The Function And Mechanism Of PHF8 On The Growth And Invasion Of Human NPC Cells

Nasopharyngeal cancer(NPC)is a cancer that originates in the epithelium of the nasopharynx.As a special type of cancer,nasopharyngeal cancer has obvious differences in epigenetic modification compared with other cancers.Among them,histone methylation plays an important role in the occurrence and development of nasopharyngeal cancer,especially the abnormal methylation of histone H3K9 in nasopharyngeal cancer patients.There are three factors that regulate histone H3K9 methylation: reader,eraser and writer.The erasers of H3K9 mainly include KDM2 B,KDM3B,KDM4 C and PHD Finger protein 8(PHF8).H3K9 is an active site for transcription regulation,and its methylation level will inhibit its activation and inhibit gene transcription at this site.A large number of studies have reported that there are many proto-oncogenes at this site,and the removal of methylation at this site will enhance the epigenetic dependent transcription of a large number of proto-oncogenes.In this way,cell proliferation,cell cycle,migration and invasion ability are enhanced,resulting in oncogenic phenotypic changes.PHF8 is a classic histone demethylation enzyme with substrates of H3K9me3 and H4K20me3.PHF8 is abnormally expressed to varying degrees in a variety of tumors,including leukemia,gastric cancer,breast cancer,prostate cancer,glioma,cervical cancer,colon cancer,laryngeal cancer,lung cancer and esophageal cancer.Objective:We conducted a "The function and mechanism of PHF8 on the growth and invasion of human NPC cells",namely,the expression of PHF8 is abnormal in NPC,including the abnormality of DNA,m RNA and protein levels,promotes the abnormality of the biological behavior of NPC cells,including proliferation,cell cycle,apoptosis,migration and invasion.PHF8 mediate the removal of H3K9 and H4K20 methylation levels in NPC cells,activate key molecular signaling pathways,and ultimately induce the occurrence and development of NPC.To test this hypothesis,three modules were used in this study:(1)The expression of PHF8 in human NPC cells and tissues;(2)The biological function of PHF8 in promoting the growth and invasion of human NPC cells;(3)Molecular mechanism of PHF8 promoting growth and invasion of human NPC cells.To clarify the cancer-promoting effect of PHF8 demethylase on NPC cells and the specific mechanism of its participation in the growth and invasion of NPC cells,in order to provide a new theoretical basis and experimental basis for the treatment of new targets of NPC.Methods:The ectopic expression of PHF8 in DNA,m RNA and protein levels were analyzed based on the multiple public databases.The differential expression of PHF8 m RNA in immoralized nasopharyngeal epithelial cell lines and NPC cell lines was tested by RT-PCR.IHC was used to detect the PHF8 protein levels in NPC,paracancer and normal nasopharyngeal epithelium tissues.Build HONE1 and HK1 cell lines with PHF8 low expression and then select MTT experiment,Ed U,clone formation assay,scratch mark experiment,Transwell migration experiment,experiment,flow cycle flow apoptosis,RT-PCR,Western blot,molecular biology technology such as cell immunofluorescence to analysis PHF8 in terms of its biological function in the development of NPC.The possible molecular mechanism of PHF8 in NPC was analyzed by bioinformatics.Western blot,RT-PCR and dual luciferase reporter gene were used to verify the bioinformatics analysis results.Results:(1)Bioinformatics indicated that the DNA and m RNA levels of PHF8 were not significantly different in the data set of head and neck squamous cell carcinoma(P>0.05).Its protein levels were highly expressed in NPC data sets.(2)RT-PCR indicated that PHF8 m RNA was highly expressed in NPC cell lines,but was relatively low in immortised nasopharyngeal epithelial cells(P<0.05).(3)IHC staining indicated that the expression of PHF8 protein in NPC cells was significantly higher than that in normal nasopharyngeal epithelium and paraccancerous tissues(P<0.05).PHF8 was strongly positive in 77.3% of NPC samples,and the expression of PHF8 was significantly different from that of normal nasopharyngeal epithelium samples(23.5%)(P<0.001).When comparing PHF8 expression with clinicopathological parameters,we found a significant correlation between PHF8 expression level and lymph node invasion(P=0.0044).(4)Western blot confirmed the successful construction of HONE1 and HK1 cell lines with low expression of PHF8.MTT assay showed that the cell viability of sh-PHF8 #5 and sh-PHF8 #6 groups was significantly lower than control group and Vector group(P<0.05).In addition,XX knockdown of PHF8 could significantly inhibit the clone formation ability of NPC cell lines HONE1 and HK1.EDU assay showed that when PHF8 expression was inhibited,the DNA replication ability of NPC cells was also significantly decreased(P<0.05).Western blot analysis showed that the expression levels of proliferation markers PCNA and Ki-67 were also decreased with the decrease of PHF8 expression.Immunofluorescence staining indicated that the expressions of PCNA and Ki-67 were positively regulated by PHF8,but their cell localization was not affected by PHF8.TCGA database analysis showed that PHF8 was positively correlated with Ki-67 and PCNA(P<0.05).These results suggest that PHF8 knockdown can inhibit the proliferation of NPC cells.(5)Flow cytometry indicated that PHF8 knockdown significantly induced G1 stagnation of NPC cell lines(P<0.05).Western blot analysis showed that the expression levels of Cdc25 A and CCNE1 were also decreased with the decrease of PHF8 expression.Immunofluorescence staining indicated that Cdc25 A and CCNE1 expression were positively regulated by PHF8,but their cell localization was not affected by PHF8.TCGA database analysis showed that PHF8 was positively correlated with Cdc25 A and CCNE1(P<0.05).This suggests that PHF8 knockdown can induce NPC cell cycle arrest.(6)Flow cytometry showed that when PHF8 was knocked down in NPC cell lines,the level of apoptosis was significantly increased.Western blot analysis showed that the expression level of apoptosis marker Bax was increased with the decrease of PHF8 expression,and the expression level of anti-apoptotic marker Bcl-2 was decreased with the decrease of PHF8 expression.Immunofluorescence staining indicated that the expression of Bax was increased with the decrease of PHF8 expression,and the expression of Bcl-2 was decreased with the decrease of PHF8 expression.TCGA database analysis showed that PHF8 had a significant positive correlation with Bcl-2(P<0.05)and a significant negative correlation with Bax(P<0.05).These results suggest that PHF8 knockdown can promote the apoptotic ability of NPC cells.(7)Wound healing assay showed that PHF8 knockdown significantly inhibited the migration of NPC cells(P<0.05).Transwell migration and invasion assay showed that PHF8 knockdown significantly inhibited the migration and invasion ability of NPC cells(P<0.05).Western blot analysis showed that the expression levels of N-cadherin and Vimentin were also decreased with the decrease of PHF8 expression.Immunofluorescence staining indicated that the expressions of N-cadherin and Vimentin were positively regulated by PHF8,but their cell localization was not affected by PHF8.TCGA database analysis found no significant correlation between PHF8 and N-cadherin or PHF8 and Vimentin(P>0.05).These results suggest that PHF8 may promote the migration and invasion of NPC cells.(8)Subsequently,we constructed a plasmid with high expression of PHF8 and transfected it into immortified nasopharyngeal epithelial cells NP69.The successful construction of NP69 cells with high expression of PHF8 was confirmed by Western blot.MTT assay showed that overexpression of PHF8 significantly increased the proliferation of NP69cells(P<0.05).EDU assay showed that PHF8 significantly promoted DNA replication in NP69 cells(P<0.05).Western blot showed that PHF8 could up-regulate the expression levels of the proliferation markers Ki-67 and PCNA(P<0.05),which suggested that overexpression of PHF8 could promote the proliferation of immoralized nasopharyngeal epithelial cells.(9)Flow cytometry indicated that PHF8 overexpression could reduce G1 cell arrest in NP69 cells.Western blot showed that PHF8 could upXXII regulate the expression of Cdc25 A and CCNE1(P<0.05).This suggests that PHF8 can accelerate the cell cycle of NP69.(10)Both wound healing assay and transwell migration and invasion assay showed that PHF8 overexpression promoted the migration and invasion of NP69 cell lines(P<0.05).Western blot showed that PHF8 promoted the expression of N-cadherin and Vimentin.This suggests that overexpression of PHF8 may up-regulate the migration and invasion ability of NP69 through the EMT process.(11)Based on TCGA database of head and neck squamous cell carcinoma data set,PHF8-related genes were extracted,and volcano map and heat map were visualized by R language.PCA principal component analysis showed that this group of genes could significantly distinguish between head and neck squamous cell carcinoma and head and neck squamous cell carcinoma paracancerous samples.These results suggest that these genes play an important role in the molecular biology of head and neck squamous cell carcinoma.This set of genes was further imported into the STRING database to construct the PPI protein interaction network,and the key genes were screened by Cytoscape software for extension optimization.The key genes were imported into David database for GO and KEGG enrichment analysis,and the possible molecular biological effects caused by PHF8 were predicted.The results suggested that PHF8 may be directly involved in the mechanism of transcriptional regulation,DNA binding,histone binding and cell cycle regulation.GSEA enrichment analysis further revealed that PHF8 may be involved in cell tight junctions and other functions,suggesting that PHF8 may be directly involved in the regulation of migration and invasion mechanisms.Structural analysis of the protein revealed that PHF8 had a Jmj C domain(regulating histone demethylation)and a zinc finger domain(a transcription factor characteristic involved in targeted chelation of DNA promoter regions).It is suggested that PHF8 can be enriched in the promoter region of target genes through the form of transcription factors,regulate histone methylation level,and then change the degree of chromatin tightness,thereby regulating the transcription of target genes.(12)Ch IP-sequence analysis showed that PHF8 had significant peaks in the promoter regions of Vimentin,Cdc25 A and DVL2 based on GEO database.RT-PCR indicated that knockdown of PHF8 could significantly reduce the transcription levels of Vimentin,Cdc25 A and DVL2.(13)Western blot showed that knockdown of PHF8 significantly increased the levels of H3K9me1 and H4K20me1.These results suggest that PHF8 can promote transcription by removing histone methylation levels in the promoter regions of Vimentin,Cdc25 A and DVL2,reducing the degree of chromatin compression in the promoter regions.Western blot showed that PHF8 knockdown inhibited the protein expression of DVL2 and the nuclear translocation of β-catenin.The activation degree of Wnt/β-catenin was detected by TOPFlash assay with dual luciferase reporter gene.Transcriptional changes of target genes downstream of the Wnt/β-catenin signaling pathway were detected by RT-PCR.These results suggest that PHF8 can up-regulate the expression of DVL2 through transcriptional regulation,promote the nuclear translocation of β-catenin,and then activate the Wnt/β-catenin signaling pathway,and accelerate the occurrence and development of NPC.(14)By xenograft tumor model,PHF8 knockdown could inhibit the growth of NPC cells.Immunohistochemical staining showed that PHF8 knockdown inhibited the protein levels of Ki-67,PCNA,Cdc25 A,CCNE1,Bcl-2,N-cadherin,Vimentin and DVL2.Enhanced protein expressions of BAX,H3K9me1 and H4K20me1;Inhibition of nuclear translocation of β-catenin.These results suggested that PHF8 up-regulated the expression of DVL2,promoted the nuclear translocation of β-catenin and activated the Wnt/β-catenin signaling pathway,and regulated the expression levels of proliferation,cycle,apoptosis,migration and invasion proteins to promote the growth of NPC in vivo.Conclusion:(1)The expression level of PHF8 protein in NPC tissue samples was significantly higher than that in normal nasopharyngeal epithelium.(2)PHF8 promotes cell proliferation,accelerates cell cycle;promotes cell migration and invasion,and inhibits apoptosis in NPC cells.(3)Histone demethylation enzyme PHF8 may promote transcriptional activation of Vimentin,Cdc25 A,and DVL2 by decreasing the levels of H3K9me1 and H4K20me1 at histone methylation sites in the Vimentin,Cdc25 A,and DVL2 promoter regions,which activates the Wnt/β-catenin pathway.