The Role And Mechanism Of CENP-N/AKT1 Promoting Malignant Biological Behavior Of Nasopharyngeal Carcinoma Cells

Background and objective: Nasopharyngeal carcinoma(NPC)is a highly malignant head and neck cancer that originates from the epithelial cells of nasopharynx.According to international cancer surveys,most NPC patients have significant geographic and ethnographic clustering characteristics.It is prevalent in Thailand,Vietnam,southern China and North Africa,while in other places,increased incidence has been reported.These epidemiological and etiological features show the importance of genetic dysregulation in the occurrence and development of NPC.The overall incidence and mortality rate of NPC has decreased with the advancement of screening and treatment techniques.However,in epidemic areas,the risk of these malignant tumors to human health cannot be ignored.And in situ recurrence and distant metastasis are still the major factors for mortality in patients with NPC.So far,the mechanisms of the occurrence and progression of NPC have not been fully understood yet.Therefore,an in-depth study on the mechanism of gene dysfunction in the occurrence and progression of NPC is of profound significance to our further understanding and prevention of this disease.In this study,we firstly started from the sequencing of NPC tissue microarray and combined with transcriptomics and proteomics public database analysis to screen for CENP-N,a potential target gene that plays a role in the progression of NPC.Secondly,we combined gene-editing technology to interfere with CENP-N expression in NPC cells and then investigated its effects on the biological functions of NPC cells in vitro and in vivo.Finally,we explored the molecular mechanism of CENP-N affecting malignant biological behavior in NPC.The present study is divided into the following three parts:Part 1: Validation of the expression and significance of oncogene CENP-N in NPC based on public data screening and clinical samplesObjective: To Explore differentially expressed genes in NPC and control tissues to screen for target genes that affect the survival of patients with NPC.Methods: 1.Two NPC tissue microarrays selected from the GEO database were subjected to GEO2 R analysis to search for candidate genes that are differentially expressed in NPC.Subsequently,the differentially expressed candidate genes were further screened for Hub genes that might affect NPC by protein interaction network analysis and Cytoscape visible operation.2.The Kaplan-Meier Plot and GEPIA database were used to analyze the correlation between these Hub genes and the prognosis of head and neck squamous carcinoma patients.3.Immunofluorescence staining,protein immunoblotting(Western blot)and immunohistochemical staining were used to test the expression levels of CENP-N in NPC samples and nasopharyngitis(NPG)tissues.4.The relationship between CENP-N expression and the uptake of 18F-FDG by PET/CT examination of patients with NPC was analyzed.The prognostic correlation between CENP-N expression and NPC patients was analyzed too.5.Western blot was used to detect CENP-N protein expression in different NPC cell lines(HK-1,5-8F,SUNE-1,CNE-2Z,6-10B)and immortalized epithelial cell line NP460.Results: 1.A total of 568 common differentially expressed genes were screened from the 2 microarrays,of which 207 genes were up-regulated,while 361 genes were down-regulated.37 candidate Hub genes were screened based on PPI and visible analysis.2.20 Hub genes associated with prognosis in head and neck squamous cancer patients were screened in Kaplan-Meier plotter database.Finally three core differential genes were selected in GEPIA database: ANLN,CDK1 and CENP-N.3.The results of immunofluorescence,Western blot and immunohistochemistry confirmed high expression of CENP-N in NPC tissues(P< 0.05).4.Compared to patients with low expression of CENP-N,those with high expression of CENP-N had increased glucose uptake in the primary lesion(P< 0.05).Among NPC patients,those with high expression of CENP-N mostly have a poor prognosis(P< 0.05).5.Compared with NP460,CENP-N expression was elevated in multiple NPC cell lines(P< 0.05).Conclusion: CENP-N expression was significantly elevated in both NPC tissues and cells.CENP-N was negatively associated with survival time in NPC patients.Glucose uptake was increased in tumors of NPC patients with high expression of CENP-N.Part 2: CENP-N promotes glucose metabolism,cell proliferation,cell cycle and apoptosis resistance of NPC cells in vitro and in vivoObjective: To investigate the effects of changing CENP-N expression on glucose metabolism,cell proliferation,cell cycle and apoptosis in NPC.Methods: 1.The lentivirus-packaged plasmid containing CENP-N-sh RNA was used to infect and construct stable transfected NPC cells with CENP-N gene knockdown.2.Transcriptome sequencing was used to detect the effects of CENP-N gene knockdown on functional gene expression,signaling pathway regulation and biological functions of NPC cells.3.The effects of CENP-N knockdown on m RNA and protein expression of genes related to biological functions in NPC strains were examined by q RT-PCR and Western blot assays.Glucose assay,lactate assay,CCK8,clone formation and flow cytometry were used to detect the effects of CENP-N knockdown on glucose metabolism,cell proliferation,cell cycle and apoptosis in NPC cells in vitro.4.Nude mice model with NPC subcutaneous transplanted tumor and small animal PET/CT were used to detect the effects of CENP-N knockdown on proliferation,glucose uptake and glycolysis.Immunofluorescence staining,immunohistochemical staining and Western blot were used to test the effects of CENP-N knockdown on glucose metabolism,cell cycle and apoptosis related protein expression in NPC cells in vivo.Results: 1.A null set group and two groups of cells with CENP-N transcript knocked down,which were named sh NC,sh CENP-N1 and sh CENP-N2,were constructed separately in 5-8F and CNE-2Z cell line.2.Genes affected after CENP-N knockdown were enriched in cellular functions related to glucose metabolism and cell cycle.3.Compared with sh NC cells,after CENP-N knockdown in 5-8F and CNE-2Z cells HK2,GLUT1,ENO1,PFKFB2,PFKFB3,c-MYC,Ki67,PCNA,CDK2,Cyclin D1,Cyclin E1 and Bcl-2 m RNA expression were down-regulated,while Bax and Caspase-3 m RNA expression were up-regulated(P< 0.05).Compared with sh NC cells,CENP-N knockdown resulted in down-regulation of HK2,GLUT1,Ki67,PCNA,CDK2,Cyclin D1 and Bcl-2 protein expression and up-regulation of Bax protein expression in NPC cells.Compared with the sh NC group,both sh CENP-N1 and sh CENP-N2 in knockdown NPC cells exhibited reduced glucose uptake,decreased lactate production,reduced cell survival and proliferation ability,cell cycle G0/G1 phase arrest and up-regulated apoptosis ratio(P< 0.05).4.Compared with the sh NC group,in vivo condition 5-8F and CNE-2Z NPC cell line with CENP-N knockedown both exhibited significant down-regulation of tumor growth rate,final subcutaneous graft tumor weight,maximum standardized tumor glucose uptake,mean standardized tumor glucose uptake and total lesion glycolysis in nude mice(P< 0.05).Compared with the sh NC group,two NPC cell lines exhibited down-regulation of HK2,GLUT1,Ki67,PCNA,CDK2,Cyclin D1 and Bcl-2 expression and up-regulation of Bax expression after CENP-N knockdown in vivo(P< 0.05).Conclusion: CENP-N gene knockdown significantly inhibited aerobic glycolysis,cell survival,proliferation and promoted cell cycle arrest and apoptosis of NPC cells both in vitro and in vivo.Part 3: The molecular mechanism of CENP-N/AKT1 promoting malignant biological behavior of NPC cellsObjective: To investigate the molecular mechanisms of CENP-N involved in regulating glucose metabolism,cell survival and proliferation,cell cycle and apoptosis in NPC cells.Methods: 1.The effects of CENP-N knockdown on AKT,JNK and P53 signaling pathways were examined by Western blot.2.q RT-PCR was used to detect the changes of m RNA expression in AKT isoforms after CENP-N knockdown.Then,Immunofluorescence,immunohistochemistry and Western blot were used to detect the changes of AKT1 S473 phosphorylated protein expression after CENP-N knockdown.3.Cellular immunofluorescence was used to detect the subcellular localization of CENP-N and AKT1.4.Immunoprecipitation and GST-pull down assays were used to detect the protein interaction between CENP-N and AKT1 in NPC cells under in vivo conditions.5.Glucose assay,lactate assay,CCK-8,clone formation,and flow cytometry assays were used to detect whether the AKT1 inhibitor MK-2206 could block the changes in glucose metabolism,cell proliferation,cell cycle and apoptosis in NPC cells after over-expression of CENP-N.6.Bioinformatics methods were used to predict transcriptional regulator of CENP-N.7.NPC cells with IRF2 knockdown were constructed in 5-8F and CNE-2Z cell lines,and changes of CENP-N protein expression levels in NPC cell lines after intervention with IRF2 were detected by Western blot.8.The relationship between IRF2 and CENP-N promoter sequence was examined by chromatin immunoprecipitation(Ch IP)assay.9.The transcriptional regulation of CENP-N by IRF2 was examined by double luciferase reporter assay.Results: 1.AKT,MAPK and P53 signaling pathways changed in sh CENP-N1 and sh CENP-N2 groups compared with sh NC group.And the most significant change was in AKT signaling pathway,in which the main effect was on AKT1 S473 phosphorylation site.2.m RNA amounts in AKT1,AKT2,and AKT3 were down-regulated in both sh CENP-N1 and sh CENP-N2 groups compared with the sh NC group,with the most significant change in AKT1(P< 0.05).CENP-N knockdown in NPC cells significantly reduced the expression of phosphorylated proteins at AKT1 S473 site(P< 0.05).3.Cellular immunofluorescence showed protein co-localization expression of CENP-N and AKT1 in two NPC cell lines.4.There was a direct protein-protein interaction between CENP-N and AKT1 in cells.5.MK2206 was able to block the effects of overexpression of CENP-N on promoting aerobic glycolysis,cell survival and proliferation capacity,cell cycle and inhibiting apoptosis in two NPC cell lines(P< 0.05).6.IRF2 may have binding sites with the upstream promoter sequence of CENP-N.IRF2 expression was positively correlated with CENP-N(P< 0.05).7.In both IRF2 knockdown NPC cell lines,CENP-N protein expression levels were downregulated in the sh IRF2 group compared with the sh NC group(P< 0.05).8.IRF2 could directly bind to the promoter sequence of CENP-N.9.IRF2 could promote the transcription of CENP-N in NPC cells.Conclusion: CENP-N promotes aerobic glycolysis,proliferation,cell cycle and apoptosis resistance by regulating phosphorylation at AKT1 and S473 sites in NPC cells.IRF2,a CENP-N transcription factor,promotes CENP-N gene expression in NPC cells.