IL-17-STAT3 Pathway In Regulating Invasive Behaviors Of Oral Squamous Carcinoma With Clinical Correlations

Objectives:Oral squamous cell carcinoma(OSCC)is one of the most common types of head and neck cancer.Local recurrence,cervical lymph node metastasis,and advanced clinical stage are considered to be the main causes of poor prognosis.Interleukin-17,as an important component of the tumor inflammatory microenvironment,has been shown to be pivotal in a variety of malignancies.It has been proved that in OSCC,IL-17 is present in the tumor microenvironment.It could be secreted by a variety of cells and then plays an important role in the invasion and metastasis of OSCC.However,the underlying mechanism remain largely unknown.Previous studies have found that the emergence of tumor budding in OSCC is closely related to its prognosis,but the mechanism is still unclear.Through bioinformatics analysis,correlation between IL-17 combined with tumor budding and OSCC prognosis and affect of IL-17 on OSCC cancer cell invasion,this study intends to determine a new factor for the evaluation of OSCC prognosis and explore the regulatory effect of IL-17 on OSCC invasion and its mechanism.Material and methods:Three datasets of oral squamous cell carcinoma were selected in the GEO database,and differentially expressed genes(DEGs)were obtained by analysis.Gene Ontology(GO),pathway enrichment analysis and protein interaction network analysis(PPI)were performed respectively.Candidate genes and signaling pathways that may affect OSCC were filtrated finally.Further analysis showed that IL-17-STAT3 pathway may play an important role in the progress of OSCC.A total of 110 OSCC cases were enrolled.The tumor tissues,tumor margins and normal tissues were obtained.The IL-17 level was detected by ELISA and immunohistochemical staining were performed to evaluate the presentation of tumor budding and expression of IL-17 in tumor invasion front(TIF).Then evaluate the relationship between the expression of IL-17 and presentation of tumor budding in the OSCC TIF,as well as analyze the correlation between OSCC prognosis and co-expression of IL-17 and tumor budding.CD44~+/ALDH1~+and CD44~—/ALDH1~—oral squamous cell carcinoma cells were sorted by flow cytometry.Cell colony formation,migration/invasion and subcutaneous tumorigenic ability of nude mice were observed by in vivo and in vitro assays.After the addition of exogenous IL-17,the variation in the invasive ability of CD44~—/ALDH1~—subpopulations was also evaluated.The role and mechanism of IL-17 in regulating the invasiveness of oral squamous cell carcinoma were detected by immunoblotting,Polymerase Chain Reaction(PCR)and migration/invasion assays.Results:Through the bioinformatic analysis of three groups of OSCC datasets,a total of 129differential genes(DEGs)were obtained.After gene ontology,pathway enrichment and protein interaction network analysis(PPI)analysis,genes such as STAT,SPARC,APP,SNAI2,CXCL8,MMP13,SPP1,COL1A2,ITGA6 and IFI27were significantly elevated in OSCC,and pathway such as ECM-receptor interaction,pathways in cancer,transcriptional misregulation in cancer,PI3K-Akt-m TOR pathway and Jak-STAT pathway were also significantly enriched.It was suggested that interleukin and its related pathway may be involved in the development of OSCC.In clinical correlation analysis,ELISA assays showed that IL-17 expression was significantly increased in tumors and tumor margins compared with normal tissues,IL-17expression in tumor margins was higher than tumors but no significant difference.In immunohistochemical staining sections,IL-17 expression was also found to be elevated in tumor and tumor margins,and IL-17 expression level was positively correlated with the presentation of tumor buddings.In multivariate survival analysis,the IL-17 combined with tumor budding was highly correlated with local recurrence rate and poor prognosis.In vitro assays such as immunoblotting,migration/invasion,and colony formation showed that the invasiveness of CD44~+/ALDH1~+subpopulation cells was higher than that of CD44~—/ALDH1~—subpopulation cells.These cells could be considered as a subpopulation of oral squamous cell carcinoma with high/low invasive potential,respectively.After the addition of exogenous IL-17,the ability of CD44~—/ALDH1~—subpopulation in migration/invasion,colony formation,and tumor formation in nude mice were significantly improved.This result suggested that IL-17 may contribute to the process of OSCC invasion.Western blot analysis showed that the expression of phosphorylated STAT3(p-STAT3),vimentin and N-cadherin was significantly increased in CD44~—/ALDH1~—subpopulation cells after addition of exogenous IL-17,while E-cadherin was decreased.When STAT3 inhibitor(Stattic)was added,not only the increased migration/invasive ability,but also the expression levels of vimentin and N-cadherin were significantly decreased.Western blotting,PCR and Transwell results suggested that after treatment of IL-17,the expression of STAT3 was elevated,and that blocking STAT3 could reduce the invasiveness of tumor cells.These results suggested that IL17-STAT3 could play an important role in regulating the invasion of OSCC.Conclusion:Our findings demonstrated that IL-17 combined with tumor budding could be a valuable prognostic marker in OSCC.IL-17 could also regulate tumor cell invasion by STAT3 and play an important role in tumor development.Therefore,targeting IL17-STAT3 may provide new insights for the comprehensive treatment in the patients with OSCC.