The Mechanism Of PKP1 Promoting Proliferation,invasion And Migration Of Nasopharyngeal Carcinoma Via Activating ERK1/2 Pathway

Objectives:(1)To explore the significantly differentially expressed genes(DEGs)in nasopharyngeal carcinoma(NPC)and their biological functions and roles in immune infiltration.(2)To investigate the role and mechanism of PKP1(Plakophilin 1)in the proliferation,invasion and migration of NPC,and to provide a theoretical basis for the development of NPC.Methods:(1)The DEGs from bulk RNA sequencing data of NPC from Gene Expression Omnibus(GEO)database were analyzed and annotated.Then,the selected differential genes were performed GO(Gene Ontology)enrichment and KEGG(Encyclopedia of Genes and Genomes)pathway analysis.The Metascape network server was used to construct protein-protein interaction network.Candidate genes were selected and performed clinical prognosis analysis using GSE102349 dataset.Singlecell RNA sequencing data were used to analyze the expression and location of the candidate genes.The Immune Cell Abundance Identifier tool was used to analyze immune cell infiltration.The correlation with core genes was analyzed as well.In addition,the effect of the core genes and immune cells on the prognosis of NPC was evaluated.(2)Real-Time quantitative Polymerase Chain Reaction(RT-q PCR)and Western Blot(WB)were performed to identify the expression of PKP1 in several NPC cell lines.Immunohistochemistry was performed to detect the expression and localization of PKP1 in NPC and normal tissues.Immunofluorescence was used to determine the location of PKP1 in NPC cells.PKP1-interferring lentiviral infection of HONE1 cells generated interfered stable cell line.PKP1-overexpressing lentiviral infection of S18 cells generated overexpressed stable cell line.The effect of PKP1 on cell proliferation was determined by plate clonal formation assay.Nude mice were injected subcutaneously with PKP1 overexpressed stable cell line to observe the effect of PKP1 on tumorigenesis.Wound-healing assay and transwell assay were used to evaluate PKP1 expression on the ability of migration and invasion of NPC cells.Phalloidin was used to observe the effect of altered PKP1 expression on the cytoskeleton of NPC cells.WB assay was performed to determine the proteins involved in proliferation,survival and epithelial-mesenchymal transition(EMT)after PKP1 knockdown and overexpression.Stable cell lines overexpressing PKP1 were performed bulk RNA sequencing and enriched by Gene set enrichment analysis(GSEA).WB was performed to detect activation of the ERK1/2 pathway.The above mentioned phenotypic and molecular changes were further investigated after treatment with the ERK/MAPK pathway inhibitor of PD98059.In addition,the interaction between PKP1 and Vimentin was examined by co-precipitation(Co-IP)and immunofluorescence colocalization to explore the underlying mechanism by which PKP1 acts.Results:(1)A total of 1026 up-regulated genes and 273 downregulated genes were identified from two databases,GSE68799 and GSE118719.The upregulated differential genes were preferentially enriched in biological processes such as epidermal development and extracellular matrix tissue.Cellular components such as cell-cell junctions,keratin filaments and intermediate filaments and the molecular functions such as integrin binding,cell adhesion mediator activity were enriched as well.KEGG analysis showed that PI3K-AKT signaling pathway and extracellular matrix receptor interaction were the major enrichment pathways.The downregulated differential genes were mainly enriched in B cell-related events,such as B cell activation and proliferation.A gene set of 13 genes,including PKP1 and LOR,was selected as the candidate genes after the PPI network analysis.Survival analysis showed that five genes,including ITGA11 and PKP1,were significantly associated with the prognosis of NPC patients,among which PKP1 was mainly localized and highly expressed in NPC cells.In addition,the tumor microenvironment(TME)of NPC was characterized by increased infiltration of dendritic cells(DC)and γδ T cells,but less infiltration of B cells.All three cell types were associated with the prognosis and expression of PKP1.(2)PKP1 was highly expressed in NPC tissues and localized to cytoplasm and nucleus.PKP1 promoted clonal formation,tumorigenesis,migration and invasion,and was associated with skeletal remodeling and protrusion formation in NPC cells.WB showed that PKP1 increased the expression of proliferation-associated molecule of PCNA and decreased it after PKP1 knockdown.On the other hand,overexpression of PKP1 decreased Ecadherin and increased N-cadherin,Slug and Vimentin,with the opposite results after PKP1 knockdown.GSEA results of the RNA sequencing data showed that the MAPK pathway was significantly enriched in the samples.WB showed that PKP1 increased the level of p-ERK1/2.The level of pERK1/2 was significantly suppressed after S18 PKP1 cells treated with PD98059.And the expression of PKP1,PCNA,Bcl-2,Vimentin,Ncadherin was also significantly decreased,and while the amount of Ecadherin was increased.The ability of invasion and migration of NPC cells was significantly accelerated by the addition of PD98059.Co-IP showed that PKP1 could co-immunoprecipitate with Vimentin,suggesting a direct interaction between them.Immunofluorescence showed that both endogenous and exogenous PKP1 co-localized with Vimentin in the perinuclear region.Conclusions:(1)High PKP1 expression is significantly associated with poor prognosis in NPC patients and is a key gene for assessing immune cell infiltration in NPC.(2)PKP1 is highly expressed in NPC.PKP1 activates the ERK1/2 signaling to promote cell proliferation,survival,invasion and migration of NPC.Figures: 35,Tables:16,References: 148...