Super Enhancer Related Gene SCARB1 Regulates The Proliferation,Apoptosis,Migration And Invasion Of Gastric Cancer Cells Through PI3K/AKT Pathway

Objective:This study aimed to clarify the expression of super enhancer-related gene SCARB1 in gastric cancer cells,and to explore its effects on proliferation,apoptosis,migration and invasion of gastric cancer cells,so as to provide possible new targets for the treatment and prognosis analysis of gastric cancer.Methods:1.Bioinformatics methods(1)The super enhancer map was drawn by using the gastric cancer cell MKN45 Ch IP-seq data in the GEO database,and the super enhancer related gene SCARB1 was preliminarily determined as the research target molecule by GO analysis.(2)KM plotter database was used to explore the relationship between SCARB1 expression level and prognosis of gastric cancer.(3)The expression of SCARB1 in gastric cancer tissues and adjacent normal tissues was compared by GEPIA and TNMplot database.(4)Expression Atlas database was used to analyze the expression of SCARB1 in human gastric cancer cells AGS,HGC27 and MKN45.2.The expression of SCARB1 in human gastric epithelial cells GES1 and human gastric cancer cells AGS,HGC27 and MKN45 was analyzed and detected by RT-q PCR and Western blot.3.AGS,HGC27 and MKN45 were all randomly divided into three groups: si-SCARB1 group transfected with SCARB1 small interfering RNA(experimental group),scramble group transfected with negative si RNA sequence(negative control group)and mock group without transfection of si RNA(blank control group).The cells in each group were treated for 48 h to verify the interference efficiency.4.The effect of SCARB1 on the proliferation of gastric cancer cells was detected by colony formation assay and CCK-8 assay.5.The effect of SCARB1 on apoptosis of gastric cancer cells was detected by flow cytometry.6.The effect of SCARB1 on the migration and invasion of gastric cancer cells was detected by cell scratch test and Transwell migration and invasion test,and the expression of MMP2 and MMP9 proteins was detected by Western blot.7.Western blot was used to detect the relative expression of AKT,p-AKT(ser473),PI3 K and evaluate the phosphorylation level of AKT.Results:1.The results of bioinformatics analysis showed that :(1)There were 489 super enhancers and 892 super enhancer-related genes in MKN45 cells.SCARB1 was associated with super enhancer 5＿MACS＿peak＿7443＿loci Stitched.GO analysis suggested that SCARB1 may be related to epithelial cell migration and proliferation;(2)KM plotter database analysis showed that the 5-year overall survival rate,5-year first progression survival rate and 5-year post progression survival rate of gastric cancer patients with high expression of SCARB1 were lower than those of patients with relatively low expression;(3)GEPIA and TNMplot database analysis showed that the expression of SCARB1 in gastric cancer tissues was higher than that in adjacent normal tissues;(4)Expression Atlas database analysis showed that SCARB1 was highly expressed in AGS,HGC27 and MKN45.2.RT-q PCR results showed that the relative expression of SCARB1 m RNA in AGS,HGC27 and MKN45 was higher than that in GES1(P<0.05).Western blot results were consistent with RT-q PCR results(P<0.05).3.Compared with the scramble group,the expression of SCARB1 in si-SCARB1 group of AGS,HGC27 and MKN45 cells decreased(P<0.05).Western blot results were consistent with RT-q PCR results(P<0.05).4.Clone formation assay showed that the colony formation rate of AGS,HGC27 and MKN45 cells decreased after down-regulation of SCARB1 expression(P<0.05);CCK-8 assay showed that the cell viability of AGS cells in si-SCARB1 group was lower than that in scramble group at 24 h,48h and 72h(P<0.05),and the cell viability of HGC27 and MKN45 cells in si-SCARB1 group was lower than that in scramble group at 48 h and 72h(P<0.05).There was no significant difference in clone formation rate and cell viability between scramble group and mock group(P>0.05).5.Flow cytometry results showed that the apoptosis rate of AGS,HGC27 and MKN45 cells increased after down-regulation of SCARB1 expression(P<0.05).6.Scratch test showed that the average scratch width of AGS cells in si-SCARB1 group at 24 h and 48 h was larger than that in scramble group(P<0.05),and the average scratch width of HGC27 and MKN45 cells in si-SCARB1 group at 48 h was larger than that in scramble group(P<0.05).Transwell migration assay showed that the number of migrated AGS,HGC27 and MKN45 cells decreased after down-regulation of SCARB1 expression(P<0.05).There was no significant difference in the average scratch width and the number of migrated cells between the scramble group and the mock group(P>0.05).7.Transwell invasion assay showed that the number of invasion of AGS,HGC27 and MKN45 cells decreased after down-regulation of SCARB1 expression(P<0.05).8.Western blot results showed that the expression of MMP2,MMP9,AKT,p-AKT(ser473)and PI3K(p110)in AGS,HGC27 and MKN45 cells decreased after down-regulation of SCARB1 expression,and the phosphorylation level of AKT decreased(P<0.05).Conclusions:1.SCARB1 is associated with super enhancer named 5＿MACS＿peak＿7443＿loci Stitched in MKN45 cell line.2.Bioinformatics suggests that gastric cancer patients with high expression of SCARB1 have poor prognosis.3.SCARB1 is highly expressed in AGS,HGC27 and MKN45,and SCARB1 promote the proliferation,migration and invasion and inhibit apoptosis of gastric cancer cells AGS,HGC27 and MKN45 through PI3K/AKT signaling pathway,participating in the occurrence and development of gastric cancer.