The Effects And Mechanisms Of SEMA4A And ILT4 In Tumorigenesis And Progression Of Human Non-small Cell Lung Cancer

BackgroundAs one of the most common malignant tumors in humans,lung cancer has ranked number one in terms of its high morbidity and mortality,which have seriously threatened human health.According to the histopathological characteristics,lung cancer is divided into non-small cell lung cancer(NSCLC)and small cell lung cancer.,of which NSCLC accounts for about 80-85%of lung cancer.Because of lung cancer metastasis is the main cause of treatment failure in patients,and the mechanism is not completely clear.Therefore,further elucidation of the molecular mechanisms underlying the metastasis and find new biomarkers or therapeutic targets in treatment of NSCLC.is urgently required for improving NSCLC treatment.Semaphorin 4A(SEMA4A)is a member of the Semaphorein Ⅳ family which has four classical receptors:Plexin D family,Plexin B family,Tim-2 and Nrp-1 family.It was initially found to play a role of growth-promoting factor in the development of synapses.Because of SEMA4A is mainly expressed on the surface of immune cells,it is also called "Immunosemaphorin".Recent studies have shown that SEMA4A play a crucial role in many physiological processes,including intercellular interactions,immune cell activation,differentiation and migration.Some studies have found that SEMA4A play a role of oncogene in breast and liver cancer,which promote tumor progression and malignant biological behavior,while others have reported that SEMA4A in inhibiting tumor growth and proliferation.However,the specific function of SEMA4A in NSCLC still need to be further explored.Immunoglobulin-like transcript 4(ILT4)is an immune checkpoint molecule mainly expressed in myeloid cells(including monocytes,macrophages,dendritic cells and granulocytes),which can inhibit the infiltration of immune cells in tumor microenvironment.Our previous studies have shown that ILT4 is overexpressed in diverse malignancies,which is involved in regulating the malignant biological behavior of tumor cells and tumor immune escape as well as poor prognosis of patients.Recent studies have reported that SEMA4A as ligands,can induce Th2 cell differentiation by binding to receptor ILT4,indicating SEMA4A plays its role by binding to the ILT4.To sum up,In view of the important role of SEMA4A/ILT4 in tumorigenesis and tumor development of NSCLC.,we speculate that SEMA4A may modulate tumor cell growth and tumorigenesis via interaction with ILT4.To our knowledge,no researches have reported the co-expression and interaction mechanism of SEMA4A-ILT4 in NSCLC.ObjectiveDetermine the co-expression of SEMA4A/ILT4 in NSCLC tissues and cell lines.Analyze the effect of co-expression on the prognosis of NSCLC patients.Explore the effect of SEMA4A on the malignant biological phenotype of NSCLC cells.Determine the effect of the SEMA4A-ILT4 interaction on the malignant biological phenotype of NSCLC cells.To further explore the molecular mechanism of the SEMA4A-ILT4 interaction.Methods1.The expression of SEMA4A and ILT4 in 87 NSCLC tissues as well as adjacent normal tissues were detected by Immunohistochemical staining,the correlation between their expression levels in NSCLC tissues and the relationship between their expression level and clinicopathological parameters were analyzed.2.Follow-up data of 87 NSCLC patients were collected,survival curves were made via Kaplan-Meier method,log-rank test was used to analyze the relationship between co-expression level of SEMA4A and ILT4 and overall survival time.3.The expression level of SEMA4A and ILT4 in five NSCLC cell lines(H1299、H1975、H1650、A549、H226)and normal lung epithelial cells(BEAS-2B)were detected by real-time quantitative PCR(RT-PCR)and Western blot assay.The cell lines with high expression SEMA4A and low expression SEMA4A were screened for the following experiments.4.NSCLC cell lines with high SEMA4A expression(H1299 and H1975)were downregulated by transfection of siRNA(siSEMA4A)constructed,Using transient transfection of overexpression plasmids to upregulate the low SEMA4A expression cell lines(H1650 and A549).RT-PCR and Western Blot were used to detect the ILT4 expression level in the corresponding cell lines.At the same time,the CCK-8 assay was used to test the cell proliferation ability,Transwell invasion assay was used to test the invasion ability and wound-healing assay was used to test the cell migration ability.5.Different concentration gradients(0,50,100,200,500,1000 ng/ml)of SEMA4A recombinant protein were used to stimulate A549 cells.RT-PCR and Western Blot assay were used to detect ILT4 expression.A549 cells were stimulated by SEMA4A recombinant protein at different concentrations(0 ng/ml and 200 ng/ml).The downstream molecular pathway proteins expression level of p-P38 MAPK and p-ERK were detected by western blot assay.Cell proliferative,migratory,and invasive capacities were assessed by CCK-8,wound-healing assay and Transwell invasion assays,respectively.6.A549 cells was treated anti-ILT4 blocking antibody for twelve hours,then stimulated with 200 ng/ml SEMA4A recombinant protein.The downstream molecular pathway proteins expression level of p-P38 MAPK and p-ERK were detected by Western Blot assay.The ability of cell proliferation,migration and invasion were tested by CCK-8,wound-healing assay and Transwell invasion assays,respectively..Results1.The expression level of SEMA4A and ILT4 in NSCLC tissues were detectedThe IHC assay showed that 55.17%(48/87)tissue samples had high expression of SEMA4A and 52.87%(46/87)tissue samples were overexpressed.The expression levels of SEMA4A and ILT4 were significantly higher in carcinomatous than in adjacent normal tissues.The expression of SEMA4A and ILT4 shows significantly positive correlation in NSCLC tissues.(P<0.001,R=0.476).2.Co-expression of SEMA4A and ILT4 in NSCLC tissues are closely associated with clinicopathological parameters and prognosis.As compared with SEMA4A-group,SEMA4A+group was associated with bigger tumor volume(p=0.002),increased lymph node metastasis(p<0.001),advanced TNM stage(p<0.001)and poor prognosis(p<0.001).High ILT4 expression significantly predicted older age(p=0.044),bigger tumor volume(p=0.025),increased lymph node metastasis(p=0.03),advanced TNM stage(p=0.004),and poor prognosis(p<0.001),respectively.3.Co-expression of SEMA4A/ILT4 are associated with clinicopathological parameters and poor prognosis of NSCLC patients.As compared with SEMA4A/ILT4 co-expression group,SEMA4A-/ILT4-group was associated with small tumor volume(p=0.002),decreased lymph node metastasis(p=0.001),early TNM stage(p=0.001),and favorable prognosis(p=0.001),SEMA4A-/ILT4+group was associated with early TNM stage(p=0.017)and favorable prognosis(p=0.027),SEMA4A+/ILT4-group was associated with favorable prognosis(p=0.033).4.SEMA4A and ILT4 were co-expressed in NSCLC cell lines.RT-PCR and Western Blot assay showed that the expression levels of SEMA4A and ILT4 in NSCLC cell lines(H1299、H1975、A549、H226)were much higher than those in normal lung epithelial cells(BEAS-2B),and the expression levels of SEMA4A and ILT4 was quite consistent.5.In NSCLC cells,over-expression of SEMA4A resulted in up-regulated expressions of ILT4 and promoted NSCLC cell proliferation,migration,and invasion.The overexpression of SEMA4A upregulated the mRNA and protein expression of ILT4 in H1650 and A549.Furthermore,overexpression of SEMA4A promoted NSCLC cell proliferation,migration and invasion in vitro.Similarly,The expression of ILT4 was markedly downregulated when SEMA4A expression was decreased in H1299 and H1975 at mRNA and protein level.Simultaneously,downexpression of SEMA4A inhibited NSCLC cell proliferation,migration and invasion in vitro.6.SEMA4A recombinant proteins can lead to an increased ILT4 expression.The A549 cell lines was stimulated by different concentration gradient SEMA4A recombinant protein.RT-PCR and Western Blot assay showed that the SEMA4A recombinant protein markedly upregulated the expression of ILT4 in a dose-dependent manner at mRNA and protein level.7.The combination of SEMA4A and ILT4 promotes the invasion,migration and proliferation of NSCLC cells byactivating P38 and ERK signaling proteins.The ERK and P38 proteins were significantly activated by SEMA4A recombinant protein stimulation in NSCLC cell lines,and the phosphorylation levels of P38 and ERK were significantly increased,and the invasion,migration and proliferation of NSCLC cells were significantly increased.The expression of NSCLC cell ILT4 was first reduced by anti-ILT4 blocking antibody then up-regulated by SEMA4A recombinant protein.Phosphorylation of ERK and P38 pathway protein was inhibited and no significant increase in p-P38 MAPK and p-ERK was observed,and the increased invasion,migration and proliferation of NSCLC cells were also counteracted.ConclusionsSEMA4A is highly expressed in NSCLC cell lines and tissues and is associated with disease progression and poor prognosis.SEMA4A and ILT4 are co-expressed in NSCLC cell lines and the interaction can promote malignant biological phenotypes such as invasion,migration and proliferation of NSCLC cells.SEMA4A recombinant protein induces the phosphorylation of downstream pathway protein ERK and P38 MAPK and play a tumor-promoting role on NSCLC cells through bind to its receptor ILT4.