The Function Of Copine-3 Protein In Lung Adenocarcinoma Cells

Copine-3,a calcium-dependent protein is encoded by the CPNE3 gene,and is one of the members of the copines protein family.The members of this protein family are highly conserved in evolution and functional,but the mechanisms of these proteins are not yet clear.Studies have shown that copine-3 protein has a correlation with the occurrence and development of diseases such as tumors,which may play an important role in biological functions,especially in tumor cells.In this study,we found that copine-3 protein was widely expressed in the lung adenocarcinoma cell lines and the clinical samples of the lung adenocarcinoma patients by western-blot and immunohistochemistry(IHC),and IHC showed that the copine-3 protein was mainly distributed in the cytoplasm and also had the localization in the nucleus.The pEGFP-N1/CPNE3 eukaryotic expression vectors were transfected into 293T and H1299 cells respectively.The detection of subcomponents of the cells by western-blot and the confocal laser confocal observation showed that the copine-3 protein was located in cytoplasm and nucleus.Then,the CPNE3 gene was interfered by transfected the small interference RNA(Small interfering RNA;siRNA)in A549 cells,and the transwell experiment showed that the cell migration ability was significantly enhanced(P<0.01).51 differential proteins were identified in A549 cells after the siRNA interfered CPNE3 gene by using differential proteomics technology based on stable isotope labeling(stable isotope labeling by amino acids in cell culture,SILAC)based on cell culture.Among them,the expression of SQSTM1/p62(encoded by SQSTM1 gene)was significantly down-regulated after the CPNE3 gene was silenced.And the SQSTM1 gene was interfered thought siRNA in A549 cells could enhanced migration of cell,and western-blot detection showed that the expression of E-cadherin was down-regulated,and vimentin was up-regulated.This suggests that after the CPNE3 gene was silenced,the expression of SQSTM1/p62 downregulated,and promoting cell migration by promoting the occurrence of Epithelial-mesenchymal transition(EMT).At the same time,in order to explore the function of copine-3,we successfully silenced the expression of CPNE3 gene in lung adenocarcinoma cell lines A549,H1299 and SPC-A1 using lentivirus-mediated shRNA.Panel cloning and soft agar cloning experiments showed that the stable silencing of CPNE3 gene could promote the proliferation of lung adenocarcinoma cells.In addition,we have successfully constructed an overexpression cell line of CPNE3 gene in human bronchial epithelial cells(HBE),the results showed that the overexpression of CPNE3 gene could inhibit the proliferation of HBE cells.Using differential proteomic techniques based on SILAC,we identified differentially expressed proteins that stably silence the CPNE3 gene in A549,and obtained 59 differentially expressed total proteins and 209 differential nuclear protein.Finally,the bioinformatics analysis was performed on the differential proteomic data of the siRNA interference and the stably silencing of the CPNE3 gene in A549 cells by SILAC technology.Gene Ontology analysis and KEGG Pathway enrichment showed that these differential proteins participate in multiple biological processes and pathways.At the same time,the analysis of the interaction network of differential proteins also has been completed,which provides clues for the subsequent experimental research of signal transduction pathway.It also provides a preliminary basis for further exploring the biological function of copine-3 protein and clarifying the mechanism of copine-3 protein to regulate the migration and proliferation of tumor cells.