MiR-145 Targets ZEB2 To Regulate P53-mediated Glycolytic Pathway To Improve Liver Fibrosis

Liver fibrosis is a pathological development of diffuse hepatocyte damage resulted from various acute and chronic liver diseases,identified with the accumulation of extracellular matrix（ECM）.The proliferation and activation of hepatic stellate cells have been confirmed to be the core events in the occurrence and development of liver fibrosis.MiR-145 is a kind of small non-coding RNA with regulatory effect,which can influence the disease process in many ways.It has been reported that miR-145 can be involved in the process of glucose metabolism in diseases.Previous research has confirmed that miR-145 is strongly associated with the occurrence and development of liver fibrosis,and found that miR-145 plays an important role in the apoptosis and aging process of hepatic stellate cells.In this paper,we will deeply study the correlation between HSCs cell function changes and metabolic patterns,study the effect of miR-145 on HSCs glycolysis,explore the potential molecular mechanism to provide new ideas for the prevention and treatment of liver fibrosis.Objectives:This project aims to explore the effect of miR-145 on the glycolysis and related molecular mechanisms in the process of CCl4-induced hepatic fibrosis mice and TGF-β1-induced activated LX-2 cells.Methods:1.Changes of miR-145 and glycolysis in CCl4-induced hepatic fibrosis mice and TGF-β1-activated LX-2 cells.In vivo,C57BL/6J mice（4-week-old,male,weight 20-22 g）were assigned into vehicle group and model group（n=20 per group）randomly.Model group were intraperitoneally injected with a mixture of CCl4 and olive oil[1:9（V/V）]（0.01 ml/g）for 4 weeks,twice a week.At the same time,vehicle group were intraperitoneally injected with the same volume of 100%olive oil.4 weeks later,all mice sacrificed,serum and liver tissues were collected.Degree of liver injury was evaluated via hematoxylin eosin（HE）staining,HA,ALT and AST tset.Collagen deposition in liver tissues was detected via Masson Trichrome and Sirius red staining.Level of miR-145 was detected by one-step qRT-PCR.Lactic acid kit detected lactate level in mice serum.qRT-PCR and Western blot detected the mRNA and protein of key glycolysis enzymes PKM2 and HK2,myofibroblast markers α-SMA and COL1A1.In vitro,LX-2 cells were stimulated with TGF-β1（10 ng/ml）for 24 hours.Level of miR-145 was detected by one-step qRT-PCR.Extracellular lactate level of LX-2 were detected by lactic acid kit.qRT-PCR and Western blot were applied to detect the mRNA and protein expression of PKM2,HK2,α-SMA and COL1A1.2.miR-145 ameliorates liver fibrosis by inhibiting glycolysisMice were exposed to liver-specific adeno-associated virus AAV9-miR-145-luc through tail vein.A week later,CCl4-induced hepatic fibrosis model started to establish.The degree of liver injury was evaluated via HE staining,HA,ALT and AST test.Collagen deposition in liver tissues was detected via Masson Trichrome and Sirius red staining.the level of extracellular lactate was evaluated via Lactic acid kit.Immunohistochemistry,qRT-PCR and Western blot detected the expression of liver fibrosis related indicators α-SMA,COL1A1 and key glycolytic enzymes PKM2,HK2.The immunofluorescence double staining was used to detect the co-localization of the glycolytic protein PKM2 and the liver fibrosis related indicators α-SMA.We established overexpression or downexpression of miR-145 by using miR-145 mimic and miR-145 inhibitor in TGF-β1-induced LX-2 cells.Lactic acid kit detected the level of extracellular lactate.qRT-PCR and Western blot detected the expression of PKM2,HK2,α-SMA and COL1A1.Co-localization of PKM2 and α-SMA was detected by the immunofluorescence double staining.CCK-8 assay detected the influence of the proliferation on LX-2 cells.Wound healing was used to detect the influence of migration on LX-2.3.miR-145 regulates glycolysis via p53 in activated HSCsAfter overexpression of miR-145,qRT-PCR and Western blot detected the expression of p53 in vivo and in vitro.Next,LX-2 cells were treated with p53-plasmid and p53 small interfering RNA（p53-siRNA）to establish an overexpressing and silencing p53 cell model,and then TGF-β1 stimulated for 24 h.Lactic acid kit detected the level of extracellular lactate.qRT-PCR and Western blot detected the expression of PKM2,HK2,α-SMA and COL1A1.Double-IF staining detected the co-localization of PKM2 and α-SMA.CCK-8 assay detected the influence of the proliferation on LX-2 cells.Wound healing detected the influence of migration on LX-2.4.miR-145 targets ZEB2 to regulate p53-mediated glycolytic pathway to ameliorate liver fibrosisBioinformatics software analysis results show that miR-145 has a targeting relationship with ZEB2.Firstly,LX-2 cells were treated with ZEB2 small interfering RNA（ZEB2-siRNA）to establish a silencing cell model,and then TGF-β1（10 ng/ml）stimulated for 24h.Experiments were conducted to dectect the lactate acid,key glycolysis enzymes,liver fibrosis related indicators,cell function of proliferation and migration.Nextly,expression of ZEB2 was detected by qRT-PCR and Western blot after miR-145 overexpressed and silenced in vivo and in vitro.Co-transfection of miR-145 inhibitor and ZEB2-siRNA were conducted in TGF-β1 activated LX-2 cell.Lactic acid kit detected the level of extracellular lactate.Western blot detected the expression of PKM2,HK2,α-SMA and COL1A1.Fllowing,expression of p53 was detected by qRT-PCR and Western blot after ZEB2 silenced in activated LX-2 cell.Co-transfection of ZEB2-siRNA and p53-siRNA were conducted in TGF-β1 activated LX-2 cell.Lactic acid kit detected the level of extracellular lactate.Western blot detected the expression of PKM2,HK2,α-SMA and COL1A1.Results:1.Expression of miR-145 was decreased and the level of glycolysis increased significantly in CCl4-induced hepatic fibrotic tissues and TGF-β1-induced LX2 cells.2.Liver-specific miR-145 overexpressed can inhibit glycolysis,alleviate liver injury,and improve liver fibrosis in mice.Meanwhile,in TGF-β1 stimulated LX-2 cells,miR-145 overexpressed can reduce the level of extracellular lactate,reduce the expression levels of key glycolysis enzymes PKM2,HK2 and liver fibrosis related indicators α-SMA,COL1A1,inhibit the cell function of proliferation and migration.Silencing of miR-145 got opposite results.3.Overexpression of miR-145 significantly increased p53 protein levels in vivo and in vitro.In TGF-β1 stimulated LX-2 cells,p53 overexpressed can reduce the level of extracellular lactate,reduce the expression levels of PKM2,HK2,α-SMA and COL1A1,inhibit the cell function of proliferation and migration.Silencing of p53 got opposite results.MiR-145 may regulate glycolysis via p53 in activated HSCs.4.Silencing of ZEB2 can reduce the level of extracellular lactate,reduce the expression levels of key glycolysis enzymes and liver fibrosis related indicators.Overexpression of miR-145 can significantly inhibit the protein and mRNA level of ZEB2,while silencing of miR-145 got opposite results.Increased glycolysis and liver fibrosis markers were down-regulated after silencing ZEB2 on the basis of silencing miR-145 again in TGF-β1 stimulated LX-2 cells.Meanwhile,silencing of ZEB2 increased p53 mRNA and protein levels significantly.We also found that inhibited glycolysis and liver fibrosis markers were up-regulated after silencing p53 on the basis of silencing ZEB2 again in TGF-β1 stimulated LX-2 cells.Conclusion:The above results suggest that miR-145 inhibits p53 mediated HSCs glycolysis by targeting ZEB2,thereby improving liver fibrosis.