In Vivo Experimental Study On Ant-fak Short Hairpin RNA For Metastasis Of Colorectal Cancer

Background and ObjectivesColorectal cancer (CRC) remains one of the most common cancer, and its incidence appears to be rising. At present, surgery is still the main treatment of colorectal cancer, supplemented by radiotherapy and chemotherapy combined treatment in the same time, But for the people who has been in the later period and who has been tranfered, The clinical outcomes was poor. The death of patients with colorectal cancer is mainly caused by tumor metastasis. Tumor metastasis is a complex process. The tumor cell invade and break through the extracellular matrix is the basic steps, then enter blood, Reach distant organs through the blood,break blood vessels into the target organ for settlement and proliferation.Studies shown that focal adhesion kinase as an important intracellular signaling molecule, mediating intracellular signaling network of cross-linking, who play a key role in cells' movement and invasion. The expression of FAK in primary cancer and liver metastases more than in normal colorectal mucosa. Therefore, Interventing the expression of FAK could provide a new way to treat the colorectal cancer. RNA interference(RNAi) has a strong role in inhibition of the targeted gene, which is more than Antisense oligonucleotides.RNAi is considered as a landmark after the PCR method of genetic engineering.We have proofed in vitro that, the silence of FAK inhibits the growth and metastasis of human colon cancer cell line sw620。For the further study in vivo, we are about to transplant the colon cancer cells into the cecum of nude mouse, built orthotopic transplantation mouse model, for the advantage of resemble the human metastatic disease most closely, providing a model for study of the treatment of metastases. Methods1. For production of lentiviruses,293FT cells were tansfected by Turbo transfect,using pLL3.7 FAK, lentivirus-containing media were harvested and used to infect SW620 cells. Infection efficiency were obtained by the expression of GFP, and maintained as a pool population. Expression of FAK were detected by RT-PCR and Western blot.2. Interfere SW620 cells, untreated group and negative plasmid control group cells were injected into the cecum of nude mouse and the subcutaneous to built nude mouse xenograft model.3. To determine the different between FAK RNAi group, untreated group and negative plasmid control group cells in the sizes of the tumor growth and metastasis.Results1. Lentiviruses were successfully producted with over 90% infecting rate; cells stable expressing GFP. Expression of FAK were detected significantly reduced by RT-PCR and Western blot.2. The orthotopic transplantation and subcutaneous transplantation models were successfully built and providing a model for future study of the nude mouse xenograft.3. The suppression of FAK expression did not affect the growth subcutaneously, however silencing of FAK resulted in the inhibition of tumor metastasis and strongly support the therapeutic targeting of FAK in the treatment of cancer.Conclusions1. FAK stable silencing SW620 cells were established.2. The orthotopic transplantation and subcutaneous transplantation models were successfully built.3. Silencing of FAK resulted in the inhibition of tumor metastasis.