Construction Of PcDNA3.1-CD74-ROS1 Recombinant Plasmid And Effect Of CD74-ROS1 Expression On Proliferation And Migration Of Lung Cancer Cells

ObjectiveTo construct the pcDNA3.1-CD74-ROS1 recombinant plasmid and CD74-ROS1 stable expression cell lines.To investigate the effect of the overexpression of CD74-ROS1 fusion gene on the cell morphology,cell proliferation,migration and invasion in the non-small cell lung cancer(NSCLC)A549 cells.Cd74-Ros1 expression can be used as a molecular marker for early warning of the occurrence and prognosis of ROS1 positive non-small cell lung cancer,and can be used as a new biomarker and potential therapeutic target for gene therapy of non-small cell lung cancer.MethodsWith H2228 cDNA as the template,CD74 and ROS1 segments were obtained by PCR.The CD74-ROS1 fusion gene was constructed by fusion of CD74 and ROS1 through fusion PCR.We inserted the fusion gene into the eukaryotic expression vector pcDNA3.1 to construct the recombinant plasmid pcDNA3.1-CD74-ROS1 by molecular cloning technology,and transfected the pcDNA3.1-CD74-ROS1 recombinant plasmid into A549 cells by liposome transfection technology,and screened the transfected cells by G418 antibiotics.The m RNA or protein expression of CD74-ROS1 was detected by real-time PCR or Western blot.The difference of cell proliferation before and after transfection was observed by MTT method.The cell morphological was tested by crystal violet staining.Then we used western blot or immunofluorescence technique to detect the influence of CD74-ROS1 on the epithelial cell marker protein E-cadherin or mesenchymal cell marker protein N-cadherin or Vimentin.Wound healing and Transwell assay were used to detect the effect of the expression of CD74-ROS1 on lung cancer cell migration and invasion abilities.ResultsThe results of double enzyme digestion and DNA sequencing showed that pcDNA3.1-CD74-ROS1 recombinant plasmid was successfully constructed.Real-time PCR and western blot results showed a significant increase in the expression of CD74-ROS1 m RNA and protein in the positive cells Clone 4 and Clone 6.The overexpression of CD74-ROS1 enhanced the cell proliferation,inducing the cells changed from epithelial cells to mesenchymal cells.Western blot and immunofluorescence showed that the expression of epithelial marker protein E-cadherin is reduced,but the expression of mesenchymal cells marker protein N-cadherin or Vimentin was increased,indicating that CD74-ROS1 induced cells EMT.In addition,the results of wound healing and transwell showed that CD74-ROS1 overexpression enhanced the migration and invasion abilities of lung cancer cell.ConclusionspcDNA3.1-CD74-ROS1 recombinant plasmid is successfully constructed and expressed stably in A549 cells.CD74-ROS1 overexpression could promote cell proliferation,inducing EMT of lung cancer cells,and enhance the migration and invasion abilities of lung cancer cells.