NAP1L1 Targeting Suppresses The Proliferation Of Nasopharyngeal Carcinoma

1.Background and Purpose:Nasopharyngeal carcinoma(NPC)is a type of head and neck squamous cell carcinoma(HNSCC),with high morbidity in the south of China.EBV infection,smoking,passive smoking at home,air pollution,eating nitrosamine-rich preserved food,drinking alcohol,oral health and oral microbes,and genetic susceptibility are all etiopathogenic factors of nasopharyngeal carcinoma.These factors play a role in the occurrence and development of nasopharyngeal carcinoma by regulating the expression of tumor-related genes.exploring the underlying pathogenesis of nasopharyngeal carcinoma and finding effective molecular therapeutic targets is one of the most active frontier research hotspots and difficulties in the field of nasopharyngeal carcinoma.NAP1L1,a member of the nucleosome assembly protein family,is involved in DNA replication,regulation of chromatin formation,and promotion of cell gro wth.In the past,N AP1L1 was considered as a potential tumor promoter,and this idea has been confirmed by the present study.NAP1L1 is involved in the pathogenesis of various cancers,including colorectal cancer,kidney cancer,liver cancer,pancreatic neuroendocrine tumor and so on.However,the role and molecular mechanism of NAP1L1 in nasopharyngeal carcinoma have not been reported.In The GEPIA(Gene Expression Profiling Interactive Analysis)database and The Human Protein Atlas database,The research team found that NAP1L1 was elevated in both Gene and Protein levels in head and neck squamous cell carcinoma.Nasopharyngeal cancer is the most common squamous cell cancer of the head and neck.We detected and analyzed the high expression of N AP1L1 in tissues of nasopharyngeal carcinoma and its association with poor prognosis,which is consistent with the results in the atabase.Subsequently,we confirmed in vitro and in vivo that NAP1L1 can promote the proliferation of nasopharyngeal carcinoma as a cancer-promoting factor.In further mechanistic studies,we found that NAP1L1 can promote the proliferation of nasopharyngeal carcinoma by promoting G1 transformation through HDGF/C-Jun/CCND1 signaling axis.Our study fistly reveals the role and molecular basis of NAP1L1 in nasopharyngeal carcinoma,providing a new molecular targets for clinical treatment of nasopharyngeal carcinoma.2.Research contents and methods:2.1 Bioinformatics Analysis(GEPIA and THE HUMAN PROTEIN ATLAS DATABASE)hint that the differential expression of NAP1L1 in head and neck squamous cell carcinoma.The interaction proteins of NAP1L1,including HDGF,MYC and FBXW7,were searched in BioGRID,providing ideas for subsequent experiments.2.2 NAP1L1 overexpression promotes the NPC’s proliferation.2.2.1 161 nasopharyngeal carcinoma tissues(including 57 pairs of cancer and adjacent tissues)were selected.Immunohistochemistry showed that NAP1L1 was highly expressed in nasopharyngeal carcinoma tissues and low in adjacent tissues.Survival analysis showed that overexpression of NAP1L1 was an adverse factor in reducing the overall survival time of patients with nasopharyngeal carcinoma.2.2.2 transient transfection of NAP1L1 and stable virus infection were carried out in nasopharyngeal carcinoma cell line.Cell function experiment such as plate cloning,MTT and EDU confirmed that NAP1L1 promote the nasopharyngeal carcinoma cells’ proliferation;The cell cycle experiment was carried out with the nasopharyngeal carcinoma cell line stably infected with NAP1L1 virus.It was found that the nasopharyngeal carcinoma cell line stably knockdown NAP1L1 was blocked in G1 phase.The nasopharyngeal carcinoma cell line stably infected with NAP1L1 virus was used for subcutaneous tumorigenesis in nude mice.The in vivo experiment further confirmed that NAP1L1 promoted the proliferation of nasopharyngeal carcinoma cells.2.3 Transcriptome analysisTranscriptome analysis of nasopharyngeal carcinoma cell lines with stable knockdown of NAP1L1 was sequenced,and the differential gene expression was collected.Based on the differential gene expression,Advanced GSEA analysis was performed based on the RNA-seq data,and found that the nasopharyngeal carcinoma cell lines with knockdown of NAP1L1 were inhibited in cell differentiation,activation and growth,and the significant differential genes related to proliferation were CCND1 and E2F1.It was further verified by qPCR and Western Blotting that CCND1,CDK4 and E2F1 were down regulated in stably knocking down NAP1L1nasopharyngeal carcinoma cell lines.2.4 NAP1L1 binds to HDGF to recruit c-JUN and stimulates CCND1,CDK4 and E2F1 to promote proliferation of Nasopharyngeal carcinoma cells2.4.1 Interaction between NAP1L1 and HDGF:According to the database analysis results,exogenous immunoprecipitation experiment confirmed the interaction between NAP1L1 and HDGF.Confocal laser microscopy showed that the binding site of NAP1L1 and HDGF was mainly in the cytoplasm of nasopharyngeal carcinoma cells.2.4.2 HDGF recruits c-JUN:CO-IP analysis shows that c-JUN and HDGF interact in nasopharyngeal carcinoma.2.4.3 Transfection of HDGF increased c-JUN/CCND1/CDK4/E2F1 signal and reversed the proliferation of nasopharyngeal carcinoma cells inhibited by NAPIL1 knockdown:qPCR and Western blot analysis verified that the up-regulation of HDGF.Cell proliferation and EDU staining ability were significantly restored.The expression of c-JUN,CCND1,CDK4 and E2F1 was significantly increased by WesternBlot.2.4.4 In nasopharyngeal carcinoma cells with stable knockdown of NAP1L1,transfection of c-JUN plasmid enhanced CCND1/CDK4/E2F1 signal and reversed the inhibitory effect of shRNAN-NAP1L1 on proliferation of Nasopharyngeal carcinoma cells:shRNAN-NAP1L1 nasopharyngeal carcinoma cells were transfected with cJUN-cdna plasmid.QPCR and WesternBlot analysis confirmed that c-JUN expression was up-regulated,and cell proliferation and EdU staining ability were significantly restored.WesternBlot results showed that c-JUN,CCND1,CDK4 and E2F1 protein expression were significantly increased.2.5 After HDGF knockdown,the expression of c-JUN/CCND1/CDK4/E2F1 in nasopharyngeal carcinoma cells decreased,but there was no change in NAP1L1.NAP1L1 cDNA plasmid was transfected into HDGF knockdown nasopharyngeal carcinoma cells to restore the ability of cell proliferation.3.Conclusion:3.1NAP1L1 highly expressed in nasopharyngeal carcinoma.High expression of NAP1L1 is an adverse factor for clinical progression,short survival and poor prognosis of nasopharyngeal carcinoma.3.2 NAP1L1 promotes the proliferation of nasopharyngeal carcinoma cell line.3.3 Transcriptome analysis and GSEA analysis showed that knockdown of NAP1L1 significantly inhibited the cell activation,differentiation and growth of nasopharyngeal carcinoma cell line.The differential genes significantly related to proliferation were CCND1 and E2F1.3.4 NAP1L1 interacts with HDGF,HDGF interacts with c-JUN,induce the expression of cell cycle promoter CCND1/CDK4/E2F1 and promote G1 phase transformation of NPC cells,thereby inducing the growth of NPC cells.