Overexpression Of Twist Genes Inducing Lung Adenocarcinoma NCI-H1299Cells EMT And The Impact On Proliferation, Migration And Invasion

Object:Epithelial-mesenchymal transitions (EMT) refers to epithelial cells losing polarity and cell tight junctions and adhesion in some situations, acquiring invasion and migration, changing into cells with mesenchymal cell morphology and characteristics. The loss of the epithelial phenotype and mesenchymal characteristics obtained is the main features of EMT. This transition of the phenotype of the tumor cells causes lack of intercellular adhesion, and more invasion and metastasis. Twist is an inhibitor of apoptosis protein. Upregulation of Twist may contribute to the malignant transformation of the cell phenotype, which may be the key of the tumorigenesis and development of malignant tumors. Twist was originally considered as a key transcription factor in embryonic development, which is widely involved in the regulation of organ development and tumorigenesis, cell proliferation and differentiation process. Twist could directly induce EMT, and indirectly affect other EMT activators of transcription. In this study, we overexpressed Twist gene to induce human lung cancer cell line NCI-H1299to EMT, and then observed the impact of tumor cell proliferation, migration and invasion.Methods:We use the adenosis venom carrying the Twistl gene and negative to infect human lung cancer NCI-H1299cells as the infected group, the control group and blank control group, then observe the morphological changes; qPCR detected five target genes:Twistl, E-cadherin, N-cadherin, Vimentin and Fibronectin expression; WesternBlot detected five proteins: Twistl, E-cadherin, N-cadherin, vimentin and Fibronectin expression; then draw CCK-8experiment cell growth curve, scratch test to observe cell migration, Transwell experiments to detect the invasion.Results:1. The adenosis venom carrying Twistl gene successfully infected NCI-H1299cells. 2. In qPCR detection, the expressions of Twistl, N-cadherin, vimentin and Fibronectin in infection group are higher than control group, while E-cadherin expression had no significant difference between the two groups.3. In WesternBlot detection, the levels of Twistl, N-cadherin, vimentin and Fibronectin in infection group are higher than control group, and E-cadherin level is lower in infection group.4. CCK-8experiment to draw cell growth curve, the proliferation of the infected group is slower than the control group.5. In scratch test, the number of cell migration in infected group is higher than the control group.6. In transwell test, the migration ODs is higher in infection group than in the control group.Conclusion:1. Overexpression of Twist gene could induce NCI-H1299cells to EMT.2. Overexpression of Twist gene could decrease the level E-cadherin of the NCI-H1299cell but increase the levels of mesenchymal genes and proteins.3. Overexpression of Twist gene could inhibit the proliferation of NCI-H1299cells.4. Overexpression of Twist gene could enhance the NCI-H1299cell migration ability.4. Overexpression of Twist gene could enhance the NCI-H1299cell invasion ability.