PKM2-dependent Histone H3 Phosphorylation Promotes EGF-mediated DKK1 Transcription And Molecular Mechanism Of Hepatocellular Carcinoma Metastasis

Background:Hepatocellular carcinoma is the most common primary malignant tumor in the liver.Hepatocellular carcinoma is the sixth largest malignant tumor type in the world,and the fatality rate ranks third among all cancer deaths.It ranks fifth in fatality among men and sixth in women,and more than half of new cases and deaths occur in China.Although surgery can significantly improve the survival rate of patients with early hepatocellular carcinoma,the survival rate of patients with metastasis cannot be significantly improved.In recent years,many papers have reported that DKK1 is highly expressed in the tissues of patients with hepatocellular carcinoma,and this abnormal expression can promote the metastasis of hepatocellular carcinoma.informal,the mechanism of DKK1 up-regulation in hepatocellular carcinoma is still unclear,so clarifying the mechanism of DKK1 up-regulation may provide potential targets for the treatment of hepatocellular carcinoma.Studies have shown that the expression of EGFR in hepatocellular carcinoma tissues is significantly up-regulated,and the abnormally activated EGFR is positively correlated with the metastasis of hepatocellular carcinoma cells.Therefore,this study proposes a scientific hypothesis that the abnormal expression of DKK1 in hepatocellular carcinoma cells may be mediated by the activation of EGFR.If EGFR mediates abnormal expression of DKK1,what is its mechanism? These are a scientific problem to be solved in this subject.Methods:First,we used Western blot and Real time-PCR to detect the expression of DKK1 protein and mRNA in Hep G2 and SMMC-7721 human hepatocarcinoma cells induced by EGF;Gefitinib that is inhibitors of the EGFR,and PD98059 that is inhibitors of ERK1/2 were applied to cells prior to EGF treatment,and the expression of DKK1 protein and mRNA was detected by Western blot and real time PCR.The levels of p-ERK,p-PKM2,p-H3 and other proteins were detected by Western blot.;immunofluorescence assay was used to detect the expression of DKK1 protein and the localization of PKM2 protein in hepatocellular carcinoma cells;Transwell chamber and Matrigel assay were used to detect the metastasis and invasion of hepatocellular carcinoma under EGF induction;The interaction between PKM2 and ERK1/2,PKM2 and H3 protein induced by EGF was detected by Co-immunoprecipitation assay.The expression of DKK1 and H3 protein in rat liver cancer tissues induced by DEN was detected by immunohistochemistry.The levels of DKK1 and EGF in rat serum were detected by ELISA,and the levels of p-PKM2,p-H3 and DKK1 protein in rat hepatocellular carcinoma tissues was detected by immunoblotting.Results:1.Western blot and Real time PCR showed that EGF activated EGFR could up-regulate the expression of DKK1 protein.2.Transwell experiment showed that EGF could promote the migration of hepatocellular carcinoma cells.3.Western blot assay showed that the levels of p-ERK1/2 was significantly up-regulated after EGF induction in hepatocellular carcinoma cells.Gefitinib,an EGFR inhibitor,and PD98059,a MEK-ERK1/2 pathway inhibitor,reversed the up-regulation of p-ERK1/2 and DKK1 induced by EGF.Transwell chamber test and Matrigel test showed that EGF could promote the migration and invasion of HCC cells,and the same inhibitor could reversemigration and invasion.4.Western blot showed that phosphorylation of p-PKM2 was up-regulated significantly under EGF stimulation.Through Co-IP experiment,we found that PKM2 and histone H3 interacted after EGF treatment.Western blot and immunofluorescence experiments showed that EGF induced PKM2 to enter the nucleus.When Gefitinib and PD98059 were used,the amount of PKM2 entered the nucleus significantly reduced.5.The rat model of hepatocellular carcinoma induced by DEN was successfully established.After 16 weeks of DEN treatment,obvious and uniform nodules were found in the liver tissue induced by DEN.HE staining showed that DEN-induced liver tissue had obvious hepatocellular carcinoma characteristics.6.The expression of DKK1 protein and DKK1 gene in rat hepatocellular carcinoma tissues detected by Western blot and real time PCR was significantly higher than that in negative control group.7.The serum levels of EGF and DKK1 in DEN-induced hepatocarcinoma rats were significantly higher than those in the control group in ELISA assays.8.Western blot assay showed that p-PKM2 and p-H3 protein were highly increased in rat hepatocellular carcinoma tissues.9.Immunohistochemical assay showed that p-PKM2 and p-H3 protein were highly expressed in rat hepatocellular carcinoma tissues.Conclusions:1.Activation of EGFR in hepatocellular carcinoma cells promotes transcription of2.The ERK1/2 pathwaydownstream of EGFR phosphorylates PKM2 and promotes nuclear translocation,which in turn causes transcription ofDKK1.3.PKM2-dependent histoneH3 phosphorylation promotes EGF-inducedDKK1 transcriptionanddevelopment ofhepatocellularcarcinoma.