Application Of Secretory Proteome For Identifying Plasma Biomarkers In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma (HCC) is the major malignant cancer with high morbidity and mortality. The incidence is particularly high in China, take up almost55%among the cases of HCC, and increasing year by year. Treatment of small HCC by surgical resection is the best treatment for decreasing recurrence and improving prognosis. However, most of cases lost those chances due to the advanced stage at diagnosis, owing to the limitation of current surveillance programs. Alpha-fetoprotein (AFP) has been widely used during high-risk population screening, but its limited performance is not sufficient. Thus, improvements in early detection and more accurate markers are urgent needed. Proteomics approaches with high-throughput present new possibilities for researching liver cancer related proteins and screening novel biomarkers.Objective:The aims of this study are to establishment secretory/releasing proteome of HCC and to screening novel biomarkers in the circulating plasma. The biomarker candidates will assist to improve AFP performance in diagnosis HCC. Meanwhile, screening biomarkers that will used to predict HCC prognosis.Experiment design:Based on the novel model previously developed in our laboratory. For the primary tissue cultures, the cancerous tissues and the surrounding noncancerous hepatic parenchyma were obtained from six primary HCC patients with chronic HBV infection who had underwent surgical resection. One-dimensional SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) separated CM proteins from cultures HCC tissues. Then, LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) based proteomic analysis was used to establish high-quality secretory/releasing proteome. Subsequently, the basic properties of the CM proteins were analyzed with GOFFA, Array Track, STRING tools. The biomarker candidates were selected form CM database according to bioinformatics analysis. After that, we validated seven biomarker candidates in plasma samples that including179HCCs,80liver cirrhosis, and103healthy controls by ELISA assays. Mann-Whitney test compared the difference of the plasma levels of selected proteins. The ROC curves were built to estimate performance of those biomarker candidates in diagnosis HCC. Kaplan-Meier method used to calculate disease free survival and overall survival rates.In this study, the role of pregnancy specific glycoprotein9(PSG9) in tumor angiogenesis was preliminary exploration. Real-time quantitative PCR (qPCR) used to analyze the PSG9and VEGFA mRNA levels in HCC cancerous and surrounding non-cancerous tissues. Western blot used to detect the PSG9protein levels in nine HCC cell lines. The effects of PSG9in regulation VEGFA expression was investigated by up-regulation and down-regulation in SMMC7721cell lines Dual-luciferase assay was applied to investigate the interaction of PSG9with VEGFA promoter. Chromatin immunoprecipitation (ChIP) assay was used to detected PSG9binding site in VEGFA promoter region. Cell-proliferation assay was performed to analyze the proliferation potential of stable up-regulation and down-regulation PSG9SMMC7721cells using Cell-Counting Kit (CCK)-8. HUVEC forming tube and Matrigel plug assays were also enrolled to examine the role of PSG9in promoting angiogenesis.Results:Proteomics analysis of CM proteins allowed the identification of1365proteins. The enriched molecular functions and biological processes of CM proteins consisted with liver functions as the most important metabolism organ such as hydrolase and catabolic process. Moreover,19%of which proteins were characterized to be situated on the extracellular or membrane-bound. The referred pathways of those CM proteins were classified using Array Track tools, and showed that most CM proteins involved in human disease related pathways.The suppression of TGF-P is an early event during the hepatocarcinogenesis. The candidates of plasma biomarkers associated with TGF-β will help to detect HCC in early stage. The CM proteins matrix metalloproteinase1(MMP1), MMP7, MMP9, osteopontin (OPN), PSG9and tissue inhibitor of metalloproteinase1(TIMP1) and AFP involved in TGF-β signaling pathways were validated in plasma. In general, MMP7, MMP9, and TIMP1in plasma of liver cirrhosis were increased by comparison with healthy controls and HCC. AFP, MMP1, OPN and PSG9were significantly increased in HCC patients. This would be the potential biomarkers of HCC. MMP1and OPN, but not PSG9overall performance remained greater than AFP in diagnosis HCC. The area under the receiver operating characteristic curve (AUC) values of AFP, MMP1and OPN were0.642,0.945, and0.911. Furthermore, the plasma levels of OPN and PSG9were significantly correlated with HCC poor prognosis.We focus on the function of PSG9in angiogenesis, the results indicated that PSG9was one molecular participated in angiogenesis. The mRNA expression of PSG9was confirmed positively correlated with the VEGFA mRNA in HCC tissues. The up-regulation PSG9promoted VEGFA expression while down-regulation resulted in inhibiting VEGFA expression. ChIP identified that the potential binding site of PSG9in VEGFA promoter region located in-1684/-1766. The results of HUVEC tube forming and Matrigel assays proved that PSG9regulated the process of tumor angiogenesis.Conclusions:Our study thus provides a valuable resource of HCC secretome with potential for investigation as serological biomarkers. MMP1and OPN could be used as novel biomarker for improving sensitivity of AFP. Furthermore, OPN and PSG9are the biomarkers for predicting prognosis of HCC. We found that PSG9may facilitate development of HCC by fostering angiogenesis via promote VEGFA production from cancer cells.