Expression And Functional Relevance Of ANXA1 In Hypopharyngeal Carcinoma With Lymph Node Metastasis

Purpose:This study aims to investigate the expression of Anexin A1(ANXA1)in lymph node(LN)metastasis of hypopharyngeal squamous cell carcinoma(HSCC),analyze its relationship with clinicopathological features and prognosis of patients with HSCC,study the biological function of ANXA1 in Fadu cells,and preliminarily explore the mechanism of ANXA1 in LN metastasis of HSCC.Methods:The differentially expressed genes were obtained by RNA sequencing of HSCC tissue samples from 3 patients with LN metastasis and 3 patients without LN metastasis.q RT-PCR and Western blotting were used to verify the expression of the target gene ANXA1.The expression of ANXA1 in 74 cases of HSCC and normal mucosa was detected by IHC method.The relationship between the expression of ANXA1 in HSCC and the clinicopathological parameters and the prognosis of the patients were analyzed.The ANXA1 gene in Fadu cells was silenced by si-RNA.Cell proliferation was detected by CCK-8 staining and colony formation assay,cell migration and invasion were detected by Transwell assay,and cell apoptosis and cell cycle were detected by flow cytometry.Bioinformatics was used to analyze the GOBP,KEGG and PPI of the different genes to find the possible mechanism of ANXA1.In Fa Du cells and HSCC tissues,the target gene Yap1 of ANXA1 was verified by q RT-PCR and Western blotting,and the correlation between the expression of ANXA1 and Yap1 was analyzed by Pearson correlation statistical method.The mechanism of ANXA1 and Yap1 was further verified by cell rescue experiment in vitro.Results:A total of 698 DEm RNAs were identified in the HSCC samples between patients with and without LN metastasis,including 278 upregulated genes and 420 downregulated genes.ANXA1 was one of these DEm RNAs.Further verification experiments confirmed that the m RNA and protein expression of ANXA1 in HSCC with LN metastasis was significantly lower than that in normal paracancerous mucosa and HSCC without LN metastasis;IHC analysis also showed that ANXA1 was highly expressed in normal mucosal epithelium.The expression in the group without LN metastasis was significantly higher than that in the tissue of the patients with LN metastasis(P < 0.05).The expression of ANXA1 was correlated with clinicopathological grade and LN metastasis factors of HSCC(P < 0.05),but not with age,gender and tumor stage(P > 0.05).The low expression of ANXA1 is related with the poor prognosis of patients(P<0.05).After silencing ANXA1,the proliferation,invasion and migration ability of Fadu cells were increased,and apoptosis was decreased.In the cell cycle,G0/G1 phase cells were significantly reduced,while S phase cells were significantly increased.The negative regulatory biological processes of epithelial cell differentiation,cell proliferation and apoptosis were significantly enriched.Hippo pathway was highly enriched,and Yap1 was a key factor in this pathway.Yap1 and ANXA1 participated in most of the same biological processes.The analysis of PPI network showed that ANXA1 and Yap1 were the central genes of these pathways and closely related to each other.After silencing the expression of ANXA1 in Fa Du cells,the m RNA and protein expression of Yap1 were significantly down regulated.At the same time,the m RNA and protein expression of Yap1 in HSCC with LN metastasis group was significantly lower than that in LN non-metastasis group,and the m RNA expression of ANXA1 was positively correlated with the m RNA expression of Yap1(P < 0.0001).In vitro cell rescue experiment confirmed that overexpression of Yap1 could reverse the effect of ANXA1 silencing.Conclusion:Detection of ANXA1 expression can effectively predict the LN metastasis and prognosis of HSCC,and provide a new molecular marker for predicting LN metastasis of HSCC.ANXA1 may inhibit the LN metastasis of HSCC by regulating the expression of Yap1,which provides a theoretical basis for studying the mechanism of LN metastasis of HSCC.