Intermodulation Between Forkhead Box M1 And Methionine Adenosyltransferase In Human Liver Cancers

OBJECTIVE Forkhead box M1(FoxM1)as a carcinogenic factor is linked to cancer initiation and progression.Methionine adenosyl transferase catalyzes the formation of S-adenosylmethionine,the principal methyl donor,which is also differentially expressed in liver cancer.Here we examined the roles and mechanisms of crosstalk between FoxM1 and MATs in order to identify new targets for the molecular research of liver cancer and provide new ideas.METHODS 1.Expression analysis of FoxM1 and MATs in public datasets of two independent liver cancer database gene expression profiles,real-time PCR was performed 181 clinical HCC samples,observe the regulation relationship between FoxM1 and MATs.Further analysis of the correlation between FoxM1 and MATs in clinical samples.2.Hepatocellular carcinoma(HCC)and cholangiocarcinoma(CCA)cell lines were cultured in vitro to detect the expression of FoxM1.HCC and CCA cell lines were used to silence or overexpress FoxM1,observe the changes in the expression of MATs by RT-qPCR,western blot and immunofluorescence experiments.3.Interfere with the expression of FoxM1,then use the luciferase assay to detect changes in promoter activity of the MATs.EMAS and ChIP assay was further used to determine whether Fox M1 could regulate MATs.4.FoxM1 inhibitors FDI-6 and TO901317 could inhibit the expression of FoxM1 treatments in HepG2 and MZCHA-1 cells,observe the effects of FDI-6 and TO901317 on MATs genes in HCC and CCA cells.5.The effect of TO901317 on FoxM1 and MATs genes in HCC was examined used xenograft tumor model.6.MTT and wound healing assay were used to examine the effect of MATs on Fox M1-mediated proliferation or migration in HCC cells.7.Real-time PCR and western blot were used to observe the effect of MATs on FoxM1 and the crosstalk between MATs genes.Luciferase assay was used to observe the changes of FoxM1 and MATs promoter activity.RESULTS 1.The HCC public database and clinical results showed that FoxM1,MAT2 A,and MAT2 B mRNA levels were higher in HCC than adjacent paracancerous tissues,whereas MAT1 A mRNA levels were lower in HCC.HCC clinical samples showed that FoxM1 was negatively correlated with MAT1 A and positively correlated with MAT2 A and MAT2 B.2.In HepG2 and MZCHA-1 cells,silencing FoxM1 could increased MAT1 A expression and promoter activity,decreased MAT2A/MAT2 B expression and promoter activity.Whereas over-expression of FoxM1 was reversed.3.Luciferase assays showed that silencing FoxM1 increased MAT1 A promoter activity and decreased MAT2 A and MAT2 B promoter activity.Importantly,there is no effect on mutant MAT1 A,MAT2A,and MAT2 B activity.EMSA and ChIP experiments further detected that the MATs on FoxM1 were able to bind to the nuclear proteins of HepG2 cells,and FoxM1 could directly regulate MATs.4.FoxM1 inhibitors FDI-6 and TO901317 significantly reduced the expression of FoxM1,MAT2 A and MAT2 B,increased the expression of MAT1 A.5.The growth rate of xenograft tumor in the group treated with TO901317 was significantly lower than the control group.The tumor size was smaller and the weight was lighter in the group of TO901317.6.The results of MTT and wound healing assay indicated that crosstalk between FoxM1 and MATs affect the proliferation and migration in HCC cells.7.Silencing MAT1 A upregulated the expression of FoxM1,MAT2 A and MAT2 B.After silencing MAT2 A,the expression of FoxM1 and MAT2 B was down-regulated while the expression of MAT1 A was increased.After silencing MAT2 B,the expression of FoxM1 and MAT2 A decreased and the expression of MAT1 A increased.In contrast,when the MATs was overexpressed,the results was the opposite.CONCLUSIONS We have unveiled a novel crosstalk between MATs and FoxM1: MAT1 A and FoxM1 regulate each other reciprocally,whereas MAT2A/MAT2 B and FoxM1 regulate each other positively.This crosstalk can further enhance tumorigenesis.