Study Of The Effect Of Cdc42 On Proliferation,invasion And Migration Of Human Tongue Squamous Cell Carcinoma Cells And Involved Mechanism

Background and purpose: Tongue squamous cell carcinoma(TSCC)is a common malignant tumor in oral and maxillofacial region,with an incidence of about 40%.Owing to the special anatomical structure and physiological characteristics of the tongue,TSCC is prone to lymph node and distant metastasis,and the local recurrence rate is high,which leads to poor prognosis.Thus,it is of great significance for the diagnosis,treatment and prognosis of TSCC to make a thorough inquiry the relevant potential mechanisms that affect the proliferation,invasion and migration of TSCC and to find potential molecular target spot.The cell division cycle 42(Cdc42)is one of the core member of small G protein Rho subfamily.As a switch molecule of cell signal conversion,it is involved in the control of many cell physiological processes such as cell multiplication,apoptosis,invasion and migration,Similarly,it is of considerable significance in the progress of tumor.In this study,we intend to probe into the effects of silencing Cdc42 gene on the migration,invasion,proliferation and apoptosis of human tongue squamous cell carcinoma cell lines CAL-27 and SCC-4 through in vitro experiments,and provide experimental and theoretical basis for gene targeted therapy of tongue cancer against Cdc42.Research methods: 1.Human tongue squamous cell carcinoma CAL-27 and SCC-4 cells were assigned into three groups randomly and named respectively as follow Cdc42-si RNA group,negative and blank control group.Then,we devised and established three si RNA targeting human Cdc42 gene sequence,which were named Cdc42-si RNA-1,Cdc42-si RNA-2 and Cdc42-si RNA-3 respectively.Transient transfection of liposome was used to transfect Cdc42-si RNA in Cdc42-si RNA group,and transfect NC-si RNA in the negative control group,while only the same dose of transfection reagent was added in blank control group.Using q RT-PCR and Western blot,the quantity changes of Cdc42 m RNA and protein in Cdc42-si RNA group and two control groups were measured respectively,and the group with the highest silencing efficiency was screened out in Cdc42-si RNA transfection group for subsequent experiments.2.The western blot experiment was used to check the expression of Cdc42,EMT-related protein Vimentin,mesenchymal marker N-cadherin,epithelial marker E-cadherin,matrix metalloproteinase-9,MAPK JNK/p38 pathway related proteins p38-MAPK,JNK,p-p38-MAPK,p-JNK,cell cycle related G1/S specific cyclin-D1(Cyclin D1),p21.3.The invasion and migration ability of cells were evaluated by Transwell invasion test and cell scratch test by comparing the number of transmembrane cells and the healing area of scratches.Application of CCK8 method analyzed the multiplication capacity of CAL-27 and SCC-4 cells by detecting OD value.Finally,using flow cytometry checked the apoptosis ability and cell cycle changes of cells.Results: 1.The expression levels of Cdc42 m RNA and protein in Cdc42-si RNA transfection group were remarkable lower than those in the two control groups as displayed in the assay data of q RT-PCR and Western blot(P < 0.05).The silence efficiency of Cdc42-si RNA-2 group was the highest,so Cdc42-si RNA-2 group was used as the follow-up experimental group.2.Cell scratch test results showed that as compared to the two control groups,the relative healing area of cell scratches in Cdc42-si RNA-2 group was obviously decreased(P < 0.05),and the amount of cells passing through the membrane was also decreased in Transwell experiment(P < 0.05),indicating that the migration and invasion ability of cells were repressed.3.Experimental results of CCK8 demonstrated that compared with the two control groups,the multiplication ability of Cdc42-si RNA-2 group was remarkable weaker(P < 0.05).The assay data of flow cytometry indicated that the apoptosis capacity of Cdc42-si RNA-2 group was accelerated(P < 0.05)and the G1/S phase of cell cycle was blocked(P < 0.05).4.The results of Western blot demonstrated that after silencing Cdc42,the expression of EMT-related protein epithelial marker E-cadherin raised markedly(P < 0.05),but the expression of mesenchymal marker N-cadherin,Vimentin and cell invasion-related protein MMP-9 reduced(P < 0.05)in Cdc42-si RNA-2 group compared with the the control groups.The proteins of JNK and p38-MAPK did not change significantly(P > 0.05),but p-JNK and p-p38-MAPK decreased significantly(P < 0.05).Cyclin D1 expression was decreased(P < 0.05),however p21 was increased(P < 0.05).Conclusion: The expression of Cdc42 in CAL-27 and SCC-4 cells of tongue squamous cell carcinoma can be effectively reduced by silencing Cdc42,in addition,the invasion,migration and proliferation of CAL-27 and SCC-4 cells were negatively regulated,the cell cycle can be regulated and apoptosis can be accelerated.It may inhibit the epithelial-mesenchymal transition(EMT)and the invasion of TSCC by blocking MAPK JNK/p38 pathway,and negatively regulate the proliferation ability of TSCC cells by impeding cell cycle in G1 / S phase through regulating the expression of Cyclin D1 and p21.