| BackgroundNasopharyngeal carcinoma(NPC)is a malignant tumor originated from the nasopharyngeal mucosa.NPC is rare in most developed countries,with incidence rate less than 1/100,000.However,the incidence is around 10-25/100,000 in South China and Southeast Asia.The carcinogenesis of NPC is a multi-step procedure with multi-factor.The main causes of NPC includes genetic susceptibility,Epstein-Barr virus(EBV)infection and environmental carcinogenesis.At present,the main therapeutic approach for NPC is comprehensive treatment based on radiation therapy.With the continuous development and optimization of treatment methods,the total five-year survival rate of patients with NPC has reached more than 80%.But part of the patients with local recurrence and distant metastasis of tumors lead to treatment failure.Therefore,it isgreatly meaningful to explore the pathogenesis of NPC and to find new therapeutic targets and means.Abnormal energy metabolism is one of the ten hallmarks of cancer.During tumorigenesis,the energy metabolism has undergone significant alteration,including glucose metabolism,fatty acid metabolism,amino acid metabolism,tricarboxylic acid cycle(TAC),oxidative phosphorylation,etc.Among them,abnormal glucose metabolism is the most common one.Besides,abnormalities in fatty acid metabolism are also common in tumors.As one of metabolites downstream of fatty acids,ketone body plays important roles in tumorigenesis.Its influence on the malignant biological behavior of tumors is still controversial.Previously we reported that the expression of 3-hydroxy-3-methylglutaryl-Co A lyase(HMGCL),one of the key gene of the ketogenesis,was down-regulated in NPC,suggesting that there was abnormal ketone metabolism in NPC.β-hydroxybutyrate(β-HB)is one of the main components of ketone bodies and has the activity of histone deacetylase inhibitors,which could affect the transcript level of the genome by modulating the histone acetylation,which may participate in the occurrence and development of tumors.However,at present,the influence of β-hydroxybutyrate on the occurrence and development of NPC and its specific mechanism remains unclear.Our previous studies have demonstrated that overexpression of HMGCL in NPC cells increased the intracellular β-hydroxybutyrate in NPC cells,thereby inhibiting cell migration and invasion by reversing the ability of epithelial-mesenchymal transition(EMT).We also found that both endogenous and exogenous β-hydroxybutyrate significantly increased the expression of epithelial cadherin E-cadherin encoded byCDH1 gene in NPC.As a member of the cadherin family,E-cadherin plays a key role in maintaining the polarity of epithelial cells and intercellular adhesion.Down-regulation of CDH1 is also an important molecular marker of EMT,indicating malignant transformation of tumors.CDH1 gene is one of the key molecules in the development of NPC.The inactivation of CDH1 expression in NPC can be regulated by mi RNA and promoter hypermethylation.However,it is still unknown that howβ-hydroxybutyrate epigenetically regulates the transcription and expression of CDH1 gene in NPC.Objective:To investigate the effect of β-hydroxybutyrate on transcription and expression of CDH1 in NPC cell line.To screen out the genomic profiles regulated by β-hydroxybutyrate via histone acetylation in NPC cells,which might better understand the epigenetic modulation of NPC genome by cellular metabolic products.Methods:1.Analyzing the influence of β-hydroxybutyrate on the activity of histone deacetylases(HDACs)in NPC by fluorescence assay.2.detecting the regulation of β-hydroxybutyrate on the expression level of histone acetylation in NPC by Western blot assay.3.Analyzing the regulation of β-hydroxybutyrate on the transcription and expression of CDH1 in NPC by Quantitative Real-time PCR(q PCR)and Western blot assay.4.Detecting the effect of β-hydroxybutyrate on the level of histone acetylation in the CDH1 promoter region by the chromatin immunoprecipitation(Ch IP)assay.5.Combined chromatin immunoprecipitation sequencing(Ch IP-Seq)and RNA sequencing(RNA-Seq)to describe the effect of β-hydroxybutyrate on the epigenome of NPC cells.Result:1.β-Hydroxybutyrate inhibits the activity of histone deacetylase in NPC.NPC cells 5-8F,HONE1,HK1-EBV,and CNE2-EBV treated withβ-hydroxybutyrate showed significantly lower histone deacetylase activity.The fold of inhibition were 1.470(P=0.000),1.091(P=0.002),1.097(P=0.078),and 1.400(P=0.000),respectively.The data indicates that β-hydroxybutyrate can inhibit the activity of histone deacetylase.2.β-Hydroxybutyrate increases the histone acetylation in NPC.NPC cells 5-8F,HONE1,CNE2-EBV,HK1-EBV treated with different concentrations of β-hydroxybutyrate.The relative expression of acetylation histone H3(Lys9 / Lys14)(Acety-H3K9/14),acetylated lysine(Acety-Lys)was significantly up-regulated compared to untreated NPC cells.Among them,according to the increasing concentration ofβ-hydroxybutyrate concentration(5,10,20 m M),the multiples of Acety-H3(K9/14)up-regulated in 5-8F cell were 1.767,1.182,1.342 times respectively,in HK1-EBV cell were 1.195,1.537 and 1.866 times,the multiples up-regulated in CNE2-EBV cell were 1.372,1.537,1.408 times respectively,indicating that β-hydroxybutyrate promoted histone acetylation levels of NPC cells.3.β-hydroxybutyrate promotes the transcription and expression levels of CDH1.The transcriptional expression level of CDH1 in NPC cells 5-8F,HONE1,HK1-EBV,CNE2-EBV treated with β-hydroxybutyrate was significantlyup-regulated.According to the increasing concentration gradient(5,10,20 m M),the up-regulation multiples in 5-8F cells were 1.988,2.207,3.227,and 5.313 times,respectively,The up-regulation multiples in HONE1 were 1.102,1.412,1.711,1.883 times,respectively,HK1-EBV were 0.763,1.120,1.384,1.577 times,and CNE2-EBV cells were 0.835,0.996,1.286,2.615 times,respectively,indicating that β-hydroxybutyrate upregulates the transcriptional expression level of CDH1 gene in NPC cells.The protein expression level of E-cadherin in 5-8F cell was significantly increased by β-hydroxybutyrate,while no obvious alteration was found in HONE1,HK1-EBV,and CNE2-EBV cell lines.4.β-Hydroxybutyrate elevates the expression of CDH1 in NPC by histone acylation.By chromatin immunoprecipitation experiments,it was found that the Input% of β-hydroxybutyrate-treated NPC cells 5-8F and HONE1 were higher than the control group,and the increase was 125.093 and 1.116 times,respectively,and The p values were 0.000 and 0.494,respectively.The results showed that β-hydroxybutyrate promotes the histone acetylation in the CDH1 promoter region,which suggests thatβ-hydroxybutyrate promotes the expression of CDH1 in NPC through histone acetylation.5.Screening of β-hydroxybutyrate upregulating gene expression profile by histone acetylation in NPC cells by high-throughput sequencing.RNA-seq revealed 1294 differentially expressed genes and Ch IP-seq revealed 1295 differentially expressed genes in histone acetylation modification in NPC cells before and after β-hydroxybutyrate treatment.Combined RNA-seq with Ch IP-seq showed a total of 32 differentiallyexpressed genes in which β-hydroxybutyrate was up-regulated by histone acetylation in NPC cell 5-8F.The gene profile of β-hydroxybutyrate regulated by histone acetylation in NPC cells was initially established.Conclusion:1.β-Hydroxybutyrate inhibites the activity of histone deacetylase in NPC cells and is an inhibitor of histone deacetylase.2.β-Hydroxybutyrate promotes histone acetylation level in NPC cells.3.β-Hydroxybutyrate increases the transcription and expression levels of CDH1 in NPC cells 5-8F,HONE1,HK1-EBV.4.β-Hydroxybutyrate elevates the expression of CDH1 by histone acylation in NPC.5.Thirty-two genes are screened out as potentially regulated byβ-hydroxybutyrate,via histone acetylation.In conclusion,β-Hydroxybutyrate inhibites histone deacetylase activity in NPC cells promotes histone acetylation in NPC cells.The transcriptional and expression of CDH1 is up-regulated by promoting the level of histone acetylation in the CDH1 promoter region of NPC cells.In addition,the gene profile of β-hydroxybutyrate-targeted histone acetylation regulation is also screened. |
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