Study On Effect And Mechanism Of S100A8 And S100A9 On Prognosis Of Nasopharyngeal Carcinoma

BackgroundS100 calcium binding protein A8(S100A8)and S100 calcium binding protein A9(S100A9)are both family members of the calcium binding protein 5100 calcium binding protein.S100A8 and S100A9 are regarded to play important regulatory effect in tumor cell proliferation,apoptosis,differentiation,invasion and metastasis.At present,there are few studies about the relationship between S100A8,S100A9 and the occurrence or development of nasopharyngeal carcinoma.Objective(1)To analyze the expression level of S100A8 and S100A9 in nasopharyngeal carcinoma and its relationship with clinicopathology and prognosis,and to provide a new therapeutic target for the treatment of nasopharyngeal carcinoma.(2)To observe the effects of S100A8 and S100A9 on the expression level of nuclear factor(NF)-κB,beta-catenin,matrix metdloproteinases-7(MMP7),MMP9,MMP12 and TNF(tumor necrosis factor)-alpha in nasopharyngeal carcinoma cells,and to observe the role of S100A8 and S100A9 in the related pathways in nasopharyngeal carcinoma cells in order to reveal the role of S100A8 S100A9 in nasopharyngeal carcinoma cells,to carry out a new research direction.(3)To study the effects of S100A8 and S100A9 on proliferation,cell cycle,cell apoptosis,cell invasion and movement of nasopharyngeal carcinoma cells,and to elucidate the effect and mechanism of S100A8 and S100A9 on the invasion ability of nasopharyngeal carcinoma cells.Methods(1)A total of 92 specimens of NPC from 92 patients with nasopharyngeal carcinoma at the first time were collected from the Department of Otolaryngology,Wuhan University Renmin Hospital from January 2008 to December 2011,the clinic pathological and survival data were also collected.Another 92 cases of chronic nasopharyngitis in the same period were selected as control group.The expression levels of S100A8 and S100A9 in all the two groups were detected by immunohistochemistry,to analyze the relationship between the expression level of S100A8 and S100A9 and the clinicopathology,prognosis and overall survival.(2)Recombinant RNAi vectors and overexpression vectors of S100A8 and S100A9 were constructed and transfected into CNE-2 cell line of human nasopharyngeal carcinoma.Total RNA was extracted,and the expressions of S100A8,S100A9,NF-B,beta-catenin,MMP7,MMP9,MMP12 and TNF-alpha were detected by RT-PCR and Western blot.(3)The recombinant RNAi vector and overexpression vector of S100A8 and S100A9 were transfected into human nasopharyngeal carcinoma cell line CNE-2 successfully,MTT assay was used to detect the cell proliferation in each group,and the cell doubling time was calculated.BD flow cytometry Cytokines were used to detect the apoptosis in each group.The invasiveness of cells in each group was detected by Transwell cell invasion assay.Scratch test was used to observe the movement characteristics of each group.Results(1)The positive expression rates of S100A8 and S100A9 in nasopharyngeal carcinoma tissues were 52.17%and 72.83%,respectively.The positive rates of expression of S100A8 and S100A9 in nasopharyngeal tissues were 17.39%and 36.96%.The positive expression rate of S100A8 and S100A9 has no correlation with age or gender and in nasopharyngeal carcinoma(P>0.05),but closely correlated with the degree of differentiation,TNM stage and lymph node metastasis(P<0.05).There was a significant positive correlation between S100A8 and S100A9 expression in nasopharyngeal carcinoma(r = 0.393,P = 0.000).Patients of nasopharyngeal carcinoma with high expression of S100A8 and S100A9 had later clinical pathological stages and shorter survival time.(2)Double enzyme digestion showed that the recombinant pEGFP-N1-S100A8 overexpression vector and the recombinant pEGFP-N1-S100A9 overexpression vector were successfully constructed.Inverted fluorescence microscopy showed that each group of cells were transfected successfully.The mRNA and protein expression levels of NF-κB,β-catenin,MMP7,MMP9,MMP12,TNF-α in S100A8-RNAi and S100A9-RNAi groups were significantly lower than those in control groups.The mRNA and protein expression of NF-κB,β-catenin,MMP7,MMP9,MMP12,TNF-α in S100A8-Over expression group and S100A9-Over expression group were significantly higher than those in control groups.(3)Cell number at 24h,48h and 72h in S100A8-RNAi group and S100A9-RNAi group were significantly lower than those in control groups,but cell number at 24h,48h and 72h in S100A8-Over expression group and S100A9-Over expression group were higher than control groups.Cell doubling time was prolonged in S100A8-RNAi group and S100A9-RNAi group,but shortened in S100A8-Over expression group and S100A9-Over expression group.The number of cells transfected through Transwell membrane in S100A8-Over expression group and S100A9-Over expression group was significantly higher than that in control groups.The apoptosis rate of transfection of nasopharyngeal carcinoma CNE-2 cells in S100A8-RNAi group and S100A9-RNAi group was significantly higher than those in control groups at 24 h,48 h and 72 h.The apoptosis rate of S100A8-Over expression group and S100A9-Over expression group was significantly lower than those in control groupsConclusion(1)Nasopharyngeal carcinoma patients with higher expression lever of S100A8 and S100A9 in tissues had later clinical pathological stages and shorter survival.(2)S100A8 and S100A9 could promote the secretion of NF-κBβ-catenin,MMP7,MMP9,MMP12,and TNF-a,so as to promote the occurrence and development of nasopharyngeal carcinoma.(3)S100A8 and S100A9 could promote the proliferation,invasion,motility and inhibit apoptosis of human nasopharyngeal carcinoma CNE-2 cells,thereby promoting the invasion and migration of nasopharyngeal carcinoma cells.