| Objective:Previous studies have found that the expression of chemokine receptor CCR7 in esophageal squamous cell carcinoma cell lines is higher than that of esophageal cell lines.It is found that CCR7 may be associated with NF-kappa B signaling pathway through esophageal cancer cell lines,and the increase of VEGF-A,VEGF-C,IL-6,IL-8,TGF-?,TNF-?expression promotes angiogenesis in esophageal squamous cell carcinoma.However,it has not been verified in human tissue samples,therefore this study aims to explore the molecular mechanism of chemokine receptor CCR7 promoting esophageal squamous cell carcinoma by analyzing the sequencing database of esophageal squamous cell carcinoma and combining it with clinical samples.Methods:1.Bioinformatics analysis of TCGA esophageal squamous cell carcinoma database,screening for specific molecular markers related to CCR7 and finding the signaling pathway associated with CCR7 by GESA enrichment analysis.2.The cancer tissues and adjacent tissues of 20 patients with esophageal squamous cell carcinoma were enrolled.The expression levels of CCR7 protein and gene were detected by Western blot,IHC and RT-PCR.The protein expression levels of IKK and IkB?of samples,the key indicators of NF-kappa B signaling pathway,were detected by Western blot and IHC.The protein expression level of CD34 was detected by Western blot and the expression of Microvessel density was detected by IHC technique.The detection of VEGF-A,VEGF-C,IL-6,IL-8,TGF-?,TNF-?,HIF-1?downstream of NF-kappa B signaling pathway was carried out by Western blot,IHC and RT-PCR,to find out the cause of CCR7 promoting angiogenesis.Results:1.Using the linkedomics online TCGA database(http://www.linkedomics.org/admin.php)to initially query the difference of 20104miRNA seq in 96 cases of esophageal squamous cell carcinoma.Screening forCCR7-related genes:20104 miRNA seq expression levels,based on the miRNA seq expression levels in the data were statistically analyzed for correlation:P valueanalysis showed:<0.05,6191 genes in total;<0.01,3759 genes in total.Analysis by FDR value:<0.05,3639 genes in total;<0.01,2093genes in total.GSE was used to analyze the effects of CCR7 on other biofunctional gene sets,and KEGG PATHWAY analysis was performed on the selected 20104 genes(analytical parameters:P<0.05,Enrichment change>2.0,Count>5).The results showed that CCR7 high expression of tumor samples enriched Cell adhesion molecules(CAMs);Hematopoietic celllineage;Intestinal immune network for IgA production;Type I diabetes mellitus;Staphylococcus aureus infection;Asthma;Autoimmune thyroid disease;Allograft rejection;Graft-versus-host disease;Primary immunodeficiency;Th1 and Th2 cell differentiation;Inflammatory bowel disease(IBD);Antigen processing andpresentation;Chemokine signaling pathway;T cell receptor signaling pathway;Cytokine-cytokine receptor interaction;Viral myocarditis;Leishmaniasis;Th17 cell differentiation;NF-kappa B signaling pathway;Rheumatoid arthritis;Africantrypanosomiasis;Malaria;B cell receptor signaling pathway;Natural killer cellmediated cytotoxicity.The NF-kappa B signaling pathway,which is closely related to the tumor and consistent with the previous experimental results,was selected forsubsequent experiments.Enrichment analysis of the affected signal molecules in the NF-kappa B signaling pathway includes 38 signaling factors:ATM;BCL2;BCL2A1;BIRC3;BLNK;BTK;CCL19;CCL21;CCL4;CD40;CD40LG;CFLAR;CXCL12;CXCL2;GADD45B;ICAM1;LAT;LCK;LTA;LTB;LY96;LYN;MAP3K14;NFKB2;PLCG2;PRKCB;PRKCQ;TLR4;TNF;TNFAIP3;TNFRSF13C;TNFSF11;TNFSF13B;TNFSF14;TRAF1;TRAF5;VCAM1;ZAP70.The results showed thatthe different functions of the affected NF-kappa B signaling pathway were transmitted through two key proteins,IKK and IKB?.2.Western blot analysis showed that the expression of CCR7 protein in esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues(Z=-2.029,P=0.042).IHC detects CCR7 protein,which is localized to the cytoplasm of tumor tissues,and its positive expression is brown.In tissues adjacent to the cancer,the cytoplasm of some cells of CCR7 were expressed in white or pale yellow.The results showed that the expression of CCR7 protein in esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues(Z=-4.790,P=0.000).The expression level of CCR7 protein in tumor tissues was positively correlated with the location of the primary tumor(?~2=10.279,P=0.036)and the N stage of the tumor(?~2=12.781,P=0.012);However,there was no significant correlation with lesion size,gender,the degree of differentiation of tumor cells,age,and T stage M(P>0.05).RT-PCR was used to analyze the expression of CCR7 mRNA in esophageal squamous cell carcinoma was higher than that in adjacent tissues,Median(interquartile range)of CCR7 mRNA 2~-??CT??CT values:28.416[0.452,855.519].3.The expression levels of P-IkB?and T-IkB?in esophageal squamous cell carcinoma were significantly higher than those in adjacent tissues(P<0.05).Detected by Western blot,the relative expression level of P-IkB?/T-IkB?phosphorylation in esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues(Z=-2.236,P=0.020).The expression levels of P-IKK and T-IKK in esophageal squamous cell carcinoma were significantly higher than those in adjacent tissues(P<0.05).The relative expression of P-IKK/T-IKK phosphorylation in esophageal squamous was no significant difference between cell carcinoma and tissues adjacent to cancer(Z=-0.216,P=0.829).The positive expressions of IkB?and IKK were all located in the nucleus;the positive expressions of P-IkB?and P-IKK were all located in the cytoplasm and nucleus,and the positive expression in Esophageal squamous cell carcinoma was higher than that in adjacent tissues(P<0.05).Western blot analysis showed that CCR7 and P-IkB?/T-IkB?showed a linear relationship(R=0.610,P=0.000),and CCR7 and-log(P-Ikk/T-Ikk)also showed a linear relationship(R=0.508,P=0.001);IHC technique showed correlation between CCR7 and IkB?expression in ESCC tissues(r_s=0.581,P=0.007);The expression of CCR7 and IKK was correlated(r_s=0.756,P=0.000);The expression of CCR7 and P-IkB?was correlated(r_s=0.745,P=0.000);The expression of CCR7 and P-IKK was correlated(r_s=0.768,P=0.000).4.Western blot analysis showed that the expression of CD34 protein in esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues(Z=-2.516,P=0.012),CCR7 and CD34 showed a linear relationship(R=0.610,P=0.000).The positive expression of MVD in esophageal squamous cell carcinoma was significantly higher than that in adjacent tissues by IHC(Z=-5.419,P=0.000).5.Western blot analysis was used to detect the expression of VEGF-A,VEGF-C,IL-6,IL-8,TGF-?,TNF-?and HIF-1?:The expression levels of VEGF-A,VEGF-C,IL-6,IL-8,TGF-?,TNF-?and HIF-1?protein in esophageal squamous cell carcinoma were significantly higher than those in adjacent tissues(P<0.05).Results of VEGF-A,VEGF-C,IL-6,IL-8,TGF-?,TNF-?,HIF-1?mRNA 2~-??CT??CT detected by RT-PCR showed that the expression of cancer tissues was higher than that of adjacent tissues.Conclusion:The chemokine receptor 7 has high expression in esophageal squamous cell carcinoma,and promotes downstream promotion of angiogenic factors HIF-1?,VEGF-A,VEGF-C,IL-6,IL-8,TGF-?,and TNF-?by regulating NF-kappa B signaling pathway.The targets are expressed and secreted into the tumor microenvironment,thereby promoting angiogenesis of tumor cells. |
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