| Background:As the burden of Gastric cancer(GC)continues to grow globally,the therapeutic effect of surgical operation supplemented by systemic chemotherapy is not satisfactory.The newly emerged molecular targeted therapy has changed the therapeutic mode of gastric cancer,but more gastric cancer tumor markers and targeted pathways of anti-gastric cancer effect are still needed to be explored to improve the therapeutic effect of Gastric cancer.More and more studies suggest that erythropoietin production liver cell kinase receptor A1(erythropoietin-producing hepatocyte kinase receptor A1,EphAl)may promote the occurrence of gastric cancer development,invasion and metastasis,and is likely to become a new molecular targets for treatment,but the study of gastric cancer with EphAl lack of system in vitro studies,and there is no deep full study of its possible mechanism.The research on tumor microenvironment has become a hot topic in cancer research.Recent studies have found that a large number of cytokines secreted by cells in tumor microenvironment can change the microenvironment,which is closely related to the growth,invasion and metastasis of tumor.Both tumor cells and non-tumor cells in the tumor microenvironment can secreted a large amount of VEGF,which,as an important tumor angiogenesis promoter,exists in a large number in the tumor microenvironment and is closely related to the malignancy,invasion and metastasis potential and prognosis of gastric cancer.Eighth biological characteristics of malignant tumor of inflammation in cancer development,play an important role in the process of invasion and metastasis,tumor microenvironment in a large number of inflammatory cytokines,which can not only raise inflammatory cells to the tumor site,amplify inflammatory effect,also can promote tumor cell growth and metastasis,promoting tumor blood vessels,lymphatic vessel formation.Studies have shown that members of the Eph family not only act on tumor cells,but also play an important role in the adjustment of tumor microenvironment.However,literature review has not drawn a definite conclusion,and further analysis is needed.To sum up:the severe burden of gastric cancer calls for exploring more potential new molecular targeted therapeutic targets;Recent studies have shown that EphAl in the Eph family may promote the occurrence,development,invasion and metastasis of gastric cancer,and may become a new target for gene therapy of gastric cancer.Gastric cancer is an inflammation-related solid malignant tumor,and its growth and metastasis are inseparable from tumor angiogenesis that exists in the tumor microenvironment to provide nutrition and continuous inflammatory cell infiltration that constitutes uncontrolled chronic inflammation.Like other members of the RTK family,the Eph family has been reported to play an important role in tumor growth and metastasis by influencing tumor angiogenesis and inflammatory microenvironment.However,so far,these studies have not been conclusive,and studies on the relationship between gastric cancer,EphAl and tumor microenvironment are rare.Therefore,we hypothesized that abnormal EphAl expression may play an important role in the development,recurrence and metastasis of gastric cancer.The mechanism may be related to the regulation of tumor angiogenesis in the tumor microenvironment(such as changing the expression of the important angiogenic factor VEGF)and inflammatory cell infiltration(such as changing the expression of the key molecule il-6 in the inflammatory signaling pathway of nf-kappa B and STAT3).This study takes the relationship between EphA1 and gastric cancer as the starting point,and proposes the hypothesis that EphAl may influence tumor angiogenesis and inflammatory microenvironment by adjusting VEGF and IL-6 cytokines in the tumor microenvironment of gastric cancer,thus promoting the occurrence and development of gastric cancer.It is intended to conduct multi-dimensional research and verification from clinical specimens to the laboratory,from in vitro cell experiments to in vivo animal experiments,in order to further elucidate the correlation between EphAl and the occurrence and development of gastric cancer and its potential mechanism of action,provide new clues for the diagnosis and targeted therapy of gastric cancer,and provide candidate targets for the development of new anti-tumor drugs.Chapter 1:Expression level of EphAl in gastric cancer and its relationship with tumor microenvironmental markers VEGF and IL-6Objective:To investigate the relationship between EphA1 expression in gastric cancer tissues and the development of gastric cancer,and to explore the correlation between EphAl expression level and the expression level of VEGF and il-6,the tumor microenvironmental markers,so as to further clarify the possibility of EphAl as a new target for gene therapy of gastric cancer.Methods:1.The expression of EphA1,VEGF and IL-6 in gastric cancer tissues,para-cancer tissues and normal gastric mucosa tissues was detected by IHC method from protein level and the relationship among the three was analyzed.2.The expression of EphA1,VEGF and IL-6 in gastric cancer tissues,paracancer tissues and normal gastric mucosa tissues were detected by qRT-PCR and the relationship among the three was analyzed.3.To analyze the relationship between EphA1 expression and the clinical characteristics of gastric cancer,such as the degree of tissue differentiation,depth of tumor infiltration,lymph node metastasis and TNM staging.4.To analyze the correlation between EphAl expression and postoperative survival rate in patients with gastric cancer.Results:1.EphA1 showed a gradually decreasing trend of protein and gene expression in gastric cancer tissues,paracancer tissues and normal gastric mucosal tissues,and the expression level in cancer tissues was significantly higher than that in paracancer tissues and normal gastric mucosal tissues.2.The higher the EphA1 protein expression in cancer tissues,the worse the tumor biological characteristics,and the worse the survival prognosis(OS)of patients.Cox multivariate survival analysis showed that EphA1 was an independent survival prognostic factor for gastric cancer patients.3.The protein and gene expression of VEGF and IL-6 in gastric cancer tissues,paracancer tissues and normal gastric mucosal tissues also showed a gradually decreasing trend.The expression level of VEGF and IL-6 in the carcinoma tissues was significantly higher than that in the paracancer tissues and normal gastric mucosal tissues.4.Further correlation analysis showed that EphA1,VEGF and il-6 protein expression levels were significantly positively correlated in gastric cancer tissues and paracancer tissues.Conclusion:EphAl was found to promote the occurrence and development of gastric cancer and affect the survival and prognosis of gastric cancer patients in gastric cancer tissue samples.EphAl can be used as a new target for gastric cancer gene therapy.EphAl may promote the development of gastric cancer by increasing the secretion levels of tumor microenvironmental markers VEGF and il-6 cytokines.Chapter 2:In vitro experiments verified that EphAl promoted proliferation,invasion and migration of gastric cancer cells and up-regulated expression levels of tumor microenvironmental markers VEGF and IL-6Objective:To verified the hypothesis in vitro that EphAl can promoting the occurrence and development of gastric cancer and its potential mechanism is up-regulating the expression levels of tumour microenvironmental markers VEGF and IL-6.Methods:1.Construction of human EphA1 gene interfering human gastric cancer cell line SGC7901 lentivirus stable strain,set as silencing group(experimental group).2.Construction of human EphAl gene overexpression human gastric cancer cell line SGC7901 lentivirus stable strain,set as overexpression group(experimental group).3.MTT coloriometry,Transwell(invasion and migration ability)experiment,flow cytometry were used to detect and analyze the proliferation,invasion and metastasis ability of cells in the silencing group(experimental group),overexpression group(experimental group)and normal control group(normal gastric cancer cell line SGC7901).4.The gene expressions of VEGF and IL-6 in the silenced group(experimental group),the overexpressed group(experimental group)and the normal control group(normal gastric cancer cell line SGC7901)were detected by qRT-PCR.5.The protein expressions of VEGF and IL-6 in the silenced group(experimental group),the overexpressed group(experimental group)and the normal control group(normal gastric cancer cell line SGC7901)were detected by Western blot.Results:1.The proliferation,invasion and migration of cells in the overexpression group(the experimental group)was enhanced and the apoptotic ability was weakened compared with that in the normal control group,while the proliferation,invasion and migration of cells in the silence group(the experimental group)was weakened and the apoptotic ability was enhanced compared with that in the normal control group.2.The gene expression levels of VEGF and IL-6 in the overexpressed group(the experimental group)were higher than those in the normal control group,while the gene expression levels of VEGF and IL-6 in the silent group(the experimental group)were lower than those in the normal control group.3.The protein expression levels of VEGF and IL-6 in the overexpressed group(the experimental group)increased compared with the normal control group,while the protein expression levels of VEGF and IL-6 in the silent group(the experimental group)decreased compared with the normal control group.Conclusion:Down-regulation of EphAl gene expression can inhibit the proliferation and migration ability of gastric cancer cells and reduce the expression levels of VEGF and IL-6,while up-regulation of EphAl gene expression can promote the proliferation and migration ability of gastric cancer cells and increase the expression levels of VEGF and IL-6.EphAl can promote the proliferation and migration of gastric cancer cells,and the mechanism may be related to the up-regulated expression levels of VEGF and IL-6 in gastric cancer cells.Chapter 3:In vivo experiments verified that EphAl promoted the occurence and development of gastric cancer transplantation tumor and up-regulated expression levels of tumor microenvironmental markers VEGF and IL-6Objective:To verified the hypothesis in vivo that EphA1 can promoting the occurrence and development of gastric cancer and its potential mechanism is up-regulating the expression levels of tumour microenvironmental markers VEGF and IL-6.Methods:1.To establish a subcutaneous xenograft model of gastric cancer in nude mice:the human EphAl gene interfering human gastric cancer cell line SGC7901 lentivirus stable strain were injected subcutaneously into nude mice to construct a subcutaneous GC xenografl model,set as silencing group(experimental group);the human EphA1 gene overexpression human gastric cancer cell line SGC7901 lentivirus stable strain were injected subcutaneously into nude mice to construct a subcutaneous GC xenografl model,set as overexpression group(experimental group);the normal gastric cancer cell line SGC7901 were injected subcutaneously into nude mice to construct a subcutaneous GC xenografl model,set as normal control group(normal gastric cancer cell line SGC7901).2.Morphological observation:observing and comparing the tumorigenesis time of each group of nude mice,observing and comparing the subcutaneous tumor growth rate of each group of nude mice,observing and comparing the possible metastasis of other organs of each group of nude mice3.The gene expressions of EphAl,VEGF and IL-6 in subcutaneous transplanted tumor tissues of nude mice in the silencing group(experimental group),the overexpressed group(experimental group)and the normal control group(normal gastric cancer cell line SGC7901)were detected by qrt-pcr,and the relationships among the three groups were analyzed.4.Protein expressions of EphAl,VEGF and IL-6 in subcutaneous transplanted tumor tissues of nude mice in the silencing group(experimental group),the overexpressed group(experimental group)and the normal control group(normal gastric cancer cell line SGC7901)were detected by Western blot,and the relationships among the three groups were analyzed.Results:1.Compared with the normal control group,the overexpression group(the experimental group)had a high tumorigenesis rate,early tumorigenesis,rapid tumor growth and larger tumors,while the silence group(the experimental group)had a low tumorigenesis rate,late tumorigenesis,slow tumor growth and smaller tumors.2.The gene expression levels of VEGF and IL-6 in subcutaneous transplanted tumor tissues of nude mice were higher in the overexpression group(the experimental group)than in the normal control group,while the gene expression levels of VEGF and IL-6 in subcutaneous transplanted tumor tissues of nude mice in the silent group(the experimental group)were lower than those in the normal control group.3.The protein expression levels of VEGF and IL-6 in subcutaneous transplanted tumor tissues of nude mice in the overexpression group(the experimental group)were increased compared with the normal control group,while the protein expression levels of VEGF and IL-6 in subcutaneous transplanted tumor tissues of nude mice in the silence group(the experimental group)were decreased compared with the normal control group.Conclusion:EphA1 knockdown can significantly inhibit the occurrence and development of gastric cancer and down-regulate the expression levels of VEGF and IL-6 in the subcutaneous transplanted tumor model of nude mice,while EphAl overexpression can significantly promote the occurrence and development of gastric cancer in the subcutaneous transplanted tumor model of nude mice and up-regulate the expression levels of VEGF and IL-6.EphAl can promote the occurrence and development of gastric cancer in the xenograft nude model in vivo,and its mechanism may be related to the increased expression levels of VEGF and IL-6 in tumor microenvironmental tissues. |
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