Stathmin1 Promotes Lymph Node Metastasis In Hypopharyngeal Squamous Cell Carcinoma Via Regulation Of HIF-1α/VEGF-A Axis And MTA1 Expression

Background:Hypopharyngeal carcinoma(HC)is a highly representative and relatively poor prognosis cancer among head and neck tumors(HNC),with squamous cell carcinoma(SCC)accounting for more than 95%of its histopathological classification.Extensive neck lymph node metastasis(LNM)is an important clinical feature of hypopharyngeal squamous cell carcinoma(HSCC),and one of the important factors leading to poor prognosis of HSCC patients.Accurate identification and correct management of neck LNM is an important means to improve the survival rate of patients with HSCC.Molecular mechanisms underlying HSCC determine neck LNM in HSCC,so understanding the molecular biological characteristics of LNM in the neck of HSCC has important clinical significance for guiding its treatment.A large amount of evidence indicates that stathmin1(STMN1)is overexpressed in a variety of human cancers and promotes the appearance of malignant biological behaviors.Among them,LNM in numerous cancers is closely related to the high expression of STMN1.However,whether STMN1 promotes neck LNM in HSCC and its potential mechanism remain to be explored.Objective:In the present study,the association between STMN1 and neck LNM in HSCC and the underlying molecular mechanisms were explored.Methods:1.Eligible cases were recruited from the Second Hospital of Shandong University between January 2015 and December 2020 and from the Shandong Provincial Hospital between January 2018 and March 2021.For eligible cases of HSCC,STMN1 expression and lymphatic vessel invasion(LVI)status in tumor tissues were evaluated with IHC staining.After IHC staining,the association between the expression of STMN1 and neck LNM,and the association between the expression of STMN1 and LVI status were both analyzed.The expression levels of STMN1 in HSCC tissues and normal tissues were analyzed.The data of the gene chip were used for HSCC containing STMN1 from the Gene Expression Omnibus(GEO,http://www.ncbi.nlm.nih.gov/geo/)for this.The expression differences of STMN1 among the HSCC tissues,metastases tissues,and normal tissues were analyzed using R4.2.0 software(http://www.R-project.org).2.After analyzing clinical cases and online database data,we carried out a series of cell experiments,using human short hairpin RNA sequences(shRNA)to down-regulate the expression of STMN1 in FaDu cell line.Functional experiments on cell proliferation,invasion,and migration were conducted to assess the potential of STMN1 to promote invasion and migration.3.In order to explore the molecular mechanisms underlying the promotion of neck LNM in HSCC by STMN1,a bioinformatics analysis was performed to identify potential target genes and pathways of STMN1.The RNA sequencing(RNA-seq)data of HSCC are unavailable in The Cancer Genome Atlas(TCGA),and the enrichment pathways of STMN1 high expression group were analyzed using the Molecular Signature Database v7.5.1 package of GSEA software(P<0.05,False discovery rate(FDR)<0.25 are considered as significant enrichment pathways).At the same time,a target gene prediction analysis was performed using the Rv4.0.3 package from the Assistant for Clinical Bioinformatics website(www.aclbi.com).Reverse transcription-quantitative PCR(RT-qPCR)and western blot(WB)analyses were used to further validate these identified target genes and pathways via a series of cell assays in vitro.Results:1.A total of 117 HSCC cases from the two hospitals were eligible,as per the inclusion criteria,and were included in the present study.IHC staining analysis of the 117 eligible cases in the present study demonstrated that STMN1 was highly expressed in the majority of cases;moreover,high expression of STMN1 was evidently associated with neck LNM in HSCC(Correlation Coefficient:0.631,P<0.001).Further analysis revealed that high expression of STMN1 was also associated with LVI(Correlation Coefficient:0.588,P<0.001).The analyses of included data from gene chips GSE2379 revealed that STMN1 expression was evidently increased in HSCC tissues compared with normal hypopharyngeal tissues,and further increased in HSCC tissues of patients with metastases(P<0.01).2.In cell functional experiments,compared with the FaDu cells in the negative control(NC)shRNA group,the cells in the STMN1 shRNA groups had lower OD450 value(P<0.01);the relative migratory ratios were significantly reduced(P<0.05),and the relative number of invasive cells in the lower chamber were significantly decreased(P<0.05).3.Bioinformatics analysis revealed that the genes in the hypoxia inducible factor1alpha/vascular endothelial growth factor-A(HIF-1α/VEGF-A)pathway were enriched in the STMN1 high expression group.At the same time,the expression level of tumor metastasis-associated protein 1(MTA1)correlated well with that of STMN1.RT-qPCR and western blot analyses confirmed that STMN1 potentially promoted LNM in HSCC via regulation of MTA1 expression and the HIF-1α/VEGF-A activation.Conclusions:1.Based on the clinical sample and online database data analysis,it was identified that STMN1 is highly expressed in HSCC tissues and potentially promotes neck LNM in HSCC.2.The cell functional experimental confirmed that high expression of STMN1 significantly promoted the abilities of FaDu cells proliferation,migration and invasion.3.The studies on underlying molecular mechanisms revealed that STMN1 potentially promotes neck LNM in HSCC via the HIF-1α/VEGF-A axis and the expression of MTA1.