The Role And Molecular Mechanism Of ILT4 And HLA-G In Colorectal Cancer Progression

BackgroundColorectal cancer(CRC)is one of the most common malignancies worldwide in terms of morbidity and mortality,with recurrence rate and poor prognosis.Moreover,most patients have already had metastasis at the early stage of treatment,so finding new targets for CRC treatment and understanding the invasion and metastasis process of CRC can provide new ideas for its treatment.Immunoglolbulin-like transcript(ILT)4 is a transcription inhibitory receptor in Immunoglobulin-like transcripts family.Encoding by 19 chromosomes gene,ILT4 is mainly distributed in antigen presented cells(APC)like mononuclear cells,macrophages and dendritic cells(DC).In recent years,studies have shown that ILT4 not only plays an immunosuppressive role in immune cells,but also is highly expressed in a variety of solid tumors,such as non-small cell lung cancer,breast cancer and gastric cancer,promoting tumor progression and affecting patients’survival.In addition,in vitro and in vivo experiments in non-small cell lung cancer have also demonstrated that ILT4 can promote tumor invasion,metastasis and other malignant biological behaviors by activating ERK pathway,thus promoting tumor development.Human leucocyte antigen-G(HLA-G)is an immune tolerance molecule initially discovered in trophoblast cells of pregnancy,which plays an immune tolerance role in autoimmune diseases,organ transplantation,maternal and fetal immunity and immune escape of tumors.In recent years,a number of experiments have shown that ILT4 and HLA-G,as classical ligand receptors,play the role of the immunosuppressive axis in human immunodeficiency virus infection,recurrent transplant failure,systemic lupus erythematosus and other diseases.Previous experiments of our research group have shown that ILT4 and HLA-G can be co-expressed in non-small cell lung cancer and lead to shortened patient survival and tumor progression.However,the expression and role of ILT4 in CRC are still unclear.ObjectiveTo detect the co-expression of ILT4 and HLA-G in CRC tumor tissues and cell lines;To analyze the effect of co-expression of ILT4 and HLA-G on the prognosis of CRC patients;To explore the effect of interaction between ILT4 and HLA-G on the proliferation,invasion,migration and other malignant biological phenotypes of CRC cells;To clarify the molecular mechanism of the interaction between ILT4 and HLA-G in the promotion of CRC development.Methods1.The expression of ILT4 and HLA-G in the CRC tumor tissues and adjacent nonnal tissues of 88 patients was detected by Immunohistochemical staining,the correlation between the expression of ILT4 and HLA-G and the association between their co-expression and clinicopathological parameters of CRC patients were analyzed.2.Follow-up data of CRC patients were collected,overall survival curves were generated via the Kaplan-Meier method,and the relationship between the co-expression of ILT4 and HLA-G and prognosis was analyzed by log-rank.3.The expression level of ILT4 and HLA-G in FHC,HCT116,HT29,SW480 and SW620 cell lines were detected by RT-PCR and Western Blot assay.4.Plasmid pGPU6/GFP/Neo-shILT4-1(sh ILT4)was added to HCT116 and SW620 cells to downregulate the level of ILT4,Plasmid Pez-lv105-ILT4(ILT4)was added to HT29 and SW480 cells to upregulate the level of ILT4.Non-targeting plasmid(sh NC)and plasmid Pez-1v105(NC)were used as negative controls.RT-PCR and Western Blot were used to detect the expression of HLA-G.Cell proliferation ability was evaluated by CCK-8 assays,migration ability was investigated by wound-healing assay and Transwell migration assay,invasion ability was measured by Transwell invasion assay.5.HT29 cells were treated with HLA-G fusion protein at concentrations of 10,20,50,100,200 and 500 ng/mL.The cells were analyzed at 24 h by RT-qPCR and at 48 h by western blot assay.The ability of proliferation,migration and invasion were examined.6.HT29 cells were treated with anti-ILT4 blocking antibody for 12 h,then,cells were treated with 200 ng/mL HLA-G fusion protein.The protein levels of p-ERK and p-AKT were detected by western blot.The ability of proliferation,migration and invasion were examined.Results1.Co-expression of ILT4 and HLA-G was detected in human CRC tissues.In total,68.18%(60/88)of the tissue samples had overexpression of ILT4 and 59.09%(52/88)of the tissue samples exhibited overexpression of HLA-G.There was an evident positive correlation between ILT4 and HLA-G expression(P<0.0001),ILT4 and HLA-G expression was too weak to be observed in adjacent normal colorectal tissues.2.Co-expression levels of ILT4/HLA-G are closely associated with the clinicopathological factors and prognosis of patients with CRC.As compared with the ILT4-/HLA-G-group,ILT4/HLA-G co-expression in the ILT4+/HLA-G+ group was significantly associated with male sex(P = 0.025),increased lymph node metastasis(P = 0.041)and decreased survival probability(P =0.032).As compared with the ILT4+/HLA-G-group,ILT4/HLA-G co-expression was correlated with advanced TNM staging(P = 0.001)and decreased survival probability(P = 0.043).Older age(P = 0.042),male sex(P = 0.001),increased lymph node metastasis(P = 0.038)and advanced TNM staging(P = 0.030)were closely associated with ILT4/HLA-G co-expression when compared with the ILT4-/HLA-G+ group.3.Co-expression of ILT4 and HLA-G was detected in CRC cells.Compared with normal cells,CRC cells(HCT116、HT29、SW480、SW620)exhibited much higher levels of ILT4 and HLA-G expression,and the expression were coherent.4.Interference of ILT4 expression affects the levels of HLA-G and regulates CRC cell proliferation,invasion and migration.The overexpression of ILT4 in HT29 and SW480 cells can upregulate the expression of HLA-G in both mRNA and protein level.Also,ILT4-overexpressing HT29 and SW480 cells had a markedly increased capacity for proliferation,migration and invasion compared with the negative control.Similarly,HLA-G expression was markedly downregulated when ILT4 expression was reduced in HCT116 and SW620 cells.Downregulation of ILT4 notably reduced the proliferation,migration and invasion of HCT116 and SW620 cells.5.HLA-G fusion protein upregulates the ILT4 level in CRC cells in a dose-dependent manner.HT29 cells,which express a low level of ILT4/HLA-G,was treated with different concentrations(10,20,50,100,200 and 500 ng/mL)of HLA-G fusion protein.HLA-G fusion protein obviously upregulated the expression of ILT4 at the mRNA and protein levels in a dose-dependent manner6.HLA-G fusion protein induces the activation of AKT and ERK protein and promotes the proliferation,migration and invasion of CRC cells through binding with ILT4.To identify the underlying molecular mechanisms of ILT4/HLA-G in CRC progression,HT29 cells were treated with 200 ng/mL HLA-G fusion protein,and the expression levels of ERK and AKT were analyzed.The results demonstrated that the phosphorylation of ERK and AKT were markedly enhanced,while the total ERK and AKT levels did not alter.Simultaneously,following stimulation by HLA-G,HT29 cells exhibited a higher capacity for proliferation,migration and invasion.Then,we block the ILT4 expression in HT29 cells via anti-ILT4 antibody.Notably,the upregulation of phospho-ERK and phospho-AKT expression induced by HLA-G was no longer apparent,and the cell proliferation,invasion and migration of HT29 was markedly reducedConclusion1.ILT4 and HLA-G are highly expressed in CRC tissues and cells,and are significantly correlated with age,gender,lymph node metastasis,tumor stage and prognosis of patients.2.ILT4 and HLA-G are co-expressed in CRC cell lines,and the co-expression can promote the proliferation,migration and invasion behaviors of cells.3.HLA-G fusion protein induces the activation of AKT and ERK protein and promotes the malignant biological behaviors of CRC cells through binding with ILT4.