Experimental Study On The Relationship Between Tissue Factor And The Biological Behavior Of Gastric Carcinoma

AimsTissue factor (TF) is the initiation factor of the extrinsic pathway of coagulation. Recent studies found that it has a role in signal transduction, inflammation, atherosclerosis, embryonic development, tumor angiogenesis, tumor growth, metastasis. Gastric cancer is one of the most common cancers in the world. It is confirmed that TF is highly expressed in several tumors, including gastric cancer, and the expression of TF is correlated with a variety of biological characteristics of tumors. The aim of this study was to investigate the effect of TF on human gastric cancer SGC7901cells, to explore the role of TF in biological characteristics of gastric cancer, RNA interference (RNAi) technology was used to silence TF in SGC7901cells and evaluate its antitumor effects in vitro and in vivo, which would provide a experimental basis for the clinical application of TF in gastric cancer.MethodsThe human TFcDNA was obtained from human placenta by nest PCR, and the constructed eukaryotic expressive vector TF-pcDNA3was transfected into SGC7901cells by lipofectamine. Stable-transfected cells were screened by G418. The expression of TF on the cells was detected by RT-PCR and Western blot. Cell motility and invasion were assessed by Transwell experiments and wound-healing assays. The mRNA sequence of TF gene was obtained through the GenBank sequence investigation, design and synthesis of specific siRNA. Construction and connection of pGPU6/GFP/Neo-TF expression vector, the products were transformed into Escherichia coli DH5alpha. Extraction of positive clone by Plasmid Extraction Kit, using enzyme digestion and sequencing method to identify aim genes, and plasmid was performed the concentration and purity detection with the automatic spectrophotometer. The lipofection method was used to transfect the recombinant plasmid and the empty plasmid into cells, and Screening by G418to establish a stable cell line. The expression of TF in the cells was detected by RT-PCR and flow cytometry. Cell proliferation and chemosensitivity were measured by CCK8. The metastatic potential of SGC7901cells was determined by Transwell experiments and wound-healing assays. Cell apoptosis was assessed by Annexin-V/PI double-staining method. In vivo, the effect of TFsiRNA on the growth of SGC7901gastric cancer xenografts in nude mice was investigated.ResultsThe results showed that the eukaryotic expressive vector TF-pcDNA3was successfully constructed and transfected into SGC7901which expressed TF stably and efficiently. Compared with control cells, the expression of TFmRNA and TF antigen in the transfected SGC7901/TF cells were increased, the cell motility and invasion in vitro of these cells were enhanced. Four siRNA sequences for TF and a negative control siRNA sequence were designed and synthesized. The enzyme slotting results showed that all digested plasmids were the positive recombinant vectors, and the sequencing results showed that the synthesized plasmid sequences were correct and successfully cloned onto the selected vector, and there was no presence of abnormal bases. Ultraviolet Spectrometer Subsystem (UVS) was used to detect the concentration and purity of the extracted plasmid DNA, and the results were satisfactory for the next transfection step. The inverted fluorescence microscope was used to observe the expression of the green fluorescent protein after the transfection showed that the transfection rate on the24th,48th and72nd h were25%,40%and30%, respectively. The RT-PCR was used to detect the expression level of TF gene, and the results showed that, compared with the control group and the negative control group, the transfected expressions of pGPU6/GFP/Neo/TF in each group decreased, the expression of TF-2gene was of the lowest, and the inhibitory effect was the best, and the TF positive expression of cells detected by flow cytometry which showed the experimental group decreased versus control. CCK.-8assay was used to detect the absorbance value (A) of each group was found that the cell proliferation of the experimental group was significantly lower than the control group, and the inhibition rate of oxaliplatin towards the TFsiRNA transfected SGC7901cell was significantly increased. Cell scratch experiment and cell invasion experiment indicated that TFsiRNA could reduce the metastatic potential of gastric cancer cells. The transfected cells with Annexin-V/PI double-staining were subjected to flow cytometric analysis showed that TFsiRNA increased the apoptotic rate in SGC7901cells. The experimental results of human gastric cancer xenografts in nude mice showed that the tumor volume and tumor weight were significantly less than that of the control group in vivo.ConclusionThese findings indicate that TF can enhance the invasion and metastasis of gastric cancer cells in vitro. TF knockdown with siRNA could inhibit the growth, invasion and metastasis, reduce the chemoresistance. enhance the apoptosis, suppress the tumor growth of SGC7901cells in vivo model of gastric cancer. Down-regulation of TF using siRNA could provide a potential approach for the gene therapy against human gastric cancer.