Effects Of Combined Gene Therapy System, PcDNA(-) 3.1(-)shVEGF/yCDglyTK To The Proliferation Of Gastric Cancer Cells

Background and Purposes:Gastric cancer is one of the common malignant tumors in humans,whose incidence ranks the first in four malignant tumors over the worldwide and No.2 on cancer mortality rate. In China, the incidence and mortality of gastric cancer tops the first. Prognosis of gastric cancer is poor, for the limited effect of surgery, radiation therapy, chemotherapy. So the need for new effective treatment is urgent. From malignant transformation and tumor development in terms of molecular genetics, cancer is a multi-stage acquired genetic disease, its growth, invasion and apoptosis are all associated with genes. Currently the main ways for gene therapy are suicide gene therapy, tumor suppressor gene therapy, antisense gene therapy, immune gene therapy, and combined gene therapy,a gene therapy strategie developed on the basis of a single.For gastric cancer gene therapy, suicide gene is more studied in all anti-tumor genes, such as suicide gene CD/5-FC, HSV-TK/GCV system, have powerful effect of anti-tumor and inhibit growth of tumor. RNA interference, discovered and developed in recent years, a new gene technology blocking genes on the level of transcription, is a double-stranded RNA (double-stranded RNA, dsRNA) molecules to close the corresponding gene sequence or silence the process of the expression on the mRNA level, that is sequence-specific post-transcriptional gene silencing (post transcriptional gene silencing, PTGS).Researches shows that, RNA interference towards VEGF is an effective strategy to inhibit proliferation, migration, invasion and vascular generation of tumor cells. Tumorigenesis is processes involved complex multi-factors, multi-steps and multi-genetic events. For a single link gene therapy treatment often can not achieve satisfactory results over the past decade, people began to turn their attentions to combined gene therapy. Combined gene therapy is the combination among several target genes, gene and chemotherapy drugs, radiation and biological response modifiers, in order to get a better combination between the anti-tumor effect, while reducing as much as possible on the body produced side effects. Most of these researches showed that combined gene therapy embraced synergistic effects, which can enhance anti-tumor effect. In order to explore more powerful gene treatment for system gantric cancer, we will clone the U6-shRNA expression cassette from pGenesil-shVEGF into the vector backbone of pcDNA3.1 (-)-CV-yCDglyTK frame basis.Through two mechanisms of gene therapy systems,we hope obtain a more effective method to killi cancer cells to provide a new strategies for clinical practice.Methods:Gastric cancer cell SGC7901 were transfected by plasmids of pcDNA3.1 (-) Null, pGenesil-shVEGF, pcDNA3.1 (-)-CV-yCDglyTK and pcDNA3.1 (-)-shVEGF/yCDglyTK mediated by carriers of calcium phosphate nanoparticles. Expressions of GFP reporter gene were observed by the fluorescence microscopy, combined with flow cytometry to determine transfection efficiency; Expressions of yCDglyTK and VEGFmRNA were detected by RT-PCR; Changes of protein of VEGF and yCDglyTK were analyzed by Western-blot; The inhibition effects of 5-FC on proliferation of transfected cells were analyzed by MTT; Apoptosis of transfected cells were observed by flow cytometry. Results:1. Four kinds of plasmids could successfully transfect SGC7901 cells mediated by calcium phosphate nanoparticles, transfection efficiency measured was about 54%; 2. The groups transfected with pcDNA3.1 (-)-CV-yCDglyTK and pCDNA3.1 (-)-shVEGF/yCDglyTK expressed yCDglyTK mRNA and protein,while the control group and the remaining two cells had no expression; The expression of VEGF of groups transfected with pGenesil-shVEGF and pcDNA3.1 (-)-shVEGF/yCDglyTK plasmid was inhibited, compared to internal reference P-actin expression level, the former was 0.36±0.04, the latter was 0.41±0.04, compared with the control group,which was 0.66±0.05, there was a significantly difference (P<0.05);3. MTT tested 5 groups of cells treated by 5-FC. The survival rate showed that growth of pcDNA3.1 (-)-CV-yCDglyTK group and pcDNA3.1 (-)-shVEGF/ yCDglyTK group were significantly inhibited, compared to pcDNA3.1 (-)-CV-yCDglyTK group pcDNA3.1 (-)-shVEGF/yCDglyTK group had a higher sensitivity to 5-FC (P<0.05).4. Results of flow cytometry also confirmed pcDNA3.1 (-)-shVEGF/yCDglyTK group had a higher apoptosis rate of (67.9±4.78)%, higher than the other 4 groups(P<0.05), which were (3.24±0.40)%,(4.56±0.71)%,(21.6±1.11)%,(56.2±2.59) %.Conclusions:1. Four kinds of plasmids, pCDNA3.1 (-) Null, pGenesil-l-hVEGF4-shRNA, pCDNA3.1(-)-CV-yCDglyTK, pCDNA3.1 (-)-shVEGF /yCDglyTK could successfully transfect SGC7901 cells mediated by calcium phosphate nanoparticles.2. Combined gene therapy system pcDNA3.1 (-)-shVEGF/yCDglyTK can inhibit the expression of VEGF in gastric cancer cells.3. Combined gene therapy system pCDNA3.1 (-)-shVEGF/yCDglyTK can effectively inhibit the proliferation of gastric cancer cells, whose effect is stronger than separate RAN interference plasmid or suicide gene. There is a synergistic effect between fusion suicide gene and the VEGF interference plasmid.