Screening Of Vero Cell Adhesion Related Molecules And Their Functions Based On Transcriptomics And Proteomics

Cell-cell and cell-extracellular matrix adhesion depend on cell-specific adhesion proteins and their related regulatory signals.Vero cells are excellent cell lines for the reproduction,purification and vaccine production of influenza virus,novel coronavirus and other viruses.When cultured in vitro,they usually grow in a two-dimensional single cell layer in the medium containing serum.The biomedical research center of Northwestern University for nationalities screened the Vero cell line by serum decline method,but the cell density is low,so it is difficult to achieve large-scale high-density suspension culture.Gene modification and serum-free medium cell screening techniques have been successfully applied to the construction of suspension cell lines.In order to use genetic engineering to construct suspension Vero cells,it is necessary to screen proteins related to their adhesion function and understand how these proteins affect cell adhesion.In this study,transcriptome and tandem mass spectrometry(tandemmasstag,TMT)quantitative proteomics techniques were used to analyze the type and number of differentially expressed genes and proteins related to cell adhesion in adherent cultured Vero cells(Adh＿Vero)and suspension cultured Vero cells(Sus＿Vero),and to identify the candidate target genes related to cell adhesion.The corresponding Vero cell engineering cell lines were obtained by gene knockout technique CRISPR/Cas9(CRISPR-Cas RNA-gudidenuclease)and lentivirus transduction technique respectively.The main research results are as follows:1.A total of 3617 differentially expressed genes(DGEs)were identified in suspension and adherent Vero cells by transcriptome analysis,of which 2100 were up-regulated and 1517 down-regulated in suspension cells.The results of GO(Gene Ontology)and KEGG(Kyoto Encyclopediaof Genesand Genomes)enrichment analysis showed that DEGs could be effectively enriched in extracellular matrix,adhesion plaque,tight junction and other different signal pathways.Fifteen DEGs related to cell adhesion were further screened: polyligand proteoglycan-4(SDC4),actin regulator(ENAH),myosin light chain 12 β(MYL12β),myosin light chain kinase(MYLK),sealing protein(OCLN),IV collagen gene α 1(COL4α1),myosin heavy chain 9(MYH9),epidermal growth factor receptor(EGFR),GTP enzyme activating protein 2(IQGAP2)containing IQ structure.Cell division factor 4(DOCK4),integrin β 2 protein(ITGβ2),transforming growth factor β receptor 1(TGFβR1),nerve cell adhesion molecule 1(NCAM1),integrin associated protein(CD47)and gelatin(GSN).QRT-PCR analysis showed that the m RNA expression levels of SDC4,ENAH,MYL12β,MYLK,OCLN,Col4α1,MYH9 and EGFR 8 were significantly down-regulated in suspended Vero,while the m RNA expression levels of GSN,DOCK4 and ITG β genes were significantly up-regulated,which was consistent with the results of transcriptome analysis.2.Proteomic analysis showed that there were 190 differentially expressed proteins in suspension Vero cells,of which 116 were up-regulated and 74 were down-regulated.Protein functional cluster analysis showed that these proteins were mainly involved in molecular metabolism,distributed on the cell surface and actin cytoskeleton,and involved in actin binding,cell morphological regulation,adhesion and other functions.A total of 15 proteins related to cell adhesion function were screened according to adhesion function,expression difference and subcell population localization.Western blot assay showed that the expression of ENAH,TGFβR1 and SDC4 in suspended Vero cells was significantly down-regulated,while the expression of OCLN,DOCK4 and MYLK was significantly up-regulated.3.Knockout SDC4 cell lines were constructed by CRISPR/Cas9 gene editing technique.A stable cell line named Vero-SDC4-KD was obtained by CRISPR/Cas9 technique against the key factor SDC4 in Adh＿Vero cells.q RT-PCR,Western blot and immunofluorescence techniques were used to verify the knockout efficiency of SDC4 factor.The results of cell proliferation,intercellular adhesion and microscopic examination showed that SDC4 knockout effectively inhibited the adhesion effect of Vero cells.4.The stable overexpression of ITGβ2 cell line Vero-ITGβ2,q RT-PCR,Western blot and immunofluorescence assay showed that the overexpression of ITGβ2 factor,cell proliferation,intercellular adhesion function and microscopic examination showed that overexpression of ITGβ2 effectively inhibited the adhesion effect of Vero cells.