Molecular Mechanism Of MiR-574-5p Regulating Angiogenesis In Gastric Cancer Cells

Background:Gastric cancer is a malignant tumor originating from the gastric mucosa.Among various malignant tumors,the incidence of gastric cancer ranks first and the mortality rate ranks third.The occurrence of gastric cancer is associated with regional environment,dietary habits,Helicobacter pylori?Hp?infection,precancerous lesions,genetics and genes.Gastric cancer can occur in any part of the gastric,but it is more common in the gastric antrum,gastric small bend and gastric large curvature.Currently,surgery is the most effective treatment for gastric cancer patients.The effect of surgical treatment is better for patients with early gastric cancer,and the 5-year survival rate can reach more than 90%.Angiogenesis is a complex process involving multiple genes and involved in the development and progression of tumors.Compared with normal tissues,the amount of nutrients and oxygen required by tumor tissues is excessive.In order to meet the needs of nutrients and oxygen which were used for maintaining tumor growth,tumor tissues will promote normal dormant blood vessels to sprout new blood vessels and activate the conversion of endothelial cells phenotype and then promote the formation of new blood vessels.Studies have shown that microRNAs are important regulators of angiogenesis and play critical roles in angiogenesis of tumors.In this paper,we aimed to explore the mechanism of miR-574-5p promoting angiogenesis in gastric cancer cells,which will have important theoretical and practical significance for the future development of molecular targeted drugs for treatment of gastric cancer.Materials and Methods:Firstly,we simulated the chemical and physical hypoxia condition in gastric cancer cells using cobalt chloride?200?M?and anoxic incubator?2%O₂?respectively,and the mice were intraperitoneally injected with sodium nitrite?0.25%?to simulate the hypoxia condition of animal tissues;we detected the expression of HIF-1?and miR-574-5p in gastric cancer cells and mouse gastrocnemius muscle after simulated hypoxia condition.Secondly,we detected the expression changes of HIF-1?and its downstream proangiogenic factor VEGFA/PDGF-BB/FGF-2 after transfecting miR-574-5p inhibitor in gastric cancer cells.Then we demonstrated the regulation of miR-574-5p on tumor angiogenesis through MTT,Transwell,tubule formation and matrigel plug assay in nude mice.We predicted the regulation of miR-574-5p on pathways and its corresponding target genes by bioinformatics methods.Simultaneously,we confirmed the regulation mechanism of miR-574-5p on angiogenesis by the dual luciferase reporter gene and RNA silencing technology.Results:In the chemical and physical hypoxia model simulated by cobalt chloride and anoxic incubator,expression of HIF-1?protein induced up-regulation of miR-574-5p expression in gastric cancer cells;In the animal model under hypoxia condition induced by intraperitoneal injection of sodium nitrite for mice,expression of HIF-1?protein also up-regulated the expression of miR-574-5p in the gastrocnemius muscle of mice.The protein expression of HIF-1?in gastric cancer cells had no change after transfection of miR-574-5p inhibitor,but in the downstream of HIF-1?,the mRNA level of VEGFA was down-regulated,and the protein expression of VEGFA and p-ERK were both down-regulated.At this time,we have two hypotheses:First,miR-574-5p may influence the transcriptional activity of HIF complex by MAPK pathway to regulate the expression of VEGFA.Second,miR-574-5p plays a regulatory role in angiogenesis.The results of MTT,Transwell,tubule formation,and matrigel plug in nude mice showed that the miR-574-5p inhibitor decreased the ability of cell viability,migration and infiltration;besides,the miR-574-5p inhibitor reduced the formation of tubules and the expression of CD31.We then predicted the binding sites of miR-574-5p to its possible target gene PTPN3 with bioinformatics methods.Dual luciferase reporter gene results showed in the wild-type PTPN3reporter plasmid and the miR-574-5p mimics co-transfected group,the relative fluorescence activity reduced.RNA silencing showed that silencing of PTPN3up-regulated the protein expression levels of p-ERK and VEGFA and increased the secretion of VEGFA.Conclusion:1.miR-574-5p promotes tumor angiogenesis in vitro and in vivo.2.PTPN3 is a target gene of miR-574-5p.3.Hypoxia promotes the expression of miR-574-5p in gastric cancer cells,and miR-574-5p maybe affect HIF-1?phosphorylation and regulates VEGFA expression through MAPK/ERK pathway.