Focal Adhesion Kinase-related Non-kinase Ameliorates Liver Fibrosis By Inhibiting Aerobic Glycolysis Via FAK/Ras/c-Myc/ENO1 Pathway

Objective:To investigate the effect of focal adhesion kinase-associated non-kinase(FRNK)on aerobic glycolytic function and pro-fibrotic biological behavior of hepatic stellate cells(HSCs)during the development of liver fibrosis.Methods:The expression trends of focal adhesion kinase(FAK)and FRNK were observed in human and mouse liver fibrosis tissues;FRNK was overexpressed or knocked out in mouse primary HSCs(p HSCs)and human hepatic stellate cell line LX-2,respectively,and the expression of α-smooth muscle actin(α-SMA)was detected by Western blotting to detect the expression of phosphorylated protein at the 397 tyrosine site of FAK(p Y397-FAK)and the expression of glycolysis-related proteins monocarboxylate transporter 1(MCT-1)and enolase(ENO1).The migration of LX-2 cells was observed by Transwell assay,cell proliferation was observed by Cell Count Kit-8(CCK-8)assay,apoptosis was observed by flow cytometry after Annexin V-PE/7-AAD staining;aerobic glycolysis ability such as glucose uptake and utilization by HSCs and extracellular lactate levels was detected.Putative binding sites for c-Myc-regulated ENO1 promoter regions were predicted by bioinformatics analysis and validated by chromatin immunoprecipitation(Ch IP)and dual luciferase reporter assays.Results:pY397-FAK protein levels were up-regulated,while FRNK was down-regulated in human and mouse liver fibrotic tissues,and FRNK knockout mice had a more pronounced liver fibrosis phenotype.FRNK knockout could promote the activation,migration,proliferation and inhibit the apoptosis of mouse p HSCs,while the increased expression of MCT-1 and ENO1 proteins,their uptake and utilization of glucose and extracellular lactate levels in mouse p HSCs were increased(P<0.05).In contrast,introduction of exogenous FRNK inhibited LX-2 activation,migration,proliferation,and promoted apoptosis,and reduced MCT-1 and ENO1 protein expression,its uptake and utilization of glucose,and extracellular lactate levels in LX-2(P<0.05).After LX2 cells were activated by TGF-β1,k-Ras/c-Myc/ENO1 protein expression downstream of FAK was up-regulated and used to promote aerobic glycolysis function in HSCs(P<0.05);Ch IP and dual-luciferase reporter assay showed that c-Myc could directly bind the promoter of ENO1 and transcriptionally activate it(P<0.05).Conclusions:FRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-Myc/ENO1 pathway,thereby improving liver fibrosis.FRNK might be a potential target for liver fibrosis treatment.