Experimental Study On Ant-FAK Short Hairpin RNA And Its Effect To Sensitivity Of Chemotherapy Drugs For Colorectal Cancer

BackgroundThe incidence of CRC has been routed to the third place in the total incidenceof malignant tumors, and also is the mortality rate. However, in recent years, theyhave been increasing swiftly. The high mortality rate of CRC is not directlyassociated with the incidence of primary tumor, but it closely related with tumorrecurrence and metastasis. Tumor invasion and metastasis is a complex, multi-stepprocess, including breakthrough the extracellular matrix, infiltration of the bloodvessels or lymphatic vessels, accessing blood stream or lymphatic fluid, and finallyadhesion to target organs and proliferation. The researches shows that FAK as animportant intracellular signal molecule mediated cell signaling network system ofcross-linking, which is over expression in many invasive and metastatic tumor,including CRC and plays an important role in invasion and metastasis. It is apromising future that inhibition of FAK can control the invasion and diffusion ofcancer. It is reported that inhibition of FAK can reverse resistance to chemotherapydrug.ObjectivesTo construct recombinant vectors for RNA interference targeting FAK, and toinvestigate its influence on the proliferation, invasion and chemotherapy drugsensitization of CRC SW620cells.Methods1. For production of lentiviruses,293T cells were transfected, using pLL3.7FAK,lentivirus-containing media were harvested and applied to infect SW620cells.Infection efficiency were determined by the expression of GFP. To establish astable transfected cell line, we use the G418screening. Expression of FAK proteinwere detected by western blot. 2. Our purpose is to detect the ratio of cell cycle and apoptosis in all three groups,which are intact SW620,SW620-shCtrl and SW620-shFAK.And we intended toobserve the connection of inhibition of FAK expression and the arrest of cell cycleor the inhibition of proliferation.3. Further,we explored the role of the inhibition of FAK in tumor invasion andmetastasis.4. Three chemotherapy drugs are used to detect three groups colorectal cancerscells, which are intact SW620,sw620-shCtrl and sw620-shFAK.Sensitivity to thosechemotherapy drugs were determined respectively.Results1. Lentivirus was successfully constructed with an overall infecting rate of70%inSW620cell.All the transfected cells expressed GFP protein stably.2. FAK expression was detected by western blotting.The expression of FAK issignificantly decreased in SW620-shFAK group compared to the other groups.3. Cell cycle experiment demonstrated that inhibition of FAK correlated to the cellcycle arrest, and most of SW620-shFAK cells were blocked in G2/M phase,and aisocorrelated to the decreased ability of migration and invasion.4Also, a significant higher sensitivity to chemotherapy drugs is observed inSW620-shFAK.Conclusions1. A FAK-silencing SW620cells line was established.2. Inhibition of FAK in colorectal cancer cells could inhibits cell proliferationactivity, and and decrease ability of migration and migration and invasion inSW620-shFAK.3. Inhibition of FAK in colorectal cancer cells increase the sensitivity tochemotherapy drugs.