Construction Of Pyroptosis-Related Prognostic Signature For Cervical Squamous Cell Carcinoma And The Effect Of EphB6 In Cervical Cancer Progression

Cervical cancer(CC)is one of the common gynecological malignancies,ranking the fourth in both morbidity and mortality of cancer among women,as well as the second in developing countries.Squamous cell carcinoma is the main pathological type of cervical cancer.The prognoses of patients with recurrence or metastasis are usually poor.In order to improve clinical outcome,it has important clinical significance to further explore the new mechanism of oncogenesis and tumor progression,and find accurate prognostic biomarkers and effective therapeutic targets.Pyroptosis is a newly discovered form of programmed cell death,which results in cell swelling with ballooning bubbles and efflux of intracellular active compounds,activating strongly inflammatory responses.Pyroptosis acts as an important natural immune response,which plays an important role in antagonizing infection and endogenous danger signals.Pyroptosis is widely involved in the occurrence and development of infectious diseases,nervous system diseases and tumors.For tumor,it is a double-edged sword.It can inhibit growth of tumor by promoting cell death,and also can promote progression of tumor by forming a suitable microenvironment.Therefore,it is a new therapeutic target on inducing pyroptosis of tumor cells.Recently,the number of studies on pyroptosis in cervical cancer is still small.It will provide new ideas for clinical prevention and treatment to deeply study the effct of pyroptosis on cervical cancer.In this study,bioinformatics methods were used to construct pyroptosisrelated prognostic signature for cervical squamous cell carcinoma(CSCC)firstly.Then the relationships among the risk model,tumor mutation burden(TMB)and tumor immune microenvironment were investigated,which establish a theoretical foundation for further understanding on the role of pyroptosis in CC.We found that EphB6 in the signature was closely associated to the prognosis of not only CSCC but also CC.However,the specific mechanism of its effect in CC has not been reported.Whether EphB6 promote the progression of CC through pyroptotic pathway remains unknown.Therefore,we further studied the expression of EphB6 in CC and its relationship with clinicopathological factors and clinical outcome.The effects of EphB6 on proliferation,invasion and migration of CC cells were analyzed by up-and down-regulating expression of the gene.The mechanism of EphB6 was explored in CC preliminarily.This study provides a new theoretical basis for the molecular mechanism of pyroptosis in the occurrence and development of cervical cancer.Part Ⅰ: Construction of pyroptosis-related prognostic signature for cervical squamous cell carcinomaObjective:To establish pyroptosis-related prognostic signature for CSCC and evaluate its predictive value of prognosis.To analysis the TMB and immune microenvironment characteristics in the signature,even to predict the immune therapeutic response.Methods: Firstly,differentially expressed pyroptosis-related genes(PRGs)were screened between tumor and adjacent tissue in TCGA database.Consensus clustering based on differentially expressed PRGs was performed.Then,the differentially expressed genes(DEGs)between clusters were identified.Univariate,least absolute shrinkage and selection operator(LASSO)and stepwise multivariate Cox regression analyses were applied to establish a prognostic model.GEO database was used to validated externally.In addition,univariate and multivariate regression analyses were performed to assess the prognostic value of risk score.Independent prognostic factors were used to drew a nomogram and evaluated its predictive value.We explored functional enrichment analysis,TMB,immune microenvironment characteristics,and immunotherapy response associated with the model.Furthermore,the expressions of model genes and their relationships with prognoses of cervical cancer were explored.Results: A total of 20 differentially expressed PRGs were identified between CSCC and adjacent tissue in TCGA database.CSCC was divided into two clusters by performing consensus clustering based on differentially expressed PRGs.The DEGs between clusters were used to develop a seven-gene prognostic signature.Patients with CSCC were divided into two groups based on median risk score,and patients in the low-risk group had significantly longer survival time than those in the high-risk group.These findings were validated using GEO data.We also established a nomogram,to expand the clinical applicability of our findings.Furthermore,the risk model was linked to TMB and various molecular pathways,including immune response,inflammatory chemotaxis activity and viral protein interaction.Moreover,The characteristics of immune microenvironment in the risk model could be used to predict immunotherapy response.CST7 and EPHB6 was found to be strongly associated with cervical cancer prognosis.Conclusions: The pyroptosis-related risk signature consisting of seven genes developed here represents a potential robust biomarker for predicting prognosis and immunotherapy response in patients with CSCC.Part Ⅱ: The expression and clinical significance of EphB6 in cervical cancerObjective:To study the expression of EphB6 in cervical cancer,and the relationship with clinicopathological factors and prognosis.Methods: Real-time fluorescent quantitative PCR(RT-q PCR)and western blot were used to analyze the expression difference of EphB6 in Ect1 and Si Ha cells.Then,RT-q PCR and immunohistochemical methods were performed to explore the expression difference of EphB6 in normal cervical tissues and cervical cancer tissues.In addition,the relationships between EphB6 protein expression and clinicopathological factors and prognosis in cervical cancer was investigated.Results: On both mRNA and protein level,the expressions of EphB6 in Si Ha cells were lower than those in Ect1 cells.The similar results were observed in cervical cancer tissues comparing with normal cervical tissues,and the differences were significant(P<0.05).The expressions of EphB6 in cervical cancer were linked to clinical stage,survival time,survival status,tumor size,lymphatic vascular infiltration,and lymph node metastasis(P<0.05),but unrelated to age,pathological type and the degree of differentiation(P<0.05).Conclusions: The expression of EphB6 in cervical cancer had a lowregulation trend on the levels of both mRNA and protein.Patients with low expression of EphB6 had worse prognosis.Part Ⅲ: The effect and mechanism of EphB6 on the function of cervical cancer cellObjective:To explore the effect of EphB6 on the proliferation,invasion and migration of cervical cancer cells and explore its mechanism preliminarily.Methods: Lentiviral transfection technology was used to transfect human cervical cancer cell lines Si Ha to construct cervical cancer cell lines with stable knock-down and overexpression of EphB6.Subsequently,CCK8 and transwell tests were used to detect the effect of up /downregulation of EphB6 expression on the proliferation,invasion and migration of cervical cancer cells.cervical cancer cells Si Ha were transfected with LPS to induce pyroptosis.The morphological changes were observed by light microscope.After knockdown/overexpressing EphB6,RT-q PCR was performed to detect the expression of caspase-4 and IL-1β in cervical cancer cells.Furthermore,the key proteins of pyroptosis non-classical pathway and EMT signaling pathways were detected by western blot.Results: The proliferation,invasion and migration of cervical cancer cells were promoted while the expression of EphB6 was knock-down.Conversely,overexpression of EphB6 inhibited the proliferation,invasion and migration of cervical cancer cells.Intracellular LPS transfection stimulation resulted Si Ha cells pyroptosis.The expression of EphB6 was negatively correlated with caspase-4 and IL-1β.Down-regulating the expression of EphB6 could promote non-classical pyroptosis pathway mediating by caspase-4,resulting in increased cleavages of GSDMDs.It also could promote EMT signaling pathway to accelerate aggressive behavior of cervical cancer cells.Conclusions: EphB6 can affect the ability of proliferation,invasion and migration of crevical cancer cells.The mechanism may be achieved by regulating pyroptosis non-classical approach and EMT pathways.