Identification And Function Analysis Of Differentially Expressed Plasma Proteins In Hbv-related Hcc: An ITRAQ Quantitative Proteomic Based Study

Part Ⅰ Identification and Validation of Aberrantly ExpressedPlasma Proteins in HBV-related HCC by an iTRAQ QuantitativeProteomic AnalysisObjective: Hepatocellular carcinoma (HCC) is the sixth mostcommon cancer worldwide. In China, HCC has been ranked as the secondmost frequent fatal cancer, and chronic infection with hepatitis B virus(HBV) is one of its leading causes. The pathogenesis of HBV-related HCCis not fully understood as yet. The aim of this study was to identify andvalidate proteins that have not been implicated in HCC.Methods: We used isobaric tags for relative and absolute quantitation(iTRAQ) combined with mass spectrometry analysis to explore thedifferentially expressed proteins in pooled plasma samples from fifteenrandomly selected individuals in each group, including HBV-related HCC,liver cirrhosis (LC), chronic hepatitis B (CHB), and healthy controls. Real-time quantitative PCR, western blotting and immunohistochemical(IHA) analysis were used for further validation.Results: Totally,452unique proteins were identified and21proteinswere found to be differentially expressed in the plasma of HCC patientscompared with non-tumor controls. The total number of21aberrantlyexpressed proteins was found to represent a total of17protein classes,7molecular functions, and13biological processes. Several differentiallyexpressed proteins, including vWF, SAA, APOE and ALDOB, werevalidated by Western blot analysis of individual plasma samples. Elevatedexpressions of these candidates were also confirmed by IHA analysis ofHCC tissue specimens. Further validation by real-time quantitative PCRand immunoblotting revealed that the expression level of VWF mRNA andprotein in tumor tissue samples was significantly higher than that innon-tumor samples.Conclusion: Through an iTRAQ based proteomic approach, severaldifferentially expressed proteins were identified and validated in the plasmaof HCC patients as compared with non-tumor controls. Von Willebrandfactor (VWF), one of the aberrantly expressed proteins, was proved to beincreased in the plasma and tumor tissue of HBV-related HCC, thus may beinvolved in the pathogenesis of HBV-related HCC. Part Ⅱ The Effect of vWF on Biological Features of HCC Cell Linesand HBV ReplicationObjective: vWF is an adhesive glycoprotein, which plays major rolesin hemostasis and tissue injury. Science vWF was found to be highlyexpressed in the plasma and tumor tissue of HBV-related HCC, it might becorrelated with development of the disease. In this part of study, weinvestigated the potential effect and mechanism of vWF on characteristicbiological behaviors of HCC cell lines and HBV replication.Methods: HCC cell lines were transfected with vWF siRNA.Effective knock-down of vWF expression was verified by Western blottinganalysis at48h post-transfection. These siRNA-transfected cells were thensubjected to wound healing and matrigel invasion assays. Cell cycledistribution and apoptosis were measured by flow cytometry. Cellproliferation was detected by MTS. Expression of several key effectorsand/or signaling molecules involved in HCC tumorigenesis and metastasiswere also determined by immunoblotting. The secreted levels of HBsAg,HBeAg, and HBV DNA in culture medium, and intracellular mRNAtranscriptional expression of type I interferon and downstreaminterferon-stimulated genes in siRNA-treated HepG2.2.15cells werequantified by ELISA and RT-PCR assays, respectively.Results: Silencing vWF expression suppressed the migration and invasion of HCC cells in vitro. Subsequent flow cytometry revealed thatvWF silencing led to the G2/M cell cycle arrest of HCC cells withincreased apoptotic proportions and decreased proliferation rates. Moreover,these changes in biological features of HCC cells were paralleled by adecrease in expression levels of STAT3, pSTAT3, MMP2, MMP9, integrinαvβ3and Gal-1. Down-regulation of vWF expression in HepG2.2.15cellsalso reduced the secretion of HBsAg, HBeAg, and HBV DNA in thesupernatant, and caused increased mRNA levels of type I IFN and fivedownstream IFN-stimulated genes (ISG15, OAS1, OAS2, RNase L, andEIF-2α).Conclusion: vWF was demonstrated to play novel roles in thepathogenesis of HBV-related HCC, putatively via a network of proteinsassociated with cell migration, invasion, proliferation or apoptosis. Inaddition, vWF may affect HBV viral infection and replication through theIFN signaling pathway.Part Ⅲ Correlation between vWF Expression and HBV-relatedHCC Prediction and Clinicopathological StagingObjective: vWF expression was observed to be elevated in the plasmaand tumor tissue of HBV-related HCC. In this part of study, we aimed toevaluate the correlation between vWF expression and HCC prediction and clinicopathological staging.Methods: The plasma concentration of vWF in patients with HCC,liver cirrhosis (LC), chronic hepatitis B (CHB), as well as healthy controls,was determined by ELISA assay. The expression level of vWF in HCCtissues was detected by immunohistochemistry (IHA), and quantified usinga semi-quantitative scoring system. Receiver operating characteristic (ROC)curve analysis was performed to evaluate the ability of vWF for theprediction of HCC. The relation between vWF expression and HCCclinicopathological staging was also investigated using the Spearman’srank correlation analysis.Results: The average plasma vWF concentration in the HCC group(2322±1089mU/mL) was significantly higher than the LC group (1760±1217mU/mL, P <0.05), CHB group (866±417mU/mL, P <0.05) andhealthy controls (317±66mU/mL, P <0.05). Subsequent IHA revealedthat vWF were increased in HCC tissues with a variety of stainingintensities. ROC analyses demonstrated that plasma levels of vWF had asignificantly higher accuracy in HCC prediction (AUC=0.830; sensitivity=91.8%, specificity=71.0%) than AFP levels (AUC=0.719; sensitivity=70.2%, specificity=62.5%). Moreover, expression levels of vWF both inblood plasma (r=0.821, P <0.05) and tumor tissue (r=0.691, P <0.05)were positively correlated with TNM staging in HCC patients.Conclusion: Plasma vWF may represent a potential predictive biomarker for development and progression of HBV-related HCC. Itspossible clinical applications are worth further investigation in futuremulticenter prospective randomized blind clinical trials with a largercohort.