The Role Of Tie2 Expression In The Proliferation、migration And The EMT Process Of Oral Squamous Cell Carcinoma

Objective:To investigate the role of targeted silencing of the Tie2 gene in the proliferation,migration and EMT of oral squamous carcinoma cell lines using RNA interference technology.Method:1.WB was used to detect Tie2 expression in DOK cells and different oral squamous carcinoma cell lines(HSC-4,SCC-9,Cal-27,HSC-3),and the squamous carcinoma cell lines with high Tie2 expression(HSC-4,SCC-9)were selected as the experimental cell lines.2.ShRNA recombinant lentiviral vector with Tie2 gene silencing was transfected into HSC-4 and SCC-9 cells.WB was performed to detect the knockdown efficiency of Tie2 after successful transfection,and stable transfected cell lines with good silencing effect were screened.CCK-8 assay was used to detect the ability of cell proliferation;Clone formation assay was used to detect the ability of cell clone formation;Single cell clone culture was used to detect cell clone morphology;The ability of cell migration was examined by cell scratch assay and Transwell assay.The ability of cytoskeletal F-actin remodelling was examined by laser confocal microscope.3.WB was used to detect the expression of EMT-related signature proteins such as E-cadherin,N-cadherin and Vimentin;Laser confocal microscope was used to detect the expression of E-cadherin and N-cadherin.Results:1.Tie2 was expressed in all four OSCC cell lines,with the highest expression of Tie2 in HSC-4,followed by SCC-9,compared to DOK cells(P<0.0001).Therefore,HSC-4 and SCC-9 were selected as the experimental cell lines.2.The recombinant lentiviral vector(Tie2-ShRNA-161,Tie2-ShRNA-162,Tie2-ShRNA-163)and control sequences(Tie2-ShRNA-429)were transfected into HSC-4 and SCC-9 cells;The results of WB showed that the expression of Tie2 in the ShRNA-162 group was the lowest and significantly lower than the ShRNA-429 control group(P<0.0001),Therefore,the ShRNA-162 group was used as the experimental group and applied to subsequent experiments.3.The results of CCK-8 experiment showed that the proliferation ability of HSC-4 and SCC-9 cells in the experimental group was significantly reduced compared to the control group(P<0.05).4.The results of the clone formation experiments showed that the clonogenic ability of HSC-4 and SCC-9 cells was significantly lower in the experimental group compared to the control group(P<0.0001).5.The results of the single cell clone culture experiment showed that three different forms of clone formation were seen in HSC-4 and SCC-9 cells after 2 weeks of culture:holoclone,meroclone and paraclone;Compared to the control group,the clone formation rate of holoclone was significantly lower and that of paraclone was significantly higher in the experimental group(P<0.05,P<0.001).6.The results of the scratch assay and Transwell migration assay showed that in the HSC-4 and SCC-9 cells,the scratch healing rate and the number of cells crossing the Transwell were significantly lower in the experimental group than in the control group(P<0.0001,P<0.05).7.The results of laser confocal microscope showed that in the HSC-4 and SCC-9 cells,the green fluorescence intensity of cytoskeletal F-actin was reduced,the cell surface was smooth and the number and length of filamentous pseudopods at the leading edge of the cells were reduced in the experimental group compared to the control group(P<0.01).8.The results of WB showed that in HSC-4 and SCC-9 cells,the expression of EMT-related epithelial marker such as E-cadherin was significantly increased and the expression of mesenchymal marker such as N-cadherin and Vimentin was significantly decreased(P<0.0001)in the experimental group compared to the control group.9.The results of laser confocal microscope showed that in HSC-4 and SCC-9 cells,the expression of E-cadherin was significantly increased and the expression of N-cadherin was significantly decreased in the experimental group compared with the control group(P<0.0001).Conclusion:1.Silencing the expression of Tie2 can inhibit the proliferation、clone and migration ability of HSC-4 and SCC-9 cells.2.Silencing the expression of Tie2 significantly can inhibit the remodelling of the cytoskeletal F-actin in HSC-4 and SCC-9 cells,suggesting that the expression of Tie2 may play an important role in the remodelling of the cytoskeletal F-actin in OSCC cells.3.Silencing the expression of Tie2 could alter the expression of EMT-related signature proteins in HSC-4 and SCC-9 cells,suggesting that Tie2 may be involved in the epithelial mesenchymal transition process in OSCC.