The Molecular Mechanism Of FOXQ1 Gene In Tumor Microenvironment Regulation

Angiogenesis and inflammatory microenvironment plays an important role in tumor development and metastasis.Therefore,anti-tumor angiogenesis and inhibition of inflammatory microenvironment are important research directions in the field of anti-tumor therapy.The search for new targets is of great significance for the development of new strategies for anti-tumor angiogenesis and inhibition of inflammatory microenvironment.Previous studies found that FOXQ1,one of the important member of the Forkhead box proteins family,plays an important role in the occurrence,development and metastasis of Colorectal cancer(CRC).However,the function and mechanism for the FOXQ1 gene in CRC tumor angiogenesis and tumor microenvironment(TME)modification are still unclear.The present work aim to study the role and molecular mechanism of FOXQ1 gene in CRC tumor angiogenesis and TME modification,the result will providing a new target for CRC anti-tumor angiogenesis and TME regulation.The main research methods and results are:Firstly,DLD1-shFOXQ1 and HCT116-FOXQ1 cell lines with stable FOXQ1knockdown or overexpression,and corresponding control cells were constructed.The effects of CRC cells and Culture media(CM)on the proliferation,migration and capillary-like tube structure formation of co-cultured HUVECs cells were detected by CCK8,Transwell and microvessel morphogenesis experiments,respectively.The results preliminarily confirmed that FOXQ1 gene can promote the proliferation,migration and microvessel morphogenesis of HUVECs cells in vitro.Then the subcutaneous xenograft model of DLD1-shFOXQ1 was constructed to observe the tumor growth,Microvascular Density(MVD)count was performed by CD31 and CD34 staining,the infiltration of macrophages in the tumor was detected by F4/80staining.The results demonstrated that FOXQ1 promote the CRC tumor angiogenesis and recruitment of tumor-associated macrophages(TAMs)into TME in vivo.Secondly,gene expression changes in DLD1-shFOXQ1 were detected by using Angiogenesis,PI3K-AKT and EGF/PDGF signal pathway SuperArray.WB was performed to verify expression changes of several proteins;Autocrine chemokines of DLD1-shFOXQ1 was detected by qRT-PCR.Secretion of CCL2 and CCL16 in CM were detected by ELISA.The results showed possible molecular mechanism that FOXQ1 gene plays a role in tumor angiogenesis of CRC is that FOXQ1 promotes the secretion of angiogenic factors such as PDGF/PDGFRB,PLAUR,VEGF,EGF,HB-EGF,etc.in the EGF/PDGF signaling pathway,and simultaneously reduces the secretion of angiogenic inhibiting factors such as ANG-1 of CRC cells.In order to study autocrine and paracrine ability of DLD1-shFOXQ1 cells and its effect on HUVECs transition from vascular endothelial cells(VECs)into tumor endothelial cells(TECs).Antibody microarrays were used for detecting changes of angiogenesis factor in CM of DLD1-shFOXQ1 cells,as well as in cell lysis of HUVECs which cultured by CRC CM.FOXQ1 was found to play an important role in the transformation of HUVECs from VECs to TECs by promoting the secretion of ANGPTL4,bFGF,Leptin,CCL2,Follistatin and VEGF,as well as inhibiting the secretion of ANG-2,TIMP-1 and IL-12 by CRC cells.Twist1 over-expression plasmid or si-Twist1 was co-transfected into DLD1-shFOXQ1 and HCT116-FOXQ1cells,respectively,and secretion of CCL2 into CM was detected by ELISA.U937cellls induced into CD68~+Macrophages(M?)by PMA treatment.The effect of Twist1/CCL2 axis on the recruitment of macrophages regulated by FOXQ1 was studied by using Transwell co-culture system mimic to TME.The possible molecular mechanism for FOXQ1 gene to play a role in the recruitment of TAMs into CRC TME is that FOXQ1 promotes the secretion of CCL2 by regulating Twist1/CCL2 axis,thus promoting the recruitment of TAMs into TME.The regulatory effect of FOXQ1on CCL2 is mediated by Twist1.Finally,IHC was performed to detect the expression of FOXQ1,Twist1,CCL2,CD31 and CD68 protein by using tissue samples consisted 83 patients diagnosed with CRC and corresponding paracancerous controls.The results showed that the positive expression rates of FOXQ1,Twist1 and CCL2 were significantly higher in CRC tissue than in adjacent tissues.Correlation between FOXQ1 expression and clinicopathological parameters,between FOXQ1 and tumor angiogenesis(FOXQ1-CD31),as well as between FOXQ1 and TAMs infiltration(FOXQ1-Twist1/CCL2-CD68)was analyzed.The results showed that FOXQ1 expression was significantly correlated with lymph node metastasis and TNM staging.FOXQ1 expression was also positively correlation between FOXQ1 and Twist1,Twist1 and CCL2,and between FOXQ1 and CD68.Kaplan-meier survival rate was used to analyze the correlation and clinical significance of FOXQ1 in tumor angiogenesis and TME modification with 8-year survival rate in CRC patients.The result showed that FOXQ1 can promote tumor angiogenesis and recruitment of TAMs in TME,and is closely related to poor long-term prognosis in CRC patients.In summary,tumor angiogenesis and TAMs recruitment in TME are important factors for the development and metastasis of CRC.FOXQ1 gene plays an important role in CRC tumor angiogenesis and TAMs recruitment.The mechanism is that FOXQ1 mainly promotes the expression of angiogenic factors in EGF/PDGF signaling pathway,reduces the expression of angiogenic inhibiting factors and promotes tumor angiogenesis.The recruitment ability of CRC cells to TAMs was promoted by Twist1/CCL2 axis.FOXQ1 gene is expected to be the target of anti-angiogenesis therapy of CRC and reducing TAMs infiltration in TME.