NF-κB/miR-223-3p/ARID1A Axis Is Involved In Helicobacter Pylori CagA-induced Gastric Carcinogenesis And Progression

Background:Helicobacter pylori(H.pylori)infection causes chronic gastritis and peptic ulcer,which is considered to be a type I pathogenic bacteria that causes gastric cancer.The pathogenicity of H.pylori is mainly attributed to its various virulence factors and the most widely studied H.pylori virulence factor is the cytotoxin related gene protein CagA.CagA can be injected into cells by the bacterially-encoded type IV secretion system,activating a variety of signaling molecules in the cell,including the inflammatory factor NF-κB.After activation of NF-κB,it is closely related to many biological effects in cells,such as inflammation,immortalization of cells,cell proliferation,anti-apoptosis,angiogenesis and tumor metastasis.One of the molecular mechanisms of the biological function of NF-κB is that NF-κB can cause the deregulated expression of key miRNAs in the cells.The imbalance of these miRNAs can cause the abnormal expression of downstream target proteins,and thus cause the above biological effects and promoting the development of tumors.After the activation of NF-κB in gastric mucosal epithelial cells by the virulence factor CagA of H.pylori,it has not been reported that NF-κB promotes the development of gastric cancer by regulating which miRNAs.We found through literature that after H.pylori infection of gastric epithelial cells,31 differentially expressed miRNAs were screened by miRNAs microarray,among which miR-223-3p was the only miRNA with up-regulated expression.The software analysis revealed that the NF-κB binding site is present in the miR-223-3p promoter region.Based on the above analysis,the present study will explore the following questions:Can NF-κB bind directly to the promoter region of miR-223-3p to regulate its expression?What biological function does miR-223-3p play in gastric cancer cells?What is the molecular mechanism underlying the miR-223-3p?Whether NF-κB can participate in the H.pylori-mediated malignant transformation of gastric mucosa by regulating the expression of miR-223-3p?Objective:(1)To investigate the molecular mechanism of H.pylori virulence factor CagA causing gastric cancer tissue transformation via the NF-κB/miR-223-3p pathway,and to enrich the understanding of the molecular mechanism of gastric cancer caused by H.pylori infection;(2)To explore the biological function of miR-223-3p in gastric cancer and its downstream target genes,and to clarify its role in H.pylori-mediated malignant transformation of gastric mucosa.Methods:(1)Gastric cancer cells were treated with H.pylori(CagA+/CagA-)and CagA overexpression plasmid(pcDNA3.1-CagA)respectively,western blot and qRT-PCR were used to detect the expression of CagA and miR-223-3p;(2)Using NF-κB inhibitor BAY 11-7082 to verify whether NF-κB is involved in the up-regulation of miR-223-3p by H.pylori CagA;using qRT-PCR and dual luciferase activity assay to analyze the regulation of NF-κB on the miR-223-3p;(3)Databases were used to predict the potential target proteins of miR-223-3p;using western blot,qRT-PCR and dual luciferase activity assay to analyze the regulation of miR-223-3p on the potential target proteins;(4)The effects of CagA,miR-223-3p and potential target proteins on proliferation and migration of gastric cancer cells were detected by EdU and Transwell assays,respectively;(5)qRT-PCR was used to detect the expression level of miR-223-3p and ARID1A in gastric cancer and its adjacent tissues,and the correlation between the expression level of the two genes was analyzed.Results:1.H.pylori CagA regulates miR-223-3p expression through NF-κB pathway(1)H.pylori promotes miR-223-3p through a CagA-dependent pathway:The expression of miR-223-3p was significantly increased with H.pylori(CagA+)infection in the gastric cancer cells,while the infection with H.pylori(CagA)had no significant effect on the expression of miR-223-3p.After transfecting with CagA overexpression vector,the expression of miR-223-3p was importantly up-regulated.(2)CagA regulating miR-223-3p is dependent on NF-κB pathway:Pretreatment of gastric cancer cells with the NF-κB inhibitor BAY 11-7082 abrogated the upregulation of miR-223-3p induced by H.pylori(CagA+)infection.BAY 11-7082 also eliminates the upregulation of miR-223-3p expression induced by transfecting CagA overexpression vectors in gastric cancer cells.Transfecting NF-KB-specific siRNA reduced miR-223-3p expression.Dual luciferase activity assay showed that NF-κB binds directly to the promoter of pri-miR-223-3p to regulate its expression.2.Biological function and regulation mechanism of miR-223-3p in gastric cancer(1)Biological function of miR-223-3p in gastric cancer:(2)miR-223-3p directly targets the ARID1A 3’UTR(SWI/SNF chromatin remodeling complex family)and reduces its expression:After transfecting miR-223-3p mimics,the mRNA and protein expression level of ARID1A decreased;on the contrary,miR-223-3p inhibitor increased ARID1A mRNA and protein expression level.Dual luciferase assay results showed that the high expression of miR-223-3p inhibited the activity of the wild-type ARID1A 3’UTR,but it had no significant effect on the mutated ARID1A 3’UTR activity.(3)miR-223-3p promotes the proliferation and migration of gastric cancer cells by ARID1 A:High expression of miR-223-3p promoted cell proliferation and migration ability in gastric cancer cells.ARID1A silencing promoted cell proliferation and migration;ARID1A overexpression vector inhibited cell proliferation and migration.Overexpression of ARID1A partially restores the miR-223-3p mimics-induced the increased ability of cell proliferation and migration.Western blot revealed that ARID1A inhibited cell proliferation and migration in gastric cancer cells by regulating p21 and E-cadherin,respectively.3.NF-κB/miR-223-3p pathway is involved in the biological effect of H.pylori CagAGastric cancer cells were co-transfected with CagA overexpression vector and miR-223-3p inhibitor.Overexpression of H.pylori CagA promoted cell proliferation and migration,while miR-223-3p inhibitor partially reversed CagA-mediated biological effects.4.Detection in clinical gastric cancer specimensIn clinical gastric cancer specimens,the expression level of miR-223-3p is significantly upregulated,while ARID1A is downregulated significantly compared with the corresponding non-cancerous tissues.Moreover,a negative correlation between miR-223-3p and ARID1A expression was found in the gastric cancer tissues.Conclusion:In this study,our results show that after H.pylori infecting gastric cancer cells,the virulence factor CagA enters the cells and activates NF-κB,NF-κB then enters the nucleus and binds directly to the pri-miR-223-3p promoter,which stimulates the expression level of miR-223-3p.MiR-223-3p plays a cancer-promoting role in gastric cancer and it can directly target ARID1A 3’UTR and reduce its expression.ARID1A regulates the proliferation and migration of gastric cancer cells by regulating p21 and E-cadherin.We also detected the expression level of miR-223-3p and ARID1A in clinical gastric cancer specimens and they showd a significant negative correlation,which is consistent with the results at the cellular level.In conclusion,our results suggested NF-κB/miR-223-3p/ARID1A axis might be a possible pathway for H.pylori-induced gastric mucosa progression from chronic gastritis to gastric cancer,thereby providing a new insight into the mechanism underlying H.pylori-associated gastric diseases.