The Role And Mechanism Of NOXIN In Liver Cancer And The Effect Of LZP On Liver Lipid Metabolism

SECTION 1 THE RESEARCH ON THE ROLE OF NOXIN IN HEPTACELLULAR CARCINOMABackground &Aims: Chromosomal DNA copy number alterations are hallmarks of human malignancies,including hepatocellular carcinoma(HCC).However,which oncogenes or tumor suppressors with DNA copy number aberrations contribute to HCC initiation and progression remain uncharacterized.We performed a genome-wide DNA copy number analysis on human HCC samples to identify novel potential oncogenes or tumor suppressors with DNA copy number aberrations.Methods: Single nucleotide polymorphism microarraygenome-wide DNAcopy number analyses were performed.RT-PCR and immunohistochemical staining were employed to evaluate NOXIN expression in HCC samples.Colony formation,cell cycle analyses and in vivo tumor xenograft assays were performed to assess the role of NOXIN in HCC cells.Reciprocal co-immunoprecipitation experiments were used to detect interactions between NOXIN and DNA polymerase ? primase.Results: Genome-wide DNA copy number analyses of 43 paired HCC samples identified the smallest DNA amplification region containing NOXIN.NOXIN overexpression was significantly associated with HCC tumor stage.Enforced NOXIN expression promoted cellular proliferation,colony formation,cell migration and in vivo tumorigenicity,whereas RNA interference against NOXIN attenuated these effects.Interestingly,NOXIN overexpression accelerated the G1-S phase transition during cell cycle progression by enhancing DNA synthesis in HCC cells,as indicated by bromodeoxyuridine incorporation.Furthermore,NOXIN interacted with DNA polymerase ?,implying that NOXIN may promote de novo DNA synthesis by promoting DNA polymerase-primase complex formation.Conclusions: NOXIN overexpression,as a result of genomic DNA amplification,promotes HCC tumorigenesis by accelerating DNA synthesis and cell cycle progression,during which NOXIN functions as a DNA polymerase-primase complex cofactor by interacting with DNA polymerase ? primase.SECTION 2 THE RESEARCH ON THE ROLE OF LZP IN MICE LIVE LIPID METABOLISMBackground &Aims: Fatty liver,especial nonalcoholic fatty liver disease(NAFLD),may lead to liver injuries ranging from steatosis to steatohepatitis that may progress to cirrhosis and hepatocellular carcinoma(HCC).However,the underlying cellular and molecular mechanisms by which hepatic steatosis contributes to HCC initiation and progression remain obscure.Significantly,LZP(liver-specific zona pellucida domain-containing protein)knockout mice established by our lab exhibit the typical pathological feature of NAFLD with formation of lipid droplets and excess triglyceride(TG),although serum TG and glucose are not elevated in these mice,implying that the LZP-deficient mice could represent a subtype of NAFLD without systemic diseases such as diabetes and obesity.The LZP-deficient mice could provide an ideal animal model for addressing the cellular and molecular mechanisms involved in the course from hepatic steatosis to HCC oncogenesis.Interestingly,LZP,also named oncoprotein induced transcript 3(OIT3),was first isolated and identified by us from human,mouse and rat livers,and was validated to be specifically expressed in hepatocytes,although very weak in mouse kidney.More interestingly,we confirmed that LZP is obviously downregulated in the majority of HCC specimens,although LZP level cannot affect cell proliferation.Based on these collective data,we propose that LZP could function as a transporter or co-factor in lipid metabolism,in which LZP may transport VLDL(very low density lipoprotein)and LDL(low-density lipoprotein)from hepatocytes to blood;hepatic steatosis occurs in the absence of LZP,and then may progress to steatohepatitis and even HCC in the presence of liver injury and inflammation stimulators,such as alcohol,chemical carcinogens and bacterial toxins such as lipopolysaccharide(LPS).Methods: Evaluated the effect of LZP on lipid metabolism indicator by detecting mice blood biochemical index,Evaluated the effect of LZP on liver morphology by histology,observed the location of LZP in liver by immunohistochemistry and immunofluorescence,detected secretion of LZP and the effects on lipid transfer protein by western blotting,Evaluated interaction bettween LZP and lipid transfer protein Apo B by co-immunoprecipitation.Results: Under the condition of LZP deficiency,peripheral blood triglyceride concentration decreased,live steatosis increased,quantity of triglyceride increased in liver homegenate and decreased in serum.LZP located in the intercellular space and secreted in cell culture medium.We also found enhanced LZP expression increased Apo B in serum and cell culture medium.LZP interacted with Apo B and LZP deficiency disturbed correct folding of Apo B.Conclusions: LZP impacted the lipid transfer by interacting with Apo B to matain the correct folding of Apo B.