Experimental Study On Anti-FAK Short Hairpin RNA For Invasion And Metastasis Of Thyroid Cancer

Background and ObjectivesThyroid cancer accounting for1%of all solid tumors, is the most commonendocrine system of malignant tumor, which is also the fastest rising incidence inmalignant tumors. Some of these refractory thyroid cancer have poorer clinicalcurative effect because of the tumor invasion and metastasis. tumor invasion andmetastasis is a complex process,including tumor cells epithelial-mesenchymaltransition (EMT), formation of local diffusion from the primary tumor,invasion andbreakthrough the extracellular matrix, then entering into the vascular or lymphatic toreach distant organs or tissues,breakthrough of vascular or lymphaticinto the targetorgans or tissues for settlement and proliferation.Focal adhesion kinase(FAK)has been confirmed by studies that it is a kind ofimportant intracellular signaling molecules, mediating intracellular signaling networksystem of cross-linking, and plays a key role in cells’ proliferation,migration andinvasion. Moreover, researches have confirmed that the FAK expression level ispositively correlated with the following aspects: thyroid cancer differentiation degree,infiltration depth, lymph node metastasis and the clinical stages. Therefore,intervening the expression of FAK will likely be signaling pathways that controlmetastasis of thyroid cancer and diffusion.RNA interference(RNAi) can eliminate specific genes or close its expression, iswidely used in the exploration of gene function and malignant tumors of the genetherapy field.A study, constructing the lentivirus vector (lentiviralpLentilox3.7-shRNA FAK)to infect people metastatic colorectal cancer cell lines SW620established andconducted by our team,shows that the FAK has great genetically modified effect,stable expression, and effectively suppresses tumor(sponsored by Xiamen Technology Bureau,No.:3502Z20074007).Based on the research mentioned above, we willconstruct the lentiviral expression vector of shRNA FAK to infect people’s metastaticthyroid cancer cell lines and use GFP reporter genes to detect the transfectionefficiency,and gene expression. Through the vivo and vitro experiment, we would liketo test the expression and metastasis of target proteins in tumor tissue and aim at theapplication value of the lentiviral expression vector of shRNA FAK in the thyroidtreatment.Methods1. For production of lentiviruses pLL3.7FAK shRNA,293FT cells are tansfected byTurbo transfect. By using pLL3.7FAK shRNA, lentivirus-containing media areharvested and used to infect SW579cells. Infection efficiency is detected by theexpression of green fluorescent protein,and G418screens cell to establishing stabletrasfected cell lines.Expression of FAK is obtained by RT-PCR and Western blot.2. In the interfered group, natural group and negative plasmid control groupcells,expression of E-cad,MMP-2and MMP-9are detected by Western blot.3. Interfered group, natural group and negative plasmid control group cells areinjected into the right axilla of nude mouse to constructed tumor xenograft model.Then the differences among FAK RNAi group, untreated group and negative plasmidcontrol group will be studied the difference of the tumor formation rate and growth.Immunohistochemistry is used to detect the expression of FAK,E-cad,MMP-2andMMP-9.Results1. Targeting FAK shRNA Lentiviruses are successfully produced with over80%infection rate, and stable transfection of human thyroid carcinoma cell line SW579areobtained. Expression of FAK is detected obviously decreased by RT-PCR and Westernblot.2. Expression of E-cad was detected significantly rose by Western blot, but theexpression of MMP-2and MMP-9were significantly reduced.3. The subcutaneous transplantation nude mouse models of thyroid cancer SW579are successfully built.The FAK silencing gene meditated by shRNA significantly inhibitsthe growth of SW579in nude mice.But there are no significant diffrerence of the rateof tumor.The result of immunohistochemistry confirms that subcutaneous tumors areinduced by the transplanted thyroid cancer cells. Expression of E-cad was detectedsignificantly rose by immunohistochemistry, but the expression of MMP-2andMMP-9were significantly reduced.Conclusions1. FAK stable silencing SW579cells have been successfully obtained.2. In vitro Experimental Study, FAK interference can regulate and control expressionof invasion and metastasis related proteins to inhibit tumor phenotype.3. The subcutaneous transplantation mouse xenograft models of thyroid cancer havebeen successfully built. FAK silencing gene meditated by shRNA significantlyinhibits the growth of SW579in nude mice.