The Expression And Function Of Transcription Factor Goosecoid In Liver Fibrosis

Liver fibrosis is a common pathological process after a series of chronic damage,as well as a reversible damage-repair process,which is charactered by the excessive deposition of extracellular matrix(ECM).Some of Liver fibrosis will progress to cirrhosis and/or hepatocellular carcinoma(HCC).For the high mortality and poor prognosis,liver fibrosis is a major threat to human health.Excessive deposition of extracellular matrix(ECM)mainly derived from hepatic stellate cells(HSC).When stimulated by various pathogenic factors of liver,quiescent hepatic stellate cells(q HSC)transform into myofibroblast cells,then activated hepatic stellate cells(aHSC)happens a series of changes,such as the big fat drops,cell growth accelerated,deposition of collagen.So the research of HSC proliferation,apoptosis and migration mechanismhas a very important significance on hepatic fibrosis.Epithelial-mesenchymal transition(EMT)refers to the epithelial phenotype transformating to mesenchymal phenotype.Studies have shown that EMT have relation to embryonic development,organ regeneration,and fibrosis and tumor metastasis.EMT invoves the progression of qHSC transformating to mesenchymal cells.Goosecoid(GSC)belonging to the homeobox transcription factor,during gastrulation,development of bulla,breast cancer cells formention,GSC as one of the organizers of genes,inducing EMT,promoting cell migration.At present,GSC recognized as an transcription factor inducing EMT,for the study of EMT.GSC can induce EMT related to embryonic development and progress of tumor,however its role in organ fibrosis related to EMT has not been reported.TGF-? is a key factor for liver fibrosis,the The role of GSC in liver fibrosis is still unclear.Objective: Determine the effects of GSC on liver fibrosis and activation of HSC,and study effects of GSC on HSC activation,proliferation,apoptosis and migration.Providing new targets for treatment of liver fibrosis.Methods: Intraperitoneal injection of carbon tetrachloride(CCl4)establishing the mouse models of hepatic fibrosis and TGF-? activation of hepatic stimulator LX-2 cells,observating GSC changes in the process of hepatic fibrosis formation and activation of HSC.HE staining of liver tissue was used to observe liver inflammation.Masson staining were performed to determine collagens.By immunohistochemistry(IHC)method tests the expression of a-SMA,Collagen I and GSC.In vitro,knocking down GSC with small interfering RNA.By Real-time PCR method observing the level of a-SMA,Collagen I and GSC.CCK-8 assay and BrdU incorporation test study the effect of knocking down GSC on cell proliferation.Annexin V/PI double staining was used to examine cell apoptosis,and the activity of apoptosis related proteins caspase-3 and PARP was further examined by western blot.Cell migration was accessed by wound healing assay.Results:1 GSC expression during the formation of liver fibrosis of mice increasedCompared with the control group,HE staining showed that disorder of hepatic lobules,inflammatory cell infiltration,fatty degeneration and necrosis of hepatocytes is serious in model group;Masson staining showed that the disappearance of hepatic lobules,septa staining obvious,the formation of fibrous septum and pseudolobule in model group,whereas control group only fiber with vein wall,interlobular and septa occasionally a small amount of fibers deposition.IHC showed ?-SMA,collagen I and GSC overexpressed.These results indicate that hepatic fibrosis can be induced by CCl4 intraperitoneal injection,and increased expression of GSC in this process.2 TGF-? inducing activation of HSC can raise GSC in vitroAfter HSC cell line LX-2 intervention by TGF-?,campared with control group,Western Blot and Real-time PCR results showed a increased protein expression and level of mRNA of ?-SMA and Collagen I.The results indicate that the GSC has positive correlation with activation of HSC.3 Knocking down GSC with small interfering RNA can inhibit HSC activationReal-time PCR results showed GSC mRNA levels decreased after transfection with siGSC 48 h,and a further decline after transfection with 72 h,showing that GSC is effectively knocking down.Campared with transfected with siControl group,Western Blot and Real-time PCR results showed transfected with siGSC group a decreased protein expression and level of mRNA of ?-SMA,Collagen I and GSC.The results indicate that the GSC promote activation of HSC.4 Knockdown GSC can inhibit HSC proliferationCCK-8 assay and BrdU incorporation test testifed that HSC proliferation downregulation by transfected with siGSC campared with siControl group.The results indicate that the GSC promote proliferation of HSC.5 Knockdown GSC has no effect on HSC apoptosisWestern blot results show the activity of apoptosis related proteins caspase-3 and PARP having no differents between siGSC group and si Control group.V/PI double staining shows the same result as western blot.The results indicate that the GSC have no effect on HSC apoptosis.6 Knockdown GSC can inhibit HSC migrationWound healing test displayed HSC treated with si GSC healed much more slowly than siControl cells indicating GSC may promote HSC migration.Conclusion:1 GSC has a overexpression in the progression of liver fibrosis formation and activation of HSC.2 GSC can promote HSC activation,proliferation and migration.3 GSC have no effect on HSC apoptosis.