The Serum Proteomics Study Of Early Recurrence/Metastasis Of Hepatocellular Carcinoma(HCC) Following Radical Resection

ObjectivesBy using iTRAQ-label-based quantitative proteomic technique,the present study aims to identify and screen the differentially expressed proteins(DEPs)associated with early recurrence/metastasis of hepatocellular carcinoma(HCC)following radical resection.Besides,Gene Ontology(GO)and Ingenuity Pathway Analysis(IPA)analyses of DEPs were implemented to explore the molecular mechanisms of early recurrence/metastasis of HCC.At last,serum concentration of DEPs were analysed to explore the relationship between DEPs and early recurrence/metastasis of HCC.Methods1.After depleted the high-abundant proteins of serum by high performance liquid chromatograph and human 14 Multiple Afinity Removal System(MARS-human 14),we employed SDS-PAGE and Label-free proteomic technique to investigate the effect of removal.2.We compared the overall proteome feature among a time series of serum samples from HCC patients who underwent radical resection before the neoplasm recurrence.Meanwhile,a series of serum samples from unrecurrent HCC patients were analysed for control.To identify the DEPs,the relative protein expression values were compared among four sample time points.The proteins were considered to be differentially expressed if the i TRAQ ratios were > 1.2 or < 0.83 when the last sample time point compared with the other three sample time points,and the protein expression values had no change in the samples of control patient.The GO analysis of the DEPs were implemented using the online tool DAVID(https://david.ncifcrf.gov/).GO annotation contains biological processes,cell components and molecular functions.The biological functions and signaling pathway annotations of the differentially expressed proteins were analyzed by IPA software.3.The ELISA technique was used to examine the serum concentration of PRDX6 ? PGK1 and CA1 in 82 HCC patients underwent radical resection.Meanwhile,the serum concentration of AFP?CEA?CA125?CA199?APOA1 and TTR were collected from medical records.The concentration of these nine proteins were analysed by t-test.The area under curve(AUC)was calculated to compare the performance of different serum biomarkers as a diagnostic test by ROC analysis.Univariate and multivariate logistic regression analysis were used to screen the risk factors for early recurrence/metastasis of HCC after radical resection.At last,the Kaplan-Meier survival analysis was performed to analyze the association of serum biomarks concentration and prognosis.Results1.After depleted the high-abundant proteins(HAPs)of serum by MARS-human 14,the SDS-PAGE shows clear in the position of HAPs and thick stain stripes of low-abundant proteins(LAPs).Label-free proteomic shows that serum Albumin,Antitypsin,Transferrin,Haptoglobin,Alpha2-Macroglobulin,Alpha1-Acid Glycoprotein,Apolipoprotein AI and chains of Immunoglobulin G,Immunoglobulin A,Immunoglobulin M are depleted specificly with less loss of LAPs.2.There are 831 proteins were identified in HCC serum by using iTRAQ-based proteomics.There are 20 up-regulated DEPs such as Matrix metalloproteinase-9,Phosphoglycerate kinase 1,Peroxiredoxin-6,Superoxide dismutase and Thioredoxin and 7 down-regulated DEPs such as Apolipoprotein A-I,Apolipoprotein C-II and Apolipoprotein C-III.3.The 27 DEPs were submitted to GO annotation and IPA analysis and the results are showed as following: These 27 DEPs are mainly involved in response to reactive oxygen species and lipid metabolic process in biological processes and lipase inhibitor activity,high-density lipoprotein particle receptor binding and serine-type endopeptidase activity in molecular functions.Besides,most of these DEPs are involved in metabolic syndrome X and insulin resistance in biological functions and Glycolysis I in pathway analysis.Moreover,the dysregulated proteins involved signal pathway revealed that alternation of ERK1/2 and NF-?B signal pathways play important roles in the tumorigenesis or tumor growth for early recurrence/metastasis of HCC after radical resection.4.The ELISA technique was used to examine the serum concentration of PRDX6 ? PGK1 and CA1 in 82 HCC patients following radical resection.Meanwhile,the serum concentration of AFP?CEA?CA125?CA199?APOA1 and TTR were collected from medical records.It shows that the serum concentration of PRDX6,PGK1,AFP,CA125 in recurrent HCC are higher than that in unrecurrent HCC patients.Besides,the serum concentration of TTR in recurrent HCC are lower than that in unrecurrent HCC patients.ROC analysis shows that the AUC of PGK1,AFP,CA125 and TTR are 0.695,0.83,0.666 and 0.702,respectively.The combination of PGK1,AFP and TTR has the best diagnostic performance for early recurrence/metastasis of HCC(AUC=0.866,sensitivity=0.729 and specificity=0.957).Univariate and multivariate logistic regression analyses show that the serum concentration of AFP,PGK1 and TTR are independent risk factors for early recurrence/metastasis of HCC after radical resection.At last,the Kaplan-Meier survival analyses show that the patients who have higher serum concentration of AFP,PGK1 and CA125 and lower serum concentration of TTR are susceptible to recurrent and the patients who have higher serum concentration of AFP and CA125 and lower serum concentration of TTR have a poorer prognosis.Conclusions1.The MARS Human 14 column could delete HAP speciaficly and enrich the LAP effectively.2.The iTRAQ-based proteomic technology could be used to compare serum samples from HCC patients who underwent radical resection and to analyze the DEPs associated with early recurrence/metastasis of HCC.3.The serum concentration of PGK1,PRDX6,AFP,CA125 and TTR are predictors of the prognosis of HCC after radical resection.The combimation of PGK1,AFP and TTR has an effective diagnostic performance for early recurrence/metastasis of HCC.The serum concentration of AFP,PGK1 and TTR are independent risk factors for early recurrence/metastasis of HCC after radical resection.