| ObjectiveExploring the expression of forkhead box K1(FOXK1)in gastric cancer cell lines(HGC-27,BGC-823 and SGC-7901)and its impact on the malignant biological behavior(proliferation,migration,invasion and apoptosis)of gastric cancer cells,and preliminarily exploring whether FOXK1 can regulate NF-κB pathway is involved in gastric cancer cell apoptosis,hoping to provide experimental basis for early diagnosis and treatment of gastric cancer.Methods1.The expression of FOXK1 at mRNA level in gastric cancer tissues and normal gastric tissues was analyzed by bioinformatics.2.RT-qPCR and Western blot assays were performed to detect the expression of FOXK1 in human normal gastric mucosal cell lines(GES-1)and gastric cancer cell lines(HGC-27,BGC-823 and SGC-7901)at the mRNA and protein levels.3.Human gastric cancer cells(HGC-27,BGC-823 and SGC-7901)were transfected in vitro with chemically synthesized si-FOXK1.The experiment was divided into three groups: blank control(control),negative control(nc-FOXK1)and transfection group(si1-FOXK1,si2-FOXK1 and si3-FOXK1).Transfection efficiency was assessed by RT-qPCR and Western blot assays at the mRNA and protein levels,respectively.4.MTT assay was performed to detect the proliferation ability of gastric cancer cells after si-FOXK1 transfection.5.Clone formation assay was performed to detect the colony formation ability of gastric cancer cells after si-FOXK1 transfection.6.Scratch assay was performed to detect the migration ability of gastric cancer cells after si-FOXK1 transfection.7.Transwell assay was performed to detect the migration and invasion ability of gastric cancer cells after si-FOXK1 transfection.8.Flow cytometry was performed to detect the apoptosis rate of gastric cancer cells after si-FOXK1 transfection.9.Western blot assay was performed to detect the expression of apoptosisrelated proteins and NF-κB pathway-related proteins after si-FOXK1 transfection.Results1.The results of the GEPIA database(http://gepia.cancer-pku.cn/)showed that the mRNA expression level of FOXK1 in gastric cancer tissues was higher than that in normal gastric tissues,and the difference was statistically significant(P<0.05).2.RT-qPCR and Western blot experiments showed that the mRNA expression levels and protein expression levels of FOXK1 in gastric cancer cell lines(HGC-27,BGC-823 and SGC-7901)were higher than those in human normal gastric mucosal cell lines(GES-1),and the differences were statistically significant(P<0.05).3.RT-qPCR and Western blot experiments showed that the expression of FOXK1 mRNA level and protein level were significantly decreased in the transfected group compared with the control group,and the difference was statistically significant(P<0.05).4.MTT assay showed that the proliferation ability of gastric cancer cells in the transfected group was reduced compared with the control group,and the difference was statistically significant(P<0.05).5.Clone formation assay showed that the colony formation ability of gastric cancer cells in the transfected group was reduced compared with that in the control group(P<0.05).6.The scratch assay showed that the migration ability of gastric cancer cells in the transfected group was reduced compared with that in the control group,and the difference was statistically significant(P<0.05).7.Transwell assay showed that the migration and invasion ability of gastric cancer cells in the transfected group was significantly reduced compared with the control group(P<0.05).8.Flow cytometry showed that the apoptosis of gastric cancer cells in the transfected group was increased compared with that in the control group,and the difference was statistically significant(P<0.05).9.Western blot showed that the expression of Bax protein and CleavedCaspase-3 protein increased,and the expression of p-NF-κB p65 protein and Bcl-2 protein decreased,while the Bax/Bcl-2 ratio increased in the transfected gastric cancer cells compared with that in the control group,and the difference was statistically significant(P<0.05).ConclusionsFOXK1 was highly expressed in gastric cancer cell lines(HGC-27,BGC-823 and SGC-7901)and knockdown of FOXK1 can inhibit the proliferation,migration,and invasion ability of gastric cancer cells and regulate NF-κB pathway promotes apoptosis in gastric cancer cells.FOXK1 may be an oncogene in gastric cancer and is expected to become a target for gene therapy. |
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