Co-expression Of FOXK1 And Vimentin Promotes EMT,Migration And Invasion In Gastric Cancer Cells

Backgroud and ObjectionFOX(Fork Frame)gene family is a large family of transcription factors,characterized by a fork frame DNA binding region in the molecular structure.At present,a total of 50 members of human FOX family have been found.The main biological functions of many genes in FOX family is involved in cell regulation,epithelial differentiation,embryonic development and organogenesis.In recent years,studys found that FOX family also regulates the expression of other genes through the activity of its transcription factors,and participates in the process of tumorigenesis,growth,differentiation,proliferation,apoptosis,migration and invasion.Forkhead-box K1(FOXK1),is a member of the FOX transcription factor family.The expression of FOXK1 in brain,colorectal and lymph node tumors was much higher than that in corresponding normal tissues,considering that FOXK1 is an oncogenic gene and participates in the occurrence and development of various tumors.Our previous studies showed that FOXK1 induces the invasion and metastasis of gastric cancer as an oncogene.In order to further study the mechanism of invasion and metastasis of gastric cancer mediated by FOXK1,We predicted regulatory genes related to FOXK1 by means of bioinformatics,and selected Vimentin which related to EMT (Epithelial-mesenchymal transition)as candidate regulatory genes.In this study,we aim to investigate the interaction between FOXK1 and Vimentin,and how they influence EMT,to further elucidate the molecular mechanism of EMT regulation in the progression and metastasis of GC,in hope to find a theoretical foundation for potential therapeutic targets of gastric cancer.MethodsFirst of all,qRT-PCR experiment test the expression of FOXK1 mRNA in gastric cancer cells and gastric cancer surgical specimens,compared FOXK1 expression in gastric cancer and normal tissues by tissue microarray(TMA)and immunohistochemistry(IHC);By transfected FOXK1 to over-express the expression of FOXK1 in gastric cancer cells,and reuse Soft agar assay,CCK-8 experiment to assess its impact on the biological characteristics of gastric cancer cells.Then,Co-Immunoprecipitation(Co-IP)experiment to study whether if FOXK1 and Vimentin binding to each other in gastric cancer cells.Then,to observe the intracellular distribution of FOXK1 and Vimentin was studied by immunofluorescence.We tansfered the vector which treated with Cyclohexamide(CHX),a protein synthesis inhibitor to the Gastric cancer cell SGC7901,to investigate the effect of FOXK1 on the stability of endogenous Vimentin.Through the experiments above,we detected the effects of FOXK1 on the proliferation of gastric cancer,FOXK1 interact with Vimentin and have synergistic effects each other.Next,we studied the correlation between FOXK1 and Vimentin expression in gastric cancer by following experiments.1.Detected the expressions of FOXK1 and Vimentin in fresh GC surgical specimens by Western blot,and calculated the gray values were by software(Image J)for further statistical analysis.2.Tissue microarray technique was used to detect the expression of these two genes in gastric cancer surgical specimens and to analyze their correlation with clinical and pathological data.3.By transfected plasmid to overexpress or the expression of FOXK1 and transfected siRNA to knock down Vimentin in FOXK1 overexpressed GC cells,reuse Wound healing experiment and Transwell invasion experiment to assess their impact on the biological characteristics of gastric cancer cells..Then,by observing the cell phenotype,immunofluorescence test Rhodamine experiment and Western blot detection EMT related markers to observe the effects of FOXK1-Vimentin axis on gastric cancer cells EMT.Finally,the in vitro cell experiment and build metastatic tumor model were further validated the effect of FOXK1-Vimentin axis,take the Western blot,IHC and ISH to clarify the molecular mechanism of FOXK1-Vimentin axis on gastric cancer.Results1.An increase in FOXK1 protein in gastric cancer tissues and GC compared with that in the adjacent normal tissue.Overexpression of FOXK1 could promote the proliferation of gastric cancer cells.2.FOXK1 and Vimentin co-localize and intersect in gastric cancer cells.The N-terminal of FOXK1 is involved in the protein interaction with Vimentin.They stabilize each other and have synergistic effects.3.The expression of FOXK1 and Vimentin was up-regulated in gastric cancer tissues,which was related to the degree of differentiation,age,serosal infiltration,lymph node metastasis,and the prognosis of the co-expressed cases was the worst.4.Overexpression of FOXK1 in GC cells can inhibit the expression of E-cadherin,and knockdown of Vimentin can promote the expression of E-cadherin,promote the expression of Vimentin,snai1,MMP2 and MMP9,and knock down Vimentin can promote the expression of E-carhdrein and inhibit the expression of the other proteins mentioned above.Up-regulation of FOXK1,phosphorylation forms of ERK,APK and mTOR of EMT signaling proteins follow.The up-regulation of FOXK1 and the down-regulation of Vimentin decreased with the down-regulation of FOXK1.5.In vitro,over-expression of FOXK1 can promote EMT in gastric cancer cells,and knock down Vimentin can reverse the process of EMT.6.In vivo experiments,FOXK1 can promote lung metastasis of gastric cancer in nude mice,while knocking down Vimentin can inhibit its effect.ConclusionsFOXK1 is markly up-regulates the expression in gastric cancer tissues and promotes the proliferation,invasion and metastasis of gastric cancer cells.it play a role of tumor promoter in gastric cancer.Vimentin has a stable synergistic effect with FOXK1 in gastric cancer cells.Knocking down Vimentin can inhibit the EMT process of gastric cancer cells mediated by FOXK1,thereby inhibiting the invasion and metastasis activity of gastric cancer cells.