FOXM1 Promotes The Metastasis Of Colon Cancer By Up-regulating DNMT1/MAT2A/MMP9 Signal Axis

BACKGROUND Most colon cancers have developed into advanced stages with metastasis when diagnosed.However,the specific molecular mechanism has not yet been elucidated.Studies have shown that forkhead box（FOX）M1 abnormally highly expressed in cancer tissues,promotes the cell proliferation and up-regulates the expression of methionine adenosyltransferase（MAT）2A,all of which closely related to the poor prognosis of the patients.The main function of MAT2A is to inhibit the synthesis of S-adenosylmethionine（SAM）.As the main methyl donor,SAM lacking will lead to the abnormal methylation.The degree of MAT2A methylation in cancer tissues was significantly reduced,suggesting that MAT2A’s promotion of cancer progression may be related to DNA methylation.The main role of DNA methyltransferase（DNMT）1 is to promote the process of gene methylation.Therefore,this article mainly discusses the research of FOXM1 promoting the metastasis of colon cancer through DNMT1/MAT2A/MMP9 signal axis,which is expected to provide a new idea and method for the prevention and treatment of colon cancer.METHODS 1.Firstly,colon cancer clinical samples were collected.HE staining was used for pathological identification.Immunohistochemistry and Western blot were used to detect the localization and quantitative expression of proteins such as FOXM1,DNMT1,MAT2A and MMP9.At the same time,the mRNA expression of FOXM1,DNMT1,MAT2A and MMP9 was detected using qRT-PCR.After analyzing the TCGA and GSE databases,the different expression of FOXM1,DNMT1,MAT2A and MMP9 in colon cancer especially metastatic and adjacent colon tissues.Furthermore,the correlation among them was analyzed.2.MC38 mouse colon cancer cells were injected into the spleen to construct a model of colon cancer liver metastasis,and the next day,the FOXM1 inhibitor named FDI-6 was intraperitoneally injected.The mice were sacrificed 28 days after the drug administration.The tumor tissues were weighed,the liver was exposed,and the liver tissues were soaked in formalin and made into sections for HE staining for pathological identification.Immunohistochemistry and qRT-PCR were used to detect the expression of FOXM1,DNMT1,MAT2A and MMP9.3.HCT116 and SW480,as two kinds of human colon cancer cells,were cultured in vitro.MTT was used to detect the effect of FDI-6on the viability of cancer cells.The expression of FOXM1,DNMT1,MAT2A and MMP9 and the effects on the migratory ability of HCT116and SW480 cells were observed before and after FDI-6 treatment.Moreover,the expression of FOXM1,DNMT1,MAT2A and MMP9 and the effect on the migration were detected after RNA interference using to silence FOXM1 expression;Subsequently,MTT was tested the effect of DNMT inhibitor 5-Aza-CdR on the viability of colon cancer cells.The expression of DNMT1,MAT2A and MMP9 and the cell migration were examinated after 5-Aza-CdR or RNA interference down-regulating DNMT1 expression.At last,RNA interference knocking down MAT2A expression was detected the effect on the expression of MAT2A and MMP9 and the ability of cell migration.RESULTS 1.The results of HE staining showed that the mucosal structure of the adjacent colon tissue was clear and distinguishable,the glands of the colon were arranged neatly,and the goblet cells were normal in shape.The mucosal structure of colon cancer tissue was disordered,the glands severely destroyed,the nucleus to cytoplasm ratio of colon cells increased significantly,and a large number of goblet cells disappeared.The results of immunohistochemistry found that FOXM1,DNMT1 and MAT2A were located in the nucleus while MMP9 in the cytoplasm.And the expression of these proteins in colon cancer tissues was significantly higher than that in adjacent tissues.Western blot and qRT-PCR results showed that the expression trend was consistent with it.Online analysis of TCGA colon cancer database found that the mRNA expression of FOXM1,DNMT1,MAT2A and MMP9 in N₀ primary colon cancer,N₁ and N₂ colon cancer metastatic tissues were significantly higher than those in adjacent tissues.The screening results of GSE41258colon cancer database were consistent.Then,TCGA database was analyzed and found that high FOXM1 expression indicated a poor prognosis for colon cancer patients.Further correlation analysis through GEPIA found that DNMT1 was positively correlated with FOXM1 and MAT2A in colon cancer.2.HE results showed that the cells in the colon cancer liver metastasis model were disorderly arranged,the liver cell morphology was severely damaged,and the nucleus to cytoplasmic ratio was significantly increased.All above proved the successful construction of the mouse colon cancer liver metastasis model.The morphology of liver cells in the FDI-6 administration group was basically normal,and the structure of hepatic cord and sinusoids could be clearly distinguished.The macroscopic results of the mouse liver also found that the application of the FOXM1 inhibitor FDI-6 significantly delayed the liver metastasis of colon cancer in mice,the liver weight was significantly reduced,the survival time was significantly prolonged,and the effect was significantly better than the positive drug 5-FU treated.In addition,the expression of FOXM1,DNMT1,MAT2A and MMP9 in the liver metastasis model of colon cancer were significantly higher than those in the control group,while the expression levels of FOXM1,DNMT1,MAT2A and MMP9were significantly lower than those in the model group after FDI-6administration.While FDI-6 down-regulates the expression of FOXM1,it could also down-regulate the expression of DNMT1,MAT2A and MMP9;3.FDI-6 could significantly inhibit the viability of HCT116 and SW480human colon cancer cells,down-regulate the expression of FOXM1,DNMT1,MAT2A and MMP9,and significantly inhibit cancer cell migration.The application of RNA interference or FDI-6down-regulating FOXM1 expression or DNMT1 inhibitor 5-Aza-CdR could all significantly inhibit the viability of two colon cancer cells.RNA interference or 5-Aza-CdR sedimentation of DNMT1 expression could also down-regulate the expression of MAT2A and MMP9,and significantly inhibit cancer cell migration.RNA interference down-regulating MAT2A expression could decrease MMP9 expression and inhibit the migration of cancer cells.CONCLUSION FOXM1 can promote the metastasis of colon cancer by up-regulating DNMT1/MAT2A/MMP9 signal axis.