Silencing ILK Expression Inhibits Metastasis In Human Tongue Cancer Cells Through Repression Of Epithelial-to-mesenchymal Transition

Purpose: Using RNAi technology specifically silence integrin adapterkinase gene expression, to observe and detect the effects on human tonguesquamous cell morphology, growth, apoptosis, invasion and metastasis invivo and in vitro, and explore the molecular mechanisms regulating geneexpression EMT signaling pathway.Methods: The specific ILK siRNA expression vectors and non-homologous control vector, were stably transfected through liposomemediation into human TCA8113/Tb tongue squamous cells, and classifiedinto TCA8113group, TCA8113vector group, TCA8113siILK group, Tbgroup, Tb vector group, Tb siILK group. After G418selection, stableexpression of ILK siRNA cells were aquired. Using western blot andimmunofluorescence to detect the expression of ILK. Xenograft modelswere built. Using MTT assay tests cells proliferation. Using scratch test todetect movement and migration of cells. Using transwell assay test invasionability. Using flow cytometry to analyze the cell cycle and apoptosis. Using western blot and immunofluorescence analysis examine the expression ofILK downstream substrate molecule Akt, GSK3β, p-Akt, p-GSK3β. Usingwestern blot detection MMP2, MMP9, N-cadherin, E-cadherin, β-catenin,Snail, Slug, Vimentin and Twist. Observing tumor tissue slices formicrovessel density changes. Observing spontaneous animal model of lungtissue sections. Detecting ILK, MMP2MMP9N-cadherin E-cadherinβ-catenin Snail Slug, Vimentin and Twist protein expression byimmunohistochemistry in tumor tissues. Detecting ILK, E-cadherinβ-catenin, Snail, Slug, Vimentin and Twist protein expression of humantongue squamous cell carcinoma clinical specimens.Results: After G418selection to obtain stable cell lines, expression ofILK was confirmed silence by Western Blot and immunofluorescence.Morphology observation found that TCA8113siILK/Tb siILK cellscompared TCA8113/Tb group and TCA8113vector/Tb vector themorphological characteristics of epithelial cells is more obvious, the nucleusis small, split-phase reduction basophilic cytoplasm is weak, indicating thata low degree of malignancy. MTT results suggested growth capacity ofTCA8113siILK/Tb siILK significantly reduced. Scratch test resultsshowed that migration of TCA8113siILK/Tb siILK group weresignificantly lower than TCA8113/Tb group and TCA8113vector/Tbvector cells. Transwell experiments showed TCA8113siILK/Tb siILK cellssignificantly decreased cell invasiveness. Flow cytometry analysis revealed TCA8113siILK/Tb siILK cells arrested in G2-M phase and G0-G1phase,but no obvious apoptosis changes observed. Immunofluorescence found thatTCA8113siILK/Tb siILK cells significantly reduced the expression ofp-Akt and p-GSK3β, but there were no significant change in Akt andGSK3β expression. Western Blot showed a significant reduction of MMP2,MMP9, N-cadherin, β-catenin, Snail, Slug, Vimentin and Twist expressionin TCA8113siILK/Tb siILK group (P <0.05), while E-cadherin expressionsignificantly increased (P <0.05). Animal xenograft model found that micein the experimental group were significantly reduced tumor weightcompared with the control group (P <0.05). Tumor tissue vascular densitywas significantly decreased, while the ILK, MMP2, MMP9, N-cadherin,β-catenin, Snail, Slug, Vimentin and Twist protein expression in tumor tissuewere weakly positive or negative expression, E-cadherin showed strongexpression. No spontaneous lung metastases were found in experimentalnude mice group, while spontaneous lung metastases were found in thecontrol groups. Clinical specimens of human tongue squamous cellimmunohistochemical findings were consistent with the discovery of animalexperiments.Conclusions: Successfully established ILK siRNA plasmid expressioncells and stable expression were selected by G418. Silencing ILK inhibits itsdownstream substrate molecule Akt and GSK3β phosphorylation and inhibitMMP2and MMP9and EMT-related proteins such as N-cadherin, β-catenin, Snail, Slug, Vimentin and Twist, etc., inhibits human tongue squamous cellcarcinoma TCA8113/Tb cell migration and invasion and metastasis, and thecells arrested in G2-M phase and G0-G1phase, thereby inhibiting cellgrowth. Animal experiments confirmed silence ILK gene can effectivelysuppress the human tongue squamous TCA8113/Tb cell growth and themetastasis ability, which may be related to silencing ILK protein expressioninhibit EMT and through ILK/Akt/GSK3β/Snail pathway. ILK can be acandidate as a human tongue squamous cell carcinoma target protein.