FOXK1 Interaction With FHL2 Promotes Proliferation,EMT,Invasion And Metastasis In Colorectal Cancer

Backgroud and ObjectionThe transcription factor FOXK1 as a member of the FOX family,involved in cell growth and metabolism,may play an important role in a variety of tumor development.Studies have shown that FOXK1 was closely related to the occurrence and development of colorectal cancer.FOXK1 can promote the invasion and metastasis of colorectal cancer cells,and affect the prognosis of patients with colorectal cancer.FHL2(four anda halflim protein 2)as a cancer gene,plays an important role in the occurrence and development of gastrointestinal cancer.There are reports in the literature,the protein of FHL2 can be combined with a variety of target proteins and regulate the activity of the target protein,induces epithelial mesenchymal transition(EMT),it is a key protein in the invasion of cancer.It is highly expressed in gastrointestinal cancer,but no expression in normal tissues.At present,there are few studies on the interaction between FOXK1 and FHL2 in myogenic progenitor cells,but here is little research on the mechanism of interaction between them in gastrointestinal tumors.The research aims to explore the interaction of the FOXK1 and FHL2 in colorectal cancer,and evaluate the effect of FOXK1 and FHL2 expression on the prognosis of colorectal cancer,which provide the basic theory for exploring new therapeutic targets for colorectal cancer.Materials and methodsMaterialsSW480 cell,Caco2 cell,SW620 cell,Rabbit anti-human FOXK1 monoclonal antibody,Rabbit/mouse anti-human FHL2 monoclonal antibody,Rabbit anti-human E-cadherin monoclonal antibody,Rabbit anti-human GAPDH monoclonal antibody,Mouse anti-human Vimentin monoclonal antibody,Mouse anti-human MMP2 monoclonal antibody,Mouse anti-human MMP9 monoclonal antibody,Mouse anti-human Snail monoclonal antibody,bovine anti-mouse IgG-TR and goat anti-rabbit IgG-FITC,Rhodamine and phalloidin.Methods1.Collect colorectal cancer tissues;detect the expression of FOXK1 in colorectal carcinoma and normal tissues by qRT-PCR and immunohistochemistry.2.Detect the expression and localization of FOXK1 and FHL2 in human colorectal cancer cells by immunofluorescence.3.Verify the interaction between FOXK1 and FHL2 and investigate the relationship between them by co-immunoprecipitation.4.Objective to evaluate the prognostic value of FOXK1 and FHL2 in patients with colorectal cancer.87 cases of colorectal cancer tissue microarray were stained with FOXK1 and FHL2 respectively.Correlation analysis verified the interaction between FOXK1 and FHL2,and survival analysis to evaluate the prognosis of colorectal cancer.5.Downregulation of FHL2 verified the interaction between FOXK1 and FHL2 in colorectal carcinoma.5.1 The stable expression strain Vector and FOXK1 were established,and on the basis,transient FHL2 siRNA(FOXK1-FHL2 siRNA).5.2 EdU cell proliferation assay,scratch test,Transwell invasion experiment were used to investigate the effect of down-regulation of FHL2 on the proliferation,invasion and metastasis of colorectal cancer cells induced by FOXK1.5.3 Cell morphological changes were observed in the three groups,observe the changes of cytoskeleton by Luo Danming phalloidin staining,Western blot detect of the expression of E-cadherin,Vimentin,Snail,MMP2,MMP9 between three groups.Explore the effect of down-regulation of FHL2 on FOXK1 induced colorectal cancer cell line EMT.6.Lymph node metastasis in patients with colorectal cancer was collected,and the expression of FOXK1 and FHL2 in metastatic tumor was examined by immunohistochemistry.7.To verify the effect of FHL2 expression on the invasion and metastasis induenced by FOXK1,the model of subcutaneous tumor formation,and the model of liver metastasis under the membrane of the spleen and the lung metastasis model of tail vein in nude mice were established.Results1.The expression of FOXK1 in colorectal cancer was higher than that in normal tissues;2.FOXK1 and FHL2 were positively correlated with the prognosis of colorectal cancer;3.FOXK1 and FHL2 have synergistic effects in colorectal cancer;4.In vivo and in vitro inhibition of FHL2 inhibited FOXK1 induced proliferation,invasion,and metastasis of EMT cells.ConclusionFOXK1 and FHL2 have synergistic effects on proliferation,invasion and metastasis of colorectal cancer.Therefore,FOXK1 and FHL2 may serve as putative targets in the combined therapy of CRC.