The Role Of MicroRNA-320d/FoxM1 Nexus In The Progression Of Gastric Cardia Adenocarcinoma And The Mechanism Study

Objective:1.To study the Role of miRNA-320d/FoxM1 nexus in the Progression of GCA and the Mechanism Study.2.To investigate the influence and mechanism of proliferation and apoptosis of sophoridine on gastric cardia adenocarcinoma cell line.Methods:Real-time PCR was employed to analysize the expression profile of miRNA-320d in GCA cell lines.Meanwhile,we analysized the correlation between miRNA-320d expression and GCA development and prognosis.The data from Targetscan and related references were analysized to identify the key gene which was correlated with GCA and could be regulated by miRNA-320d expression.Immunohistochemical staining,Real-time PCR and western-blotting was performed to verify FoxMl expression in GCA and the correlation between miRNA-320d and FoxMlexpression was analysized.By using the technique of cell transfection,we verified miRNA-320d regulated FoxMl expression in GCA cell lines.Through the luciferase reporter assay,we defined the miRNA-320d/FoxM1 nexus.Additionally,the correlation beween FoxM1 expression and GCA development and prognosis was analysized.CCK-8 assay was employed to verify the effect of miRNA-320d/FoxM1 nexus to the cell proliferation.Tirdly,Wound-healing assay was used to obeserve the effect of miRNA-320d/FoxM1 nexus in cell migration.Finally,Transwell assay was performed to test the effect of miRNA-320d/FoxM1 nexus in cell invasion.OE-19 cells and SK-GT2 cells with miRNA-320d-LV/anti-miRNA-320d-LV and NC-LV/anti-NC-LV were injected into nude mice subcutaneously to analysize the effect of miRNA-320d/FoxMl nexus to the tumor growth of OE-19 and SK-GT2 xenografts.CCK-8 was used to observe the change of proliferation of GCA cell;The fluorescence microscopy was used to observe the apoptosis of GCA cell;Reactive oxygen species(ROS)detection was used to observe the change of intracellular ROS and GSH;The Real-time PCR and Western-blot was used to detect the change of the expression of FoxMl;Dual-luciferase reporter gene assay for the transcriptional activity of FoxMl.Results:The expression of miRNA-320d was down-regulated in GCA,compared to their corresponding adjacent normal tissues.The expression of FoxM1 was negatively correlated with miRNA-320d in GCA samples and cell lines.Their expression were correlated with prognosis.The result of luciferase reporter assay indicated that miRNA-320d directly targeted FoxM1 and FoxM1 was one of the target genes of miRNA-320d.Down-regualation of miRNA-320d caused the up-regulation of FoxM1 in GCA and was correlated with the advanced progression and poor prognosis of GCA.By up-regulating/down-regulating miRNA-320d expression,FoxMl expression was down-regulated/up-regulating.The CCK-8 assay showed that GCA cell proliferation was decreased by up-regulating miRNA-320d expression(down-regulating FoxM1 expression),whereas the opposite effect was obeserved.Wound-healing assay showed that GCA cell migration was decreased by up-regulating miRNA-320d expression(down-regulating FoxMl expression),whereas the opposite effect was obeserved.Similarly,GCA cell invasion were inhibited by up-regulating miRNA-320d expression(down-regulating FoxMl expression),which was shown in Transwell assays,whereas the opposite effect was obeserved.Both of tumor volume and weight of OE-19 xenografts with up-regulated miRNA-320d were decreased,compared to the control OE-19 xenografts.FoxMl expression was decreased in OE-19 xenografts with up-regulated miRNA-320d.On the contrary,SK-GT2 xenografts with down-regulated miRNA-320d were increased,compared to the control SK-GT2 xenografts.FoxM1 expression was increased in SK-GT2 xenografts with down-regulated miRNA-320d.Sophoridine can significantly inhibited the proliferation of OE-19 and SK-GT2 cell,induced cell apoptosis and caused reactive oxygen species(ROS)generation and GSH content reduction.Sophoridine also triggered significant downregulated the expression of FoxM1 and the transcriptional activity of FoxM1.Conclusion:The regulation relationship of miRNA-320d/FoxMl is one of the important mechanisms in occurrence and development of GCA.Sophoridine shows obvious anti-cancer activity on gastric cardia adenocarcinoma cell lines by inducing cell apoptosis,the mechanism is associated with the inhibition of FoxMl expression.