The Role Of Hepatic SATB1 On HBV-related Liver Fibrosis

Aims:Chronic hepatitis B virus(HBV)infection is one of the major public health problems worldwide,mostly resulting in liver cirrhosis and high morbidity and mortality worldwide.Hepatic fibrosis is a critical procedure in the progression of chronic liver disease to cirrhosis.Nowadays there exists no effective treatment to HBV-related liver fibrosis,so exploring the molecular mechanism of HBV-related liver fibrosis may provide potential targets for treatment of chronic hepatitis B cirrhosis.Special AT-rich binding protein 1(SATB1),mainly present in thymocytes,has been implicated in reprogramming chromatin organization and transcription profiles in Tcell development and many cancers.In this study,we attempt to uncover the role and mechanism of SATBlin HBV-related liver fibrosis.Methods:Immunohistochemical(IHC)staining was used to investigate SATB1 expression and presence in 58 liver tissues from chronic hepatitis B(CHB)and liver cirrhosis(LC)in HBV infected patients.Then we established a CCl4-induced fibrosis model in HBV transgenic mice,and performed real-time PCR,western blot and immunohistochemistry to determine the expression of SATB1 and a-SMA expression.In vitro,we transfected synthetic DNA plasmids containing seven viral genes of HBV into human hepatic cell line L02 to screen which viral proteins could effectively upregulate SATB1 expression.To verify the role of HBx in the modulation of SATB1,we established two stable HBx-transfected hepatic cell line and dectected SATB1 level by real-time PCR,western blot,luciferase assay.Besides,to elucidate the molecule mechanism of HBx-mediated SATB1 expression,U0126,SP600125,SB203580 and LY294002 were used to specifically render inactivate of ERK1/2,JNK,p38 MAPK and PI3k-Akt pathway,so we could screen out the most relevant signal pathway.Next,we screened a panel of transcription factors including AP-1,NFKB1,Sp1 by delivery siRNA targeting them into HBx-overexpressed cells,real-time PCR,western blot,luciferase assay were used to examine the mechanism of transcription activation of HBx on the SATB1 promoter.On the other hand,we were determined to find out whether hepatic SATB1 could exert a paracrine effect on HSCs by real-time PCR,western blot,immunofluoresence,CCK8 and Edu.To identify the factors responsible for HSC activation and proliferation,we performed a cytokine profile of CM obtained from L02 cells overexpressing SATB1 and control group,and we incubated LX-2 with conditioned media from L02-SATB1 in the presence of blocking antibodies against the cytokines to screen out the most important one.Finally,to observe the effect of SATB1 on HBV-related hepatic fibrosis,adenoviral Vector containing SATB1shRNA or NC were injected via tail vein in CCl4-induced fibrosis model in HBV transgenic mice.The therapeutic effect and mechanism of SATB1 on fibrotic rat liver were investigated by real-time PCR,western blot and immunohistochemistry.Results:We found that hepatic SATB1 expression was significantly up-regulated in fibrotic tissues from chronic hepatitis B virus(HBV)-infected patients and HBV transgenic(HBV-Tg)mouse model.Moreover,we suggested HBV encoded x protein(HBx)induced SATB1 expression through activation of JNK and ERK pathways.Enforced expression of SATB1 in hepatocytes promoted the activation and proliferation of hepatic stellate cells(HSCs)by secretion of connective tissue growth factor(CTGF),Interleukin-6(IL-6)and platelet derived growth factor-A(PDGF-AA).Silencing SATB1 in HBx-transfected cells could down-regulated the levels of PDGF-AA and CTGF.In addition,Knockdown of SATB1 in the liver significantly alleviated CCl4-induced fibrosis in HBV-Tg mouse model.Conclusion:Our findings demonstrated that HBx upregulated hepatic SATB1 which exerted pro-fibrotic effects by paracrine activation of stellate cells through secretion of profibrogenic cytokines IL-6,PDGF-AA and CTGF in HBV-related fibrosis.