Mechanism Of P300-dependent Acetylation Of H3 Contributes To EGF-mediated DKK1 Transcription And Hepatocellular Carcinoma Metastasis

Objective:Metastasis is the most important cause of death in patients with hepatocellular carcinoma(HCC),so it is of great significance to explore the molecular mechanism of metastasis in order to reduce the mortality of HCC.Dickkopf-1(DKK1),a member of the Dickkopf gene family,is abnormally expressed in hepatocellular carcinoma and DKK1 is positively correlated with metastasis and poor prognosis of HCC.However,the specific molecular mechanisms through which DKK1 is transcriptionally regulated in hepatocellular carcinoma remain unclear.By explaining the up-regulated mechanism of DKK1 in hepatocellular carcinoma and its role in hepatocellular carcinoma metastasis,this study will help to clarify the molecular mechanism of metastasis and provide a promising strategies and target for therapy of HCC.Methods:1.To explore the expression and function of DKK1 in hepatocellular carcinoma: Real-time PCR,Western blot,immunohistochemical staining were used to study the level of DKK1 in DEN-induced rats.ELISA used to examine the content of DKK1 in serum.Immunohistochemical staining were used to examine the DKK1 expression in the tumor specimens and adjacent normal specimens.Subcutaneous transplantation tumor model and the lung metastasis model were used to detect the tumor growth and metastasis.2.To explore the transcriptional regulation of DKK1 mediated by EGFR: Four hepatocellular carcinoma cell lines(Hep G2,Huh-7,SNU-368,SNU-739)were incubated with EGF,the expression of DKK1 was detected and the expression of DKK1 in the cell culture medium was detected by ELISA.Knockdown the expression of EGFR or using EGFR inhibitor Gefitinib to detect DKK1 expression.After the HCC cells were incubated with JNK/STAT3 pathway inhibitor(AG490),MEK/ERK pathway inhibitor(PD98059)and PI3K/AKT pathway inhibitor(LY294002),the expression of DKK1 were detected.3.To explore the role of AKT-mediated phosphorylation of p300 Ser1834 site in the transcriptional regulation of DKK1 in hepatocellular carcinoma: HCC cells were incubated with EGF,and Western blot was used to detect the phosphorylation of p300 Ser1834 site.And using MK2206,the phosphorylation of p300 Ser1834 site was detected by Western blot.Knockdown the expression of p300 were used to detect the expression of DKK1.p300 WT plasmid,p300 S1834 A plasmid(phosphorylation deletion)and p300 S18347 D plasmid(simulated phosphorylation)were constructed and transfected into HCC cells,and DKK1 expression was detected.4.To explore the acetylation of histone H3K9 site by p300 was involved in the transcriptional regulation of DKK1 by EGFR in hepatocellular carcinoma: HCC were incubated with EGF and the acetylation of H3K9 site was detected by Western blot.The interaction between H3 and p300 was detected by CO-IP assay,acetylation of H3K9 site was detected when knockdown p300.p300 WT plasmid,p300 S1834 A plasmid and p300 S18347 D plasmid were transfected HCC cells,and the acetylation of H3K9 site was detected by Western blot.RNAi-resistant Histone H3 WT and RNAi-resistant Histone H3-K9 R plasmids were transfected into HCC cells after Histone H3 knockdown.Ch IP assay was used to detect the interaction between H3K9 and DKK1 promoter after EGF treatment.The expression of Acetyl-H3K9 in HCC model was detected by Western blot.The expression of Acetyl-H3K9 in clinical specimens of HCC was detected by immunohistochemistry,and then the correlation between the expression of Acetyl-H3K9 and DKK1 was analyzed.Results:1.In the DEN-induced rats,the expression of DKK1 mRNA and protein was significantly up-regulated,and the content of DKK1 in serum was also significantly up-regulated.In clinical tumor specimens,DKK1 expression was also higher than adjacent normal specimens.Subcutaneous transplantation tumor model and the lung metastasis model show that overexpression of DKK1 promoted tumor growth and metastasis,while knockdown of DKK1 inhibited tumor growth and metastasis.2.After treatment with EGF,the expression of DKK1 mRNA and protein was dramatically up-regulated,and the secretion of DKK1 mRNA and protein into cell culture medium increased.Knockdown EGFR significantly reverse the expression of DKK1 mRNA and protein mediated by EGF,and Gefitinib could also reverse the expression of DKK1 mRNA and protein mediated by EGF.PD98059 and LY294002 could significantly reverse EGF-induced upregulation of DKK1 mRNA and protein expression,while AG490 had no effect on EGF-mediated DKK1 mRNA and protein expression.3.After treatment with EGF,the phosphorylation level of p300 Ser1834 increased.Knockdown the expression of p300 reverse the increase of DKK1 mRNA and protein induced by EGF.Transfection of p300 S18347 D plasmid into Hep G2 and Huh-7 could significantly up-regulate the expression of DKK1 mRNA and protein,while p300 WT plasmid and p300 S1834 A plasmid had no such effect.4.When Hep G2 and Huh-7 were incubated with EGF,the level of Acetyl-H3K9 was significantly up-regulated,and the interaction between p300 and H3 was enhanced.Knockdown of p300 significantly diminished EGF-induced H3K9 acetylation in Huh-7 and Hep G2 cells.Transfection of p300 S18347 D plasmid in Huh-7 and Hep G2 cells also enhanced the level of Acetyl-H3K9,while p300 WT plasmid and p300 S1834 A plasmid did not.EGF-induced DKK1 mRNA and protein expression decreased significantly in the H3K9 R mutant compared with that in the WT H3.DEN administration significantly increased the level of Acetyl-H3K9.Ch IP assay also suggested that EGF treatment resulted in enhanced H3K9 acetylation at the DKK1 promoter and there was a correlation between the expression of Acetyl-H3K9 and DKK1 in clinical specimens of hepatocellular carcinoma.Conclusions:EGF/EGFR phosphorylates p300 Ser1834 site through PI3K/AKT signal pathway,then mediates acetylation of histone H3K9 site and promotes DKK1 transcription,thus inducing hepatocellular carcinoma metastasis.