Study On The Molecular Mechanism Of Y-box Binding Protein 1 Regulating The Differentiation Of Hepatic Progenitor Cells Into Hepatocytes

Objective: The liver has a remarkable capacity for regeneration.Self-renewal of hepatocytes is the main mechanism responsible for liver mass homeostasis and for liver regeneration after acute(moderate)liver injury and reductionof liver mass.However,in conditions of chronic liver injury or submassive liver cell loss,such capacity for selfrenewal is overwhelmed,exhausted,or impaired,leading to liver failure or insufficiency.In those conditions,to maintain liver parenchymal homeostasis and liver function,hepatic progenitor cells,actively proliferate,and yield transit-amplifying cells.High expression of YB-1 in human fibrotic liver,however the correlation of YB-1 with fibrotic liver and liver regeneration regulated by HPC is unclear.The aims of this study are to study on the molecular mechanism of Y-box binding protein 1 regulateing the differentiation of hepatic progenitor cells into hepatocytes,and its role during liver regeneration and fibrosis regulated by HPC.Methods: Quantitative real time polymerase chain reaction,western blot and immunohistochemistry technology were used to detect expression and distribution of YB-1,Wnt3 in human and mouse livers.Double immunofluorescence histochemistry was applied for observing expression and distribution of YB-1,Wnt3 in CK19 positive HPC.HE stain was used to detect degree of liver injury and fibrosis.HPC was isolated via methods of collagenase perfusion and andensity gradient centrifugation,and then characterized HPC.Established model of differentiation of HPC into hepatocytes in three dimention in vitro,following,knock-down and ovexpress YB-1 by gene interference in HPC.Immunofluorescence technology,WB,qRT-PCR were applied to test the expression of HPC markers(CK19,AFP),hepatic markers(albumin,glutamine synthetase,hepatic nuclear factor 4 alpha),YB-1 and p-YB-1.The concentration of secreted albumin and urea in supernate of HPC was detected by ELISA.Functional assessment of differentiated HPC adopted Oil Red O stain,ingestion and storage experiments of indocyanine green,immunofluorescence technology of golgi complex,endoplasmic reticulum,mitochondria.Chromatin immunoprecipitation assays and ChIP-sequence were introduced to probe the YB-1 binding site in promotor of Wnt3 in HPC,and then ChIP-PCR was utilized to confirm that.Biluciferase reporter gene assay was used to evaluate regulator effection of YB-1 on Wnt3 promotor,WB and qRT-PCR were applied to detect the expression of Wnt3,?-catenin,c-Myc,Axin-2 and CyclinD1.On the basis of regulating YB-1,and then knockdown and ovexpress Wnt3,respectively,WB and qRT-PCR were applied to detect expression of Wnt3,?-catenin,c-Myc,Axin-2,CyclinD1,CK19,AFP,albumin,GS,HNF4?,YB-1 and p-YB-1.At last,functional assessment of differentiated HPC was done again.To observe the hepatic differentiation of HPC in liver of human and mouse,double immunofluorescence histochemistry was used to test expression and distribution of CK19,HNF4?.After HPC transplant through spleen,HE staining was utilized to detect the degree of liver injury,double immunofluorescence histochemistry was introduced to detect expression and distribution of albumin and GFP,the concentration of albumin and urea was assessed to evaluate the liver function.Results: 1.No expression of YB-1 and weak expression of Wnt3 in normal liver of human and mouse,but the expression of YB-1 and Wnt3 was significant increased in the livers of patients infected with chronic viral hepatitis B and mice with liver injury,especially in the regions of ductular reaction and CK19 positive HPC,and the expression level correlated with the severity of liver fibrosis.2.In differentiated HPC,the expression of albumin,GS and HNF4? was increased,but CK19,AFP,YB-1 and p-YB-1 was reduced.Differentiated HPC own the function of synthesizing lipid droplet,ingesting and storing indocyanine green,secreting albumin and urea.Compaired to undifferentiated HPC,the activation of golgi complex,endoplasmic reticulum,mitochondria in differentiated HPC was increased.3.YB-1 knockdown by gene technology promoted expression of Wnt3,?-catenin,c-Myc,Axin-2,CyclinD1.At the same time,the expression of albumin,GS and HNF4? was increased,CK19 and AFP was reduced.The function of synthesizing lipid droplet,ingesting and storing indocyanine green,secreting albumin and urea was furtherly increased.YB-1 overexpression reduced expression of Wnt3,?-catenin,c-Myc,Axin-2,CyclinD1.The expression of albumin,GS and HNF4?was reduced,but CK19 and AFP was increased.The function of synthesizing lipid droplet,ingesting and storing Indocyanine green,secreting albumin and urea was furtherly reduced.4.ChIP-sequence and ChIP-PCR demonstrated that YB-1 had combining site in Wnt3 promotor.Biluciferase reporter gene confirmed that knock-down YB-1 significantly increased the luciferase activity of Wnt3 promotor,but ovexpression YB-1 significantly inhibited luciferase activity of Wnt3 promotor.Furtherly,we affirmed that the combining site of YB-1 in Wnt3 promotor was located at 5'UTR39366bp by bioinformatics contrast.5.Knocking down YB-1 and Wnt3 simultaneously,qPCR and WB verified that the expression of albumin,GS and HNF4? was reduced,but CK19 and AFP was increased.The function of synthesizing lipid droplet,ingesting and storing indocyanine green,secreting albumin and urea was furtherly reduced.Over-expressing YB-1 and Wnt3 simultaneously,qPCR and WB verified that the expression of albumin,GS and HNF4?was increased,but CK19 and AFP was reduced.The function of synthesizing lipid droplet,ingesting and storing Indocyanine green,secreting albumin and urea was furtherly increased.6.Double immunofluorescence histochemistry showed that CK19 and HNF4? double positive cells appeared in fibrotic livers of patients with chronic viral hepatitis B and chronic injuried livers of mice,but the positive ratio is very low about 1-1.5%.7.The injury degree and function of mice liver treated with HPC via spleen transplant were significantly ameliorative than others untreated with HPC,the concentration of albumin in serum was significantly increased,but ALT and AST was significantly reduced.GFP and albumin double positive cells were dispersedly distributed in injuried liver.Conclusion: 1.The expression level of YB-1 is positively correlated with degree of liver fibrosis and injury.2.CK19 positive HPC can differentiate into hepatocytes.3.The binding site of YB-1 in Wnt3 promotor is located at 5'UTR 39366 bp.4.YB-1 negatively regulates Wnt3,influences Wnt/?-catenin signaling pathway,and then controls hepatic differentiation of HPC.5 Liver injury degree and liver function of mice treated with HPC through spleen are significantly ameliorative.