Lactic Acid Promotes The Proliferation,migration And Glycolysis Of Huh7 Cells Through PI3K/AKT Signaling Pathway

Objective:To explore the effects of lactate on the proliferation,migration and glycolysis metabolism of Huh7 cells and its possible regulatory mechanism.Methods:Huh7 cells were treated with lactate at the final concentration of 0,10,20,30,40 and 50mmol/L for 48h,and the viability of Huh7 cells was detected by cck-8 method.Then the final concentration of lactate was selected as 0,2.5,5,7.5 and 10mmol/L(This study is called the relatively low concentration)for subsequent experimental studies.The viability and clonogenesis of Huh7 cells were detected by CCK-8 method and plate cloning method.The migration ability of Huh7 cells was detected by cell scratch method.Glucose uptake of Huh7 cells in medium supernatant was detected by glucose kit.Western Blot(WB)was used to detect glucose transporter 4(GLUT4)and Hexokinase 2(Hexokinase 2)in Huh7 cells of different groups.HK2,Pyruvate kinase M2(PKM2),E-cadherin(E-cad),Vimentin(Vim),Phospholnositide-3 kinase,PI3K),phospho-PI3K(Ser85)[Phospho-PI3K(Ser85),p-PI3K(Ser85)],Protein kinase B(PKB),Also known as AKT)and phosphorylated AKT(Ser473)[(Phospho-Akt(Ser473),p-Akt(S473))].The following experiments were performed using PI3K/AKT signaling pathway inhibitor BEZ235(BEZ):Huh7 cells were divided into control group,BEZ(20nmol/L)group,lactate(5mmol/L)group and BEZ combined lactate group for 48h.The viability and migration of Huh7 cells in each group were detected by cck-8 method and cell scratches method.The expression levels of GLUT4,key glycolysis enzymes HK2 and PKM2,E-cad,Vim,PI3K,p-PI3K(Ser85),AKT and p-Akt(Ser435)were detected by WB.Results:Liver cancer cells were treated with 0,10,20,30,40 and 50mmol/L lactate.The results showed that the activity of Huh7 cells was significantly inhibited when lactic acid concentration was higher than 30mmol/L,while the concentration of 0-30mmol/L lactate could promote the activity of Huh7 cells.0～10mmol/L showed an obvious upward trend.Therefore,relatively low concentration lactate of 0,2.5,5,7.5 and 10mmol/L(Relatively low concentration)were selected for subsequent studies in this study.It can be seen that with the increase of lactate concentration,the activity of Huh7 cells gradually increased.The number of cell clones increased gradually.Migration capacity gradually increases.Related protein expressions detected by WB were as follows:①With the i ncrease of Iactate concentration,the expression Ievel of GLUT4,glucose transporter,and HK2 and PKM2 proteins increased gradually;②EMT-related protein:With the increase of lactate concentration,the expression of E-cad gradually decreased,and the expression of Vim gradually increased;③PI3K/AKT signaling pathway:With the increase of lactate concentration,the protein expression levels of p-PI3K(Ser85)and p-Akt(Ser435)gradually increased,however,the total protein expression of PI3K and AKT was not significantly changed,and the difference was not statistically significant.After the introduction of PI3K/AKT signaling pathway inhibitor BEZ,the experimental results were as follows:Compared with the control group,the activity and migration ability of Huh7 cells treated by PI3K/AKT signaling pathway inhibitor BEZ were decreased(all P<0.05).GLUT4,HK2 and PKM2 were down-regulated(All P<0.05).The expression of E-cad protein increased(P<0.05).The expressions of Vim,p-PI3K(Ser85)and p-Akt(Ser435)were down-regulated(P<0.05).Compared with lactate group,the activity and migration ability of Huh7 cells in BEZ combined with lactate group were significantly decreased(P<0.01).The expression levels of GLUT4,HK2 and PKM2 were down-regulated(P<0.05).The expression of E-cad protein was up-regulated(P<0.05).The expressions of Vim,p-PI3K(Ser85)and p-Akt(Ser435)were down-regulated(P<0.05).The total protein expression of PI3K and AKT was not significantly changed,and the difference was not statistically significant.Conclusion:(1)Relatively low concentration lactate(in the concentration range of l0mmol/L)can promote the proliferation,migration and glycolysis metabolism of Huh7 cells;It may be mediated by the activation of PI3K/AKT signaling pathway;(2)BEZ,an inhibitor of PI3K/AKT signaling pathway,can inhibit the proliferation,migration and glycolysis of Huh7 cells in the acidic microenvironment of Huh7 cells simulated by the lactate.