XIAP Expression In Hepatocellular Carcinoma Tissues And Roles Of XIAP In Hepatocellular Carcinoma Cell Regulation

IntroductionHepatocellular carcinoma(HCC) is the fifth commonest malignancy and the third leading cause of cancer death in the world,being responsible for 80%of the primary malignant liver tumors in adults[1].The five-year relative survival rate is about 7%and causes more than 600,000 deaths annually worldwide[2].Although the prevelance is highest in Africa and Asia,the incidence in western countries is rising, mainly due to increasing rates of alcoholic liver diseases and hepatitis C infection[3]. HCC has several interesting epidemiologic features including dynamic temporal trends;marked variations among geographic regions,racial and ethnic groups,and between men and women;and the presence of several well-documented environmental potentially preventable risk factors[4].Global gene expression profiling of HCC is a promising new technology that has already refined the diagnosis and prognostic prediction of HCC patients[6].As expected,prediction models based on gene expression profiling discriminate HCC from non-tumor livers with high accuracy successfully[7].A great deal of studies have identified differentially expressed genes in HCC that differ according to etiological factors[8],mutations of tumor suppressor genes[9],rate of recurrence [10],intrahepatic metastasis[11],and different stages[12].However,most of these studies have identified genes associated with limited aspects of tumor pathogenesis, but do not necessarily reflect the underlying biological properties likely driving tumor behavior.This is because the level of mRNA do not always reflect the accurately the levels of corresponding proteins nor do they reveal changes in epigenetic posttranscriptional modulation of proteins or changes in proteins degradation rates. Therefore,it is important to analyze difference in protein levels or modification between tumor cells and normal cells to complement studies on mRNA expression. The Pathway Array Technique is an innovative proteomic assay that allows screen globally the changes in protein expression.The focus of pathway array is to determine the signaling network that related to cancer development(initiation, promotion,invasion).The proteins selected from those highly expressed in cancer cells are functionally linked to the angiogenesis,apoptosis,cell cycle regulation, DNA repair,migration,proliferation,signaling,stem cell association and transcription activity.In our study,we will mainly use this technique to determine changes of protein levels and signaling networks in HCC.Human hepatocellular carcinomas(HCCs) usually develop as late complications of chronic liver injury,and are characterized by mononuclear cell infiltration of the parenchyma at the margin of liver lobules and apoptotic disappearance of contiguous hepatocytes.Failure of apoptosis is considered to be important for the survival of hepatocytes that are prone to undergo genetic damage and cellular transformation by an increased proliferative rate due to hepatocyte regeneration[33,34].In fact,most HCC cells show strong resistance to various stimuli that induce apoptosis[35,36]. However,the anti-apoptotic mechanisms of HCC cells have not been well elucidated.Recently,it was demonstrated that the inhibitors of apoptosis(IAP) are crucial regulators in the molecular mechanism of apoptosis.To date,6 human IAP proteins, X-linked IAP(XIAP),c-IAP1,c-IAP2,NAIP,survivin,and Apollon have been cloned[38-42].They are characterized by the presence of 1-3 copies of a 70-amino acid motif,the BIR domain,which bears homology to sequences found in the baculovirus IAP proteins and plays an important role in binding and inhibition of caspase,particularly caspase 3 and 7[43].In addition,c-IAP1 and c-IAP2 were shown to interact with tumor necrosis factor(TNF) receptor-associated factor(TRAF)-1 and(TRAF)-2 in the yeast 2-hybrid system.These results suggested that c-IAP1 and c-IAP2 are involved in regulation of TNF-induced NF-κB activation via TRAF[43].The XIAP protein consists of 3 BIR domains and 1 RING finger domain and appears to be a more potent inhibitor of caspase 3 and 7 than c-IAP1 and c-IAP2[44-46].XIAP mRNA was observed in all adult and fetal tissues examined except peripheral blood leukocytes[43],indicating that it is a ubiquitously expressed member of the IAP family[43,47].Therefore,we will use the pathway away analysis technique to evaluate XIAP expression in HCC;at the same time use XIAP siRNA to silence the specific gene expression in HCC cell lines,so as to research how the small RNA could effect HCC cell proliferation,cell cycle,cell apoptosis,and other proteins expression.Methods:1.We collected 13-paired liver resection tissue samples including tumor tissues and normal tissues,extracted proteins to run pathway array analysis,in order to determine their expression levels.2.According to the results of pathway array analysis,we used small RNA to interfere XIAP gene expression in hepG2/2.2.1 (human hepatocellular carcinoma) cell lines.By RT-PCR amplification,we could define whether XIAP had been silenced or not.3.After XIAP siRNA transfection,we proceeded cell viability analysis to make sure whether XIAP played roles in HCC cell proliferation.4.After XIAP siRNA transfection,we proceeded cell cycle analysis to make sure whether XIAP played roles in HCC cell cycle regulation.5.After XIAP siRNA transfection,we proceeded cell apoptosis analysis to make sure whether XIAP played roles in HCC cell apoptosis.6.After XIAP siRNA transfection,we extracted proteins from cells to run pathway array analysis,to determine whether XIAP silencing could affect other proteins expression.Results:1.Among 13-paired liver resection tissue samples,we screened forty-four phosphorylated and un-phosphorylated antibodies,totally detected twenty-three proteins,twenty-two proteins with more two folds changes.XIAP,BRCA1,PCNA were much more overexpressed in HCC.2.After transfection of XIAP siRNA in hepG2/2.2.1 cells,by RT-PCR amplification,we could find that XIAP gene expression had been silenced.3.After transfection of XIAP siRNA in hepG2/2.2.1 cells,compared with controls,we could find that the cell viabilities had been decreased seriously(P＜0.05);therefore,we could conclude that XIAP affected HCC cell proliferation.4.After transfection of XIAP siRNA in hepG2/2.2.1 cells, compared with controls,we could find that XIAP siRNA had induced HCC cell G1 arrest.5.After transfection of XIAP siRNA in hepG2/2.2.1 cells,compared with controls,we could find that XIAP siRNA had induced more necrotic cells in HCC cells.6.After transfection of XIAP siRNA in hepG2/2.2.1 cells,we proceeded pathway array analysis,screened sixty-six phosphorylated and un-phosphorylated antibodies,totally detected twenty-six proteins,six proteins with more two folds changes,inhibiting ETS1 expression as well as stimulating expression of cdk6, CHK1,CREB,p38 and p53.Conclusions:1.There are many changes of proteins expression in HCC;especially XIAP, BRCA1,PCNA are overexpressed in HCC tissues.2.XIAP affects cell proliferation,cell cycle and cell apoptosis regulation of HCC,inhibiting ETS1 expression as well as stimulating expression of cdk6,CHK1, CREB,p38 and p53.