Study On Silencing VEGF-C Gene With RNA Interference To Treat Bladder Cancer

Objective To observe the changes of proliferation and invasion ability in human bladder cancer cell lines T24 and ScaBER cells after down regulated VEGF-C gene with RNA interference; To observe the effect induced by the recombinant plasmid PCDNA3.1/VEGF-C, CRM-30-shVEGF-C on the expression of VEGF-C and the growth curve of tumor in nude mice model, acquire more information about the effect of VEGF-C on tumor growth in vivo and find the base for its future using in clinic and test the feasibility of make VEGF-C gene as a target gene to treat cancers; To screen the genes and possible signal transduction pathways with which VEGF-C interact and explore the mechanism of VEGF-C in bladder cancer.Methods 1. A new type of micro-based shRNA plasmid targeting VEGF-C gene CRM-30-shVEGF-C was constructed, which use Poly II type promoter CMV to promote. Recombinant plasmids CRM-30-shVEGF-C were transfected with Lipofectamine 2000 into human bladder cancer cell lines T24 and ScaBER and the transfection efficiency can be acquired by observing red fluorescence of cells. RT-PCT was used to determine if recombinant plasmids can downregulate the expression of VEGF-C mRNA. The silencing effect can also be detected by Western blot in protein levels. The change of cell morphology and VEGF-C protein expression level after transfection can also be detected by immunocytochemistry and light microscope. The cell survival and proliferation condition was determined by MTT study and cell clone. Cell cycle and apoptosis of the cells after transfection were detected with Flowcytometry (FCM). The difference of invasion and adhesion ability between the cells transfected or not were tested with transwell filter.2.①Balb-c nude mice, aged 4 weeks, were bred in the specific pathogen free (SPF) condition. CRM-30-shVEGF-C stable transfected T24 cells or untreated T24 cells were inoculated intradermally into the ventro-anterior limb of two group nude mice respectively (3.0×10~6 /mouse), then observe effect of VEGF-C RNAi on oncogenesis.②To build the bladder cancer nude mice model with the tumor from untreated T24 cells. After the diameter of xenograft tumor reach 5mm, 20 nude mice were divided into 4 groups and injected with 200μl ofPCDNA3.1/VEGF-C and lipofectamine 2000 mixture, DMEM, CRM-30-non and lipofectamine 2000 mixture, CRM-30-shVEGF-C and lipofectamine 2000 mixture respectively in tumor. The xenograft tumors' diameters of the four groups were measured every three days after injection, and so we can acquire growth curve of tumor. All the nude mice were sacrificed 7 days after treatment and every tumor was weighed and measured. RT-PCR and Western blot were used to determine if recombinant plasmids can regulate the expression of VEGF-C mRNA and protein in xenograft tumor. Immunohistochemistry was used to detect the expression of VEGF-C in the four groups.3. Oligonucleotide DNA microarray was used to screen the genome changes after knocking down expression of VEGF-C in T24 cells.Results 1. We successfully construction of three recombinant CRM-30-shVEGF-C by sequencing analysis and plasmids PCR. The recombinant CRM-30-shVEGF-C without endotoxin was extracted successfully and then was transfected into T24 and ScaBER cells respectively, the two cell lines both have a high transfection rate. Comparing with control, VEGF-C had significantly down regulate both in mRNA level and protein levels. MTT results showed that the cell survival rates of T24 and ScaBERcells had down regulated compare with blank. Cell cycle analysis showed that CRM-30-shVEGF-C induced accumulation of cells in G1 phase and a significant decrease in the percentage of cells in S-phase. in T24 and ScaBER cells relative to control. A significant apoptosis were also presented in T24 and ScaBER cells transfected with CRM-30-shVEGF-C. The invasion ability of the cells down regulated after transfacted.2.①VEGF-C RNAi can reduce the oncogenesis ability of T24 cells about 60%.②the nude mouse model of bladder cancer xenograft tumor was constructed successfully, the xenograft formation rate is 88%.③PCDNA3.1/VEGF-C plasmid can promote the growth of the tumor 9 days after treatment; the tumor volume of recombinant CRM-30-shVEGF-C group decreased significantly when comparing with DMEM group and negative control plasmid group 6 days after treatment. VEGF-C RNAi can delay or inhibit the tumors' hemorrhage and necrosis, dyscrasia of nude mice.④In CRM-30-shVEGF-C group, VEGF-C was down-regulated in mRNA level and in protein level comparing with DMEM control group, but PCDNA3.1/VEGF-C group can raise VEGF-C expression (22.05±2.57)% in mRNA level and (20.49±2.18)% in protein level comparing with DMEM control group.3. In two experiments, 1298 co-differentially expressed genes were found. Theses genes were involved in a lot of aspects, such as cell cycle, cell proliferation, signal transduction, cell apoptosis and cell differentiation, et al. After the down-regulate of VEGF-C, the Chemokines family genes (CXCL3,IL8,CCL20,CXCL6,CXCL1,CXCL5,CXCL2) were down-regulated and cytokine receptor CCR7 was up-regulated. In the family of adhesion molecules, CD44,ITGB1,ITGA2 were down-regulated, but ITGA4,ITGB3,ITGB4,ICAM2 were up-regulated. Gadd45a's down-regulated was also related VEGF-C.Conclusion 1. The sequence specific shRNA showed a block effect in down regulation of VEGF-C gene expression, inhibition of the cellular proliferation, arrestment of the cell cycle, VEGF-C was realated with tumor cells invasion and metastis. Silencing VEGF-C may be developed into a potential tool for bladder gene therapy.2. Down-regulate of VEGF-C can inhibit tumor's growth, hemorrhage and necrosis, and delay dyscrasia's appearance of the host. But high VEGF-C expression had the reverse effect. It provided a reliable foundation both in theory and experiment to make VEGF-C gene as a target for gene therapy.3. VEGF-C is an important gene to regulate cells' adhesion and invasion and its regulatory activity was certified at the gene level. After the silence of VEGF-C gene, many genes in the pathway of bladder had a significantly differential expression, and VEGF-C gene as a target for bladder cancer treatment was further certified. However, the present study still can not completely reveal the VEGF-C signal pathways and more studies are needed to further discover and confirm other mechanism.