Inhibiting Gallbladder Carcinoma Growth And Invasion By Knock Down The Expression Of VEGF Using RNA Interference

Primary gallbladder carcinoma ranked first in malignant tumors of biliary system,and ranked fifth in malignant tumors of digestive tract. The degree of malignancy of gallbladder carcinoma is very high. Because of the early non-specific clinical manifestations, many patients at the time of diagnosis has progressed to the late, and 5-year survival rate of stage III and IV patients is only 1% and 5%. The 5-year survival rate of gallbladder cancer in United States and Europe is only 5% -13%. The main treatment for gallbladder carcinoma is surgery, but only 20%-30% of patients can be radical resection. The fact that chemotherapy and radiotherapy for gallbladder carcinoma are not sensitive has brought great difficulties to the clinical treatment for gallbladder carcinoma. It is important to explore new therapy.Solid tumor is vascular-dependent, and the tumor growth, invasion and metastasis can not be separated from angiogenesis. Tumor angiogenesis is a key factor in tumor growth. Among all tumor angiogenesis factors, only VEGF is highly specific vascular endothelial cell mitogen which directly stimulated endothelial cell division and proliferation.VEGF was highly expressed in gallbladder carcinoma. VEGF expression is different in gallbladder size, lymph node infiltration in various stages. VEGF participates in not only the occurrence of gallbladder carcinoma, but also the invasion, metastasis. The anti-angiogenic treatment targeting VEGF is very important.How to effectively inhibit VEGF gene expression is the key factort of cancer treatment. The way of inhibiting VEGF gene expression includes:antisense nucleic acid technology, ribozyme technology, and RNA interference technology. The gene suppression effect of antisense technology and ribozyme technology is very short and weak, which is not suitable for long-term study of gene function. The RNAi technology is far superior to them in suppressing specific gene expression. It has strong inhibitory effect, simple technical process, short-cycle, stability, efficiency and other unique advantages.The plan of this experiment is:we observe the impact of VEGF gene silencing on growth and invasion of gallbladder carcinoma GBC-SD cell line; we use the gallbladder carcinoma GBC-SD cells to establish nude mice xenograft model and observe the change of VEGF expression after intratumoral injection of recombinant plasmid shRNA-VEGF2 in xenografts in nude mice and effects on tumor growth and angiogenesis in order to provide experimental and theoretical basis for the clinical development and application of cancer treatment. Objective:To construct VEGF-targeted small interfering RNA and its expression vectorMethods:Complementary oligo DNA strands targeting VEGF gene were designed and synthesized according to the principles of designing siRNA. After annealing, oligo DNAs were inserted into pCY/U6/GFP/Neo vector, then enzyme digestion analysis and DNA suquencillg were performed.Results:The enzyme digestion analysis and DNA sequencing showed that VEGF-targeted small interfering RNA and its expression vector were constructed successfully.Conclusion:VEGF-targeted small interfering RNA and its expression vectors were constructed successfully. Objective:To observe the impact of VEGF-targeted shRNA on the expressions of VEGF. Luciferase reporter gene was used to verify the validity of RNA interference sites. We choose the best plasmid to establish a stable transfected cell line. We observed the expression of VEGF receptors, and study the mechanism of action between VEGF and VEGF receptors.Methods:①In order to determine the inhibiting effect of VEGF, we use semi-quantitative PCR, real-time PCR and Western blot to detect VEGF mRNA and protein expression.②We design primers containing the VEGF target sites, make it connected with the luciferase reporter gene vector. The recombinant vector and the vectors of chapter I were transfected into human gallbladder carcinoma cells to observe the changes of luciferase activity, and to verify the validity of RNA interference sites.③We use the shRNA-VEGF2 plasmid and NC plasmid to establish stable transfected cell lines by G418 selection.④The mRNA expressions of the three VEGF receptors (Flt-1, KDR, NRP-1) are detected by real-time PCR. Then we added VEGF into the cell line and detecte the mRNA expressions of Flt-1 at 24,48,72 hours in order to study the mechanism between VEGF and of its receptors.Results:①shVEGF1, shVEGF2, shVEGF3, shVEGF4 interfering vectors all inhibit VEGF mRNA expression and the inhibition rate of shVEGF2 is 64%. Detected by real-time PCR,the inhibition rate of shVEGF2 is 83%. The inhibition rate of VEGF protein of shVEGF2 is 92% detected by western blotting. ②Luciferase reporter gene through In the verification experiment, we found luciferase activity decreased 75% which imply that the target site is accurate;③We successfully establish two stable transfected cell lines, named GBC-shV2 and GBC-NC cells.④Expression of Flt-1 and KDR receptors in GBC-shV2 cell lines decreased 28% and 18%, while expression of NRP-1 receptor increased 47%. When added VEGF into the cell line, the expression of Flt-1 is 75%, 183% and 411% at 24,48 and 72 hours respectively. We can concluded that the expression of Flt-1 increased significantly with the VEGF.Conclusion:①shVEGF2 can inhibit expression of VEGF in human gallbladder carcinoma GBC-SD cells;②The luciferase reporter gene experiments show that the RNA interference target site is accurate and valid;③We established stable transfected cell lines which express shVEGF2 plasmid;④There are VEGF receptors (Flt-1, KDR, NRP-1) in human gallbladder carcinoma GBC-SD cells;⑤There is VEGF autocrine mechanism in human gallbladder carcinoma GBC-SD cells;⑥VEGF can induce expression of Flt-1 receptor. Objective:To study the effects of VEGF gene silencing on capacity of growth, adhesion, migration, and invasion of human gallbladder carcinoma GBC-SD cells.Methods:In this part, we draw the cell growth curve using MTT method; To assess the adhesion ability through adhesion experiments; To assess the migration ability through migration experiments; To assess the invasion ability through Transwell chamber invasion assay.Results:①Compared with GBC-SD cells, the cell viability decreased significantly, reaching 36%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells.②Compared with GBC-SD cells, the cell's adhesion ability decreased significantly, reaching 55%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells.③Compared with GBC-SD cells, the cell's migration ability decreased significantly, reaching 61%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells (p<0.05)④Compared with GBC-SD cells, the cell's invasion ability decreased significantly, reaching 51%, and the difference was statistically significant (p<0.05). There is no difference between the GBC-NC cells and GBC-SD cells (p<0.05)Conclusion:shRNA-VEGF interfering plasmid significantly inhibited the, cell, and ability of gallbladder cancer cells. In vitro, it validate that the VEGF gene can be a good therapeutic target. Objective:We use the GBC-SD, GBC-NC and GBC-shV2 cells to established mice xenograft models to observe the change of tumorigenic capacity; We intratumoral inject recombinant plasmid shRNA-VEGF2 into the tumors to observe the VEGF expression in tumor tissue and the change of tumor growth and angiogenesis.Methods:In this study, the experiment were divided into two parts: tumor formation experiment and treatment of tumor-bearing mice experiment.①Tumor formation experiment:18 health nude mice were randomly divided into three groups of six. The GBC-SD, GBC-NC and GBC-shV2 cells are injected into the nude mice to establish mice xenograft models. We observe tumor formation time, rate of tumor formation, change of tumor volume, and draw the growth curve.②Treatment of tumor-bearing mice experiment:24 healthy nude mice were randomly divided into four groups of six. When the xenograft models are established, we inject different 200ul reagents into the tumors: group A:liposome; group B:RPMI1640; group C:empty plasmid and liposome; group D:shRNA-VEGF2 plasmid and liposome.After the animal experiments, we remove the tumor from the mice and take it for VEGF and CD34 immunohistochemistry. We use the computer-assisted image analysis system to analyze IOD and MVD to assess the expression of VEGF and angiogenesis in tumor tissue.Results:①Tumor formation experiment:The tumor formation rate of GBC-shV2 group is 67%, and the tumor volume inhibition rate is 70%. Compared with GBC-SD cells, the IOD value of VEGF decrease 59% and the MVD value decrease 64%. There is no difference between the GBC-NC group and GBC-SD group.②Treatment of tumor-bearing mice experiment:The tumor volume inhibition rate of group D is 45%. Compared with group A, the IOD value of VEGF of group D decrease 50% and the MVD value decrease 59%. There is no difference between the group A,B and C.Conclusion:RNA interference can inhibit the expression of VEGF in tumor tissue of gallbladder carcinoma. VEGF silencing can inhibit gallbladder carcinoma cell growth and angiogenesis. Objective:Making bioinformatics analysis with VEGF-A biological sequences to obtain the structure and function-related information.Methods:First of all, we draw phylogenetic trees of VEGF-A by Phylip and Treeview software; Then, analyze the amino acid composition, isoelectric point, the signal peptide cleavage point and the titration curve of VEGF-A by Anthprot bioinformatics analysis software and ExPASY website; We predict the secondary structure of VEGF-A by GOR4, HNN, SOPMA methods and analyze hydrophilicity, polarity, and return coefficient by ExPASY website; Finally, these results are compared and analyzed. We predict the tertiary and fourth structure of VEGF-A by STRING website.Results:①we can see from the phylogenetic tree that:zebra fish is the origin of the first branch, followed by dogs, horses, pigs and other branches;②VEGF is a positively charged, hydrophobic and stable proteins;③GOR4, HNN, SOPMA predict the secondary structures that the 55-60,65-70,98-108 regions may be VEGF-A function domain. Analysis of VEGF A hydrophilicity, polarity, and return coefficient can be found, the 6-12,55-65,132-138,150-157 regions may be VEGFA function of the regional domain. There are ten interacting proteins associated with VEGF:Flt-1, KDR, NRP-1, NRP-2, HIF1A, Immunoglobulin Kappa light chain C gene segment, SRC, NOS3, ARNT, VTN.Conclusion:VEGF is a positively charged, hydrophobic and stable proteins. The 55-60 regions may be VEGF-A functional domain。...