The Study Of CCR7 On Effects And Mechanism Of Angiogenesis In Esophageal Squamous Cell Carcinoma

BackgroundEsophageal cancer(EC)is a digestive system malignant tumor with a common high incidence.China has a high incidence of esophageal cancer among the world,with mortality rate occuping the second place,second only gastric cancer.Since more than 90% of the esophageal cancer were esophageal squamous cell carcinoma(ESCC),so this research mainly discussed esophageal squamous cells carcinoma.Although through early esophageal cancer resection,and subsequent radiotherapy,chemotherapy and other strategies that can improve the survival rates of esophageal squamous cell carcinoma.However,these treatments are not easy to achieve effect a radical cure effect,and at the same time,these patients suffer from different degree of regional lymph node metastasis or remote organ metastasis.Development and metastasis of tumor is a complex process with the participation of molecular and gene and various environmental factors.Tumor growth,invasion and metastasis process are closely related to the generation of new blood vessels(angiogenesis).Chemokine receptor is a kind of expression in some 7-transmembrane domain specific cell surface receptors,belonging to a member of the G protein coupled receptor superfamily.In recent years,research has shown that chemokine receptors participate in the new angiogenesis,further influence tumor generation and transfer when different structure of chemokine plays a different role in angiogenesis.Thus chemokine receptor can be a new target of tumor treatment.The current research has confirmed a variety of tumor cells were related chemokine receptor 7(chemokine receptor,when the CCR7),suggesting that chemokine receptor and the development,invasion and metastasis of tumor are closely related.VEGF was considered as vascular permeability factor,related to the proliferation,migration of endothelial cells and the formation of vessel.VEGF family includes VEGF-A,VEGF-B,VEGF-C and VEGF-D.Tyrosine kinase was activated after the binding of VEGF and VEGFR,leding a series of signaling pathway cascade reaction,then stimulating the proliferation,migration and the formation of blood vessel endothelium.NF-ΚB existed almost all animal cells and extracted from B leukomonocyte,considered as a transcription factor.NF-κB possessed various biological activation,acted as a key molecular among inflammatory reaction and tumors.Recent studies suggested that NF-κB had a close relationshio with tumor,it regulated different target genes,involved in many signaling pathway,inhibited apoptosis and promted the proliferation,invading,formation of tumor cells.NF-κB closely related to carcinogenesis.Clinical data showed that when the CCR7 high expression is closely related to the infiltration and metastasis of ESCC.However,it is rarely reported about the expression of CCR7 in ESCC,and how the abnormal CCR7 expression is related to ESCC and other clinical characteristic,or when the CCR7 abnormal expression of induction of clinical cases of ESCC mechanism.ObjectiveSo in this thesis,we first verify whether CCR7 was upregulated in ESCC,and then discussed how CCR7 abnormal expression induced the occurrence and development of ESCC,and further clarify the molecular mechanism.And in the end we will test CCR7 silencing will effectively cure ESCC.We tries to provide theoretical background for esophageal cancer metastasis and recurrence of subsequent research and lay the foundation of CCR7 as new target drugs to clinical development in the future.The first part-Study on the Expression of CCR7 in Esophageal Squamous Carcinoma CellsMethods1.Bioinformatics methods was applied for retrospective sorting and research the different reports in esophageal squamous carcinoma CCR7 expression in tumor tissues and lymph nodes;2.Western Blot(WB)method and Reverse transcription PCR applied to detect CCR7 expression in many different kinds of esophageal squamous carcinoma cells.Results1.CCR7 gene expression in the GEO PROFILE database(https://www.ncbi.nlm.nih.gov/gds/)in the search analysis,the results showed that CCR7 levels increased significantly in esophageal squamous cell carcinoma than in normal tissue.2.We found that all the 6 strains of esophageal squamous cell cancer cells system(TE,TE-1,2,TE,TE-8,TE-12 and ECA-109)when the CCR7 protein expression levels were significantly higher than that in normal human esophageal epithelial cell line HEEC.QRT-PCR results showed that at the mRNA level,when the CCR7 expression in esophageal squamous cell cancer is significantly higher than the normal cells HEEC.The second part-Study on the effects of CCR7 in Cell Proliferation、Migration and Angiogenesis of Eca109Methods1.Set up a stable Eca109 esophageal squamous cell carcinoma cells line and when the CCR7 expression was overexpressed or silenced.2.MMT method was applied to detect cell activity when the CCR7 expression was regulated in Eca109 esophageal squamous cell carcinoma cells.3.Human umbilical vein endothelial cell proliferation and migration of experiments and CAM ability experiment for the detection of blood vessels to detect cell migration ability and the ability of inducing angiogenesis when the CCR7 expression was regulated in Eca109 esophageal squamous cell carcinoma cells..4.Western Blot method was applied to detect VEGF-A and VEGF-C in Eca109 esophageal squamous cell carcinoma cells when the CCR7 expression was regulated.Results1.CCR7 in esophageal squamous cell carcinoma cells Eca109 silence and overexpression system was established in success.2.CCR7 overexpression significantly inhibit cell survival rate of Eca109 cells,significantly increases the migration of human umbilical vein endothelial cells,and enhanced the ability of culture supernatant on inducing angiogenesis;while the CCR7 silence inhibits endothelial cell migration,weakened the supernatant fluid induced angiogenesis.3.At the same time,when the CCR7 overexpression in Eca109 cells significantly increase the expression VEGF-A and VEGF-C protein.On the contrary,CCR7 expression inhibition decreased the expression of VEGF-A and VEGF-C protein.The third part-CCR7 regulates NF-kB pathway in Eca109Methods1.Luciferase report was applied to analysis and verified the CCR7 expression relation with NF-kB pathway.2.Western blot and ELISA experiments were applied to validate expression of NF-kB pathway in regulated CCR7 expression system.Results1.CCR7 protein expression can increase NF-kB report gene transcriptional activity.2.Western blot experimental results confirmed that the over expression of CCR7 in squamous cell carcinoma ECA109 can significantly improve the phosphorylation of IKK and phosphorylation of IkB alpha expression level,but not significantly change the IKK and IkB alpha total protein expression level.3.Western blot experiment and ELISA experiment results show that many NF-kB target genes(including TNF-alpha,IL-6,IL-8 and TGF-beta)were upreguated when the CCR7 expression in esophageal squamous cell carcinoma cells was overexpressed;while that was downregulated in CCR7 silence expression system.The fourth part-Downregulation of CCR7 influents the development of Xenotransplanted Eca109 Tumor ModelMethods1.To establish subcutaneous esophageal squamous cell carcinoma transplanted tumor model in mice.After the group injected with different siRNA vector,changes in the average tumor volume were observed.2.Western bot experiment analyzes the protein expression levels of NF-kB target genes(VEGF-A,VEGF-C,and TNF alpha,IL-6,IL-8,and TGF-beta)in tumor of ESCC.Results1.Silence of CCR7 gene expression in esophageal squamous cell carcinoma transplanted tumors had significantly reduced tumor volume.2.After silence of CCR7 expression,NF-kB target genes(VEGF-A,VEGF-C,TNF alpha,IL-6,IL-8,and TGF-beta)also declined significantly,and the NF-kB signaling pathway is blocked.Conclusion1.CCR7 was highly expressed in esophageal squamous cells.2.By creating a stable CCR7 expression or silence system in esophageal squamous cell carcinoma,CCR7 was proved to promote new blood capillary formation in esophageal squamous cell carcinoma.This indicated that CCR7 plays a critial role in development of esophageal squamous carcinoma angiogenesis,might be a potential therapeutical target of esophageal squamous carcinoma.3.The overexpression of CCR7 protein upregulated transcriptional activity of NF-kB reporter gene,and promoted the phosphorylation-levels of IKK and IkB.Various NF-kB target gene including TNF-,IL-6,IL-8 and TGF-βwere upregulated in CCR7 overexpression Eca109,and downregulated in CCR7 lowexpression Eca109.This result showed that CCR7 promoted NF-kB signaling pathway.4.In vivo study indicated that the average tumor volume decreased significantly in CCR7-RNAi Eca109 xenotransplanted nude mouse.The protein expression levels of VEGF-A,VEGF-C,TNF-α,IL-6,IL-8and TGF-βwere significantly decreased,the phosphorylation-levels of IKK and IkBα were decreased,and the total protein levels of IKK and IkBα had no significant change.Futher proved that CCR7 might promote ESCC through NF-kB signaling pathway.