Screening Serum Biomarkers For Hepatocellular Carcinoma By Proteomics Techniques

Hepatocellular carcinoma (HCC) is the second cancer killer in China and accounts for 53% of all HCC deaths worldwide. Despite substantial progresses made in HCC clinical and basic research, the overall prognosis of HCC remains dismal because of the late diagnosis and low resection rate. Although alpha fetoprotein (AFP) remains the best HCC biomarker, the specificity and sensitivity of which are still not satisfied. Therefore, screening novel HCC biomarkers are still needed.Tumor serum biomarkers could be categorized by their production process and nature characteristics: one category is tumor associated autoantibodies (TAAs) which produced by host immunal response against tumor, the other is proteins or peptides which secreted into blood as a result of tumor abnormal metabolism.Proteomics is becoming the hot field of biological study in the functional genomic era. The scope of proteomics is to study the expression profile and cross reactivity of all cell, tissue or organism proteins under specific circumstances. Because of the high throughput and resolving power, proteomics techniques are becoming strong tools for contemporary medical research. According to the different nature of the two categories of serum biomarkers, we adopted two techniques as serological proteome analysis (SERPA) and two-dimensional electrophoresis -mass spectrometer (2-DE-MS) to screening HCC TAAs and proteins, respectively. After proving the feasibility of applying proteomics techniques in tumor serum biomarker study, we validated some potential HCC biomarkers.Part OneScreening hepatocellular carcinoma tumor associated autoantibodies and antigens by serological proteomeanalysisTo establish SERPA technique platforms by optimizing 2-DE and WesternBlot, and then screening HCC TAAs as diagnosis biomarkers or therapy targets by SERPA.To optimize 2-DE by comparing different protein quantitation method, different 2-DE lysis buffer and rehydration buffer, different isoelctric focusing program and different gel dye methods. After optimization, the quality and reproducity of 2-DE has improved greatly: The number of protein spots increased significantly (513 vs 401 )and the resolving power improved at the same time; The match rates for three gels from different batches reached 88%.By applying optimized 2-DE platform and image analysis, we established the 2-DE reference gel map of cell line HCCLM3, in which including 603 protein spots. By Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) identification of 153 spots, we got trustable results with 103 spots which corresponding to 80 proteins. All of these results lay the basis for later establishment of HCCLM3 2-DE database.By optimizing Western Blot, we established SERPA platform with good reproducibility at last and applying it in screening TAAs in HCC, hepatitis B virus (HBV) and normal sera. The number of spots on HCC, HBV and healthy sera blotted films were 70.75 + 24.25, 68.5 + 23.44 and 41. 38 + 15. 05, respectively. HCC and HBV group had more spots than healthy group (P<0. 05) while no significance were found between HCC and HBV group. Totally, 54 immuno-reactive spots on blot films were matched to spots on 2-DE reference gel map by image analysis and identified to be 38 proteins. The corresponding occurrences of TAAs in HCC, HBV and normal sera were collected at the same time.As the first to applying SERPA in screening HCC TAAs and corresponding antigens domestically, we proved that SERPA is a high throughput and efficient technique to screen TAAs. Those newly identified HCC TAAs and corresponding antigens can be potentially useful in the early immuno-diagnosis and therapy of HCC.Part TwoScreening serum biomarkers for heaptocellular carcinoma by two-dimensional electrophoresis and mass spectrometryThe second part of this study is to screening serum proteins or peptides biomarkers for HCC using classical 2-DE—MS strategy.To smooth intrinsic individual differences and deplete high abundant proteins in serum, we optimized a serum pretreatment strategy including mixing samples of one group, sonication, albumin and immunoglobulin (IgG) depletion and desalting. After sera pretreatment, 3-4 times more sera were analyzed and more protein spots (332 vs. 218) were detected.From optimized 2-DE gel maps of HCC, HBV and normal groups, forty spots were differentially expressed and identified by MALDI-TOF-MS to be eight proteins. Overall, tranferrin and transthyretin were under expressed while a -l antitrypsi^ haptoglobin> ceruloplasin and clusterin were over expressed in HBV and HCC group compared to normal group. Only a significant overexpression of a -1 antitrypsin in HCC group was revealed compared to HBV group, and a -fetoprotein (AFP), heat shock protein 27 (HSP27) expressed significantly and constantly only in HCC group. The screening and identification of these differential expressed proteins indicated that 2-DE—MS strategy be a useful tool for tumor serum biomarker study.To confirm the protein identification and differential expression of HSP27 in HCC sera, we conducted western blot with a new set of sera samples for validation. We found that HSP27 was detected in 90% (18/20) HCC sera and 10% (2/20) HBV sera, but in none of normal sera. The detection of HSP27 in 28% (5/18) HCC patients with AFP ^20|xg/L and 39% (7/18) HCC patients with small HCC (^5 cm) implied that HSP27 could be complementary to AFP in the diagnosis of HCC though further validation is needed.Conclusion1 The establishment of 2-DE reference gel map of cell line HCCLM3 and the identification of 103 spots laid the basis for later establishment of HCCLM3 2-DE database.2 By the establishment of SERPA platform with good reproducibility, we...