptosis By Small Interfering RNA Silencing Of Tissue Factor

Objectives:It has been shown that Tissue Factor(TF) is overexpressed in the gastric cancer and associated with its pathogenesis. To better understanding of the role of TF, a siRNA expression plasmid was constructed to inhibit TF expression in the human gastric cancer cells in vitro. BGC-823, gastric cancer cell line, was stably transfected with TF siRNA plasmids. To observe the influence of TF gene on gastric cancer cells growth velocity and cell apoptosis, and explore the function of TF gene in the development and therapy of gastric cancer.Methods:1. DNA sequences with small hairpin structure were designed and synthesized. The complement form was obtained by annealing and cloned into vector pGPU6/GFP/Neo. Recombinant plasmid was transformed into strain DH5a, identified by using restriction enzyme and sequencing of nucleic acid.2. The siRNA expression plasmid was transfected into gastric cancer cells by lipofectamine. Select the positive cell clone by antibiotic G418. The expression of the tissue factor gene mRNA on the transfected cells was measured by RT-PCR.3. After the TF siRNA transfected into gastric cancer cells by lipofectamine, The cell growth velocity and apoptosis rate were measured or observed by CCK8assay and Flow Cytometry.Results:1. The recombinant plasmid were successfμlly constructed, which was proved by restriction enzyme and sequencing of nucleic acid.2. The four siRNA expression plasmids pGPU6/GFP/Neo-TF-1、2、3、4were cotransfected into BGC—823cells by lipofectamine respectively. Results from RT-PCR indicated that they could remarkably decrease the expression levels of TF gene mRNA in transfected BGC—823cells, and the effect of pGPU6/GFP/Neo-TF-2is the most remarkable.3. Transfected by recombinant plasmid, the cell proliferation of the strain BGC—823could remarkably decrease(p<0.05).4. Transfected by recombinant plasmid, the strain BGC—823could show some morph change. Its apoptosis rate could remarkably increase(p<0.05).Conclusions:1. The TFsiRNA plasmid was successfully constructed and transfected in the gastric cancer cell lines.2. The level of TF was obviously decreasing after transfecting with target siRNA vectors in gastric cancer cells. The gastric cancer cells with silenced TF gene exhibited low growth and high apoptosis ability in vitro experiment.3. TF gene may become a new target for treatment of gastric cancer.