The Mechanism Of Hypoxia-induced Aberrant Expression Of CXCL6 In Promoting Invasion And Metastasis Of Gastric Cancer

Background:Gastric cancer is one of the most common malignant tumors of digestive system.Worldwide,the incidence and mortality of gastric cancer rank among the top list of all malignant tumors.According to the global cancer statistics report in 2018,the vast majority of new cases and deaths are mainly in East Asia and developing countries,and the annual number of new cases of gastric cancer in China accounts for about 41%worldwide.Therefore,in-depth study of the key molecular mechanisms of the occurrence,development,invasion and metastasis of gastric cancer has important theoretical and Clinical I significance for the treatment of gastric cancer.Tumor invasion and metastasis is a complex process involving multiple steps,stages and genes,which depends not only on its own biological characteristics,but also on the interaction with the tumor microenvironment.In recent years,it has been found that the tumor hypoxia microenvironment and chemokines and their receptors play important roles in the invasion and metastasis of various solid tumors such as liver cancer and breast cancer.Studies suggest that the tumor microenvironment can promote tumor invasion and migration by inhibiting the activation and chemotaxis of effector T cells,inducing tumor cell dedifferentiation,and remodeling the extracellular matrix.Hypoxia is a major feature of the tumor microenvironment.Hypoxia-inducible factor-1(HIF-1 a)is a major regulator of the adaptive response of tumor cells to the hypoxic microenvironment and is a key step in the adaptation of tumor cells to the hypoxic microenvironment.Hypoxia-inducible factor-1 has been confirmed to directly regulate the expression of dozens of genes,not only to adapt cells to the hypoxic environment,but also to make tumors have stronger survival potential and invasion and metastasis ability,as well as the tolerance to chemoradiotherapy,by degrading the extracellular matrix,inducing the dedifferentiation of tumor cells,promoting angiogenesis,regulating energy metabolism,and arresting the cell cycle.To clarify the downstream target genes related to the regulation of gastric cancer invasion and metastasis by HIF-1 a is of great significance to clarify the specific mechanism of gastric cancer invasion and metastasis and to find new clinical therapeutic targets.CXCL6(C-X-C motif chemokine ligand 6),also known as granulocyte chemotactic protein 2(GCP-2),is an important member of the ELR(+)CXC chemokine family.It is secreted by macrophages,epithelial and mesenchymal cells,and has the functions of recruiting granulocytes,stimulating the secretion of metalloproteinases(MMP2,MM9),promoting angiogenesis and regulating immunity.ln recent years,some studies have shown that some low molecular weight chemokines can not only attract leukocytes to migrate to the site of infection,but also participate in the invasion,invasion and metastasis of many tumor cells.For example,KIAA1199 increases neutrophil infiltration by up-regulating the expression of CXCL6,thereby promoting the invasion and metastasis of gastric cancer.CXCL6 is highly expressed in esophageal squamous cell carcinoma tissues,and CXCL6 high expression promotes the migration of esophageal squamous cell carcinoma cells.However,the role and mechanism of CXCL6 in the occurrence and development of gastric cancer are not yet clear.Therefore,this study focuses on exploring the specific role and mechanism of HIF-1 a in regulating the invasion and metastasis of gastric cancer under the condition of hypoxic tumor microenvironment,in order to provide new ideas for clinical treatment of gastric cancer.Methods:(1)Resuscitate one human gastric mucosal epithelial cell line GES-1 and six gastric cancer cell lines(BGC-823,MGC-803,HGC-27,AGS,MKN-45,SGC-7901),and detect HIF-by qRT-PCR 1a mRNA expression level.Using GES-1 as a control,gastric cancer cell lines with relatively low expression of HIF-1 a were subjected to hypoxia treatment,and the mRNA expression levels of HIF-1 a and CXCL6 were detected by qRT-PCR.(2)Check the public database to find proteins that may interact with HIF-1 a,and verify that there may be a positive correlation between HIF-1 a and CXCL6 expression.(3)The bioinformatics method was used to analyze and predict whether there is a potential binding site of the transcription factor HIF-1 a in the promoter region of the CXCL6 gene,construct a dual luciferase reporter gene plasmid,and verify the CXCL6 promoter using the dual luciferase reporter gene system and point mutation experiments Potential HIF-1 a binding site in the subregion.(4)BGC-823 gastric cancer cell line with strong invasion and migration ability was selected and divided into three groups according to the experimental design:normal oxygen culture group,hypoxia culture group,hypoxia culture+CXCL6 neutralizing antibody group.Western Blot was used to detect the protein expression levels of HIF-1 a and CXCL6,as well as the marker molecules MMP-2 and MMP-9,which represent invasion and migration ability.The scratch and Transwell experiments were used to observe and compare the invasion and migration ability of gastric cancer cells.(5)The bioinformatics method was used to analyze and find the differences in expression of HIF-1 a and CXCL6 between gastric cancer and normal gastric tissues in public databases.and analyze the relationship between HIF-1 a,CXCL6 and some clinicopathological features of gastric cancer patients.Results:(1)The expression of HIF-1 a in gastric cancer cell line BGC-823 is relatively low.(2)Under hypoxic conditions,the mRNA levels expressions of HIF-1 a and CXCL6 in gastric cancer cells increased,and showed time dependence.(3)HIF-1 a is correlated with CXCL6 expression.(4)Under hypoxic conditions,HIF-1 a transcription regulates the expression of CXCL6.In the promoter region of the CXCL6 gene,there is a potential binding site for the transcription factor HIF-1 a.(5)CXCL6 is an important effector molecule involved in the process of mediating HIF-1 a to promote the invasion and metastasis of gastric cancer cells.(6)Biological information analysis tipsHIF-1 a and CXCL6 are highly expressed in gastric cancer tissues;HIF-1 a and CXCL6 are highly expressed,which is associated with higher clinical stage,poorer tumor grade and more lymph node metastasis.Conclusion:This study confirmed that hypoxic conditions can significantly increase the expression level of CXCL6 in gastric cancer cells.Mechanistically,in the hypoxic microenvironment of gastric cancer,CXCL6 is regulated by the transcription factor HIF-1 a.CXCL6 is involved in mediating the regulation of HIF-1 a on the invasion and migration of gastric cancer cells.HIF-1 a and CXCL6 are relatively highly expressed in gastric cancer tissues,which are associated with some clinicopathological features of gastric cancer patients.