Regulation And Mechanism Of Aberrant NF-κB In Distant Metastasis Of Gastric Cancer By Dysregulated MicroRNA-7

Objective: More than 1 million people worldwide are diagnosed with gastric cancer each year,and because the 5-year survival rate of gastric cancer is still less than 20%,more than 850,000 people die each year from gastric cancer.The expression of microRNA-7(miR-7)and the abnormal activation of NF-κB have been found in many cancers,but the expression profile,clinical relevance and dysregulation mechanism of miR-7 and NF-κB in gastric cancer metastasis remain largely unkonwn.In this paper,the expression profile,clinical relevance and dysregulation mechanism of microRNA-7 and NF-κB subunit RelA/p65 in gastric cancer were investigated,and miR-7 was explored as a potential therapeutic target for distant metastasis of gastric cancer.Methods: The relationship between miR-7 and NF-κB subunit RelA expression level,clinical relevance and survival rate in gastric cancer were analyzed using TCGA STAD and NCBI GEO databases.Real-time PCR and pre-microRNA-7 processes and binding assays were used to detect the synthesis and expression of miR-7 in gastric cancer cell lines.To obtain thegastric cancer cell lines HGC-27 and MKN-28 overexpressing miR-7,cells were infected with recombinant lentiviruses,and the cells were sorted by flow cytometry to establish cell lines stably overexpressing miR-7.CCK8,colony formation assay,cell cycle assay and migration,invasion assay were used to detect the effect of overexpression of miR-7 on the growth,proliferation and migration of gastric cancer cells.Flow cytometry,immunofluorescence,and luciferase reporter genes verified the interaction of miR-7 and NF-κB subunits RelA/p65 and changes in NF-κB downstream metastasis-related proteins.HE and immunohistochemistry were used to detect the effects of miR-7 and NF-κB subunit RelA/p65 and its downstream metastasis-related proteins on the growth,proliferation and migration of gastric cancer cells in vivo in an animal metastasis model.Results: The expression of miR-7 is negatively correlated with the expression of RelA in the clinical samples of gastric cancer.Moreover,the decreased expression of miR-7 and increased expression of RelA predict the poor survival of clinical outcome of gastric cancer patients.The formation of Dicer1 enzyme hinders the production of miR-7 and thus leads to down-regulation of miR-7 expression in gastric cancer cells.In addition,overexpression of miR-7 can inhibit the growth,proliferation and migration of gastric cancer cells.Functionally,delivery of miR-7 displays therapeutic effects to GC lung and liver metastasis by alleviating hemangiogenesis,lymphangiogenesis as well as inflammation cellsinfiltration.Mechanistically,miR-7 suppresses NF-κB transcriptional activity and its downstream metastasis-related molecules Vimentin,ICAM-1,VCAM-1,MMP-2,MMP-9 and VEGF by reducing p65 and p65-ser536 expression.Pharmacologic prevention of NF-κB activator LPS obviously restored miR-7-suppressed NF-κB transcriptional activation and significantly reverted miR-7-inhibited cell migration and invasion.Conclusion: Our data suggest loss of miR-7 in gastric cancer promotes p65-mediated aberrant NF-κB activation,facilitating gastric cancer metastasis and ultimately resulting in the worse clinical outcome.Thus,miR-7 may act as novel prognostic biomarker and potential therapeutic target for aberrant NF-κB-driven gastric cancer distant metastasis.