RNAi MMP-9 Of Mouse Melanoma B16 Cell Lines For Targeted Gene Therapy

BackgroundThe main characteristics of tumor cells are strongly vitality, infinite proliferation, non-contact inhibitory, local invasion and distant migration that are very different from normal cells. Malignant tumor with the invasion characteristic could destroy the distant tissues and cause death. Conventional cancer treatment strategies control the growth and proliferation of tumor cells which can only play a certain role, however, the inhibition of migration and invasion is very limited. A variety of metastasis-related factors resulted in tumor metastasis. Early event of malignant tumor metastasis is degradation of extracellular matrix (ECM). Matrix metalloproteinase (MMPs) could hydrolyze different types of collagen, fibronectin and laminin. MMPs are very important in tumor invasion and metastasis by degradation of ECM. MMP-9 is a principal member of MMP family, also known as gelatinase B. MMP-9 hydrolyzes collagen IV in basement membrane and stimulates tumor cell growth, migration, invasion and metastasis.There are also studies show that MMP-9 will regulate tumor angiogenesis by regulating vascular endothelial growth factor (VEGF).Malignant melanoma (MM) is one of the most immunogenic tumor, it has been the first choice for research of tumor immune therapy and gene therapy. The first case of malignant tumor gene therapy is about advanced melanoma, Rosenberg Group did a experiment, the gene of tumor necrosis factor (TNF) was transferred into advanced melanoma by retroviral vector for gene therapy. RNA interference is post-transcriptional gene silencing which is triggered by efficient double-stranded RNA. It provide a quick and convenient approach to inhibit translation from mRNAs to proteins. RNAi technology has been used in cancer gene therapy with its maturity. Targeted silencing of murine melanoma cells MMP-9 gene is feasible for inhibiting tumor cells proliferation and metastasis.Objectives1.Screening interference sequence of MMP-9 for B16 cells;2.Exploring the invasion and metastasis of B16 melanoma cells in vitro after MMP-9 down regulation by RNAi;3.Exploring the invasion and metastasis of B16 melanoma cells in vivo after MMP-9 down regulation by RNAi.Methods1.Looking for targeted interference sites of mouse MMP-9. Designing shRNA interference sequences for MMP-9. Inserting different MMP-9 RNA interference sequences between the U6 Promoter and CMV Promoter of pGensil-1 plasmid structure. Constructing interference vectors. The vectors were transfected into B16 cells by Cationic complex transfection reagents. Transfection efficiency was measured by flow cytometry. Cell form and fluorescence expression were observed by Laser confocal microscopy. Using real-time quantitative PCR to detect effects of RNAi. Choosing best one for RNAi;2.Using the optimum to complete RNAi MMP-9 of mouse melanoma in vitro. Western blotting tested the change of MMP-9 protein after RNAi. Transwell experiment detected the change of cell invasion and migration;3. Constructing mouse claw pad subcutaneous transplant invade model. Injecting RNA interference complex to in-situ solid tumor. Evaluating treatment effect.Results1. Constructing three vectors. Mmp-9-RNAi-1#,2# and 3# respectively. On the basis of carrying efficiency assurance, quantitative PCR detection showed that 2# vector down regulate MMP-9 during entire experiment process.Inhibition efficiency was 32% at 24 hour and 63% at 48 hour.2# vector is the optimum among three vectors;2. After MMP-9 RNAi, we did not detect significant difference between silent group and control group at MMP-9 protein level, but the invasion and migration of interference group showed significantly decrease;3. Injecting MMP-9 interference complex to mouse foot pad model in situ tumor, the metastasis of tumor cells was restrained after treatment.ConclusionExperiments show that 2# vector is the most effective one of the three. It can down regulate MMP-9 of mouse melanoma cells powerfully and inhibit cells migration, invasion; it plays a significantly inhibition on tumor growth in vivo.