| Objective To explore the influence of Notch1 signaling pathway on the expression and function of GLUT1 in gastric cancer cells,and to explore whether Notch1 can regulate the autophagy level of gastric cancer cells via GLUT1.Methods To research the impact of Notch1 knockout on the expression of GLUT1(SLC2A1)and glucose uptake,gastric cancer cells were divided into three groups.Blank control group(Blank group),wild-type gastric cancer cells without any treatment.Negative control group(NC group),gastric cancer cells infected with negative control virus sg RNA-CON250.Notch1 knockout group(KO group),gastric cancer cells infected with LV-Notch1-sg RNA.To study the effect of GLUT1 overexpression on the cell proliferation,migration,invasion and autophagy induced by Notch1 knockout,gastric cancer cells were divided into another three groups.Negative control group(NC ’group),NC group infected with negative control virus CON335.Notch1 knockout group(KO’ group),negative control virus CON335 infected gastric cancer cells of KO group.Notch1 knockout combined with GLUT1 overexpression group(KO + OE group),LV-SLC2A1 27247-2infected gastric cancer cells in KO group.WB and qRT-PCR were used for detecting the expression of Notch1 and GLUT1,glucose uptake was measured by glucose detection kit,cell proliferation was detected by CCK-8,cell migration was detected by scratch test,cell migration and invasion were detected by transwell experiment,and the expression of autophagy related gene was detected by WB and qRT-PCR.Results1.After Notch1 knockout,in MKN-45 and MGC-803 cells,WB test showed that the relative expression of GLUT1 protein in KO group was markedly lower than that in NC group(t = 5.296,32.843,P = 0.006,0.000).qRT-PCR suggested that SLC2A1 mRNA relative expression in KO group was lower than that in NC group(t = 22.371,8.647,P = 0.000,0.001).The glucose uptake ability of MKN-45 and MGC-803 cells in KO group was lower than that in NC group obviously(t =10.046,4.298,P = 0.001,0.013).2.On the basis of Notch1 knockout,GLUT1 was overexpressed at the same time.The proliferation ability of MKN-45 and MGC-803 cells in KO ’group was lower than that in NC’ group significantly(t = 9.925,19.713,P = 0.001,0.000),and compared to KO’ group,the proliferation capacity of KO+OE group increased obviously(t =-10.175,-3.430,P = 0.000,0.009).3.The wound healing percentage of MKN-45 and MGC-803 cells in KO’ group was markedly lower than that in NC’ group(t = 12.739,14.120,P = 0.005,0.000).And compared to KO’ group,the wound healing percentage in KO+OE group increased obviously(t =-15.377,-10.532,P = 0.000,0.000).4.Transwell assay showed that the migration and invasion abilities of MKN-45 in KO’ group were markedly decreased than those in NC’ group(t = 15.661,15.940,P= 0.000,0.004).And compared to KO’ group,these two abilities of KO+OE group increased obviously(t =-5.275,-7.560,P = 0.006,0.002).As for MGC-803 cells,KO’ group migration and invasion capabilities were significantly lower than that in NC’ group(t = 48.866,61.363,P = 0.000,0.000),and compared to KO’ group,these two abilities of KO+OE group increased obviously(t =-4.627,-11.385,P =0.010,0.000).5.WB results showed that in MKN-45 and MGC-803 cells,the expression of LC3Ⅱ increased markedly in KO’ group than that in NC’ group(t=-13.302,-8.760,p=0.006,0.001),and the protein in KO+OE group was lower than that in KO’group(t=4.601,4.551,P=0.010,0.010).In MKN-45 and MGC-803 cells,Beclin1 relative expression increased markedly in KO’ group than that in NC’ group(t=-7.329,-4.058,p=0.018,0.015),and the relative expression of KO+OE group was lower than that of KO’ group(t = 3.096,3.492,P = 0.036,0.025).In MKN-45 and MGC-803 cells,Atg16L1 relative expression increased markedly in KO’ group than that in NC’ group(t=-8.091,-20.345,p=0.015,0.002),and the relative expression of KO+OE group was lower than that of KO’ group(t = 9.400,7.074,P= 0.001,0.002).The results of qRT-PCR suggested that in MKN-45 and MGC-803 cells,the relative expression of Atg7 mRNA increased significantly in KO’ group than that in NC’ group(t=-13.400,-12.045,p=0.006,0.000),and the relative expression of KO+OE group was lower than that of KO’ group(t= 3.632,3.575,p=0.022,0.023).In MKN-45 and MGC-803 cells,ATG12 mRNA relative expression increased markedly in KO’ group than that in NC’ group(t=-14.807,-12.487,p=0.000,0.006),and the relative expression of KO+OE group was lower than that of KO’ group(t = 2.966,5.920,P = 0.041,0.004).Conclusions Notch1 knockout can inhibit the expression of GLUT1 in MKN-45 and MGC-803 cells,and then inhibit cell proliferation,migration,invasion and glucose uptake,and enhance the level of autophagy.GLUT1 mediates the regulation of autophagy by Notch1 Signaling Pathway in gastric cancer cells. |
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