| Backgrounds and Objectives Although there has been a long time for searching for tumor antigens, major progress has only recently been made in the molecular definition of human tumor antigens. In 1995, Sahin developed an approach that can be used to identify tumor antigens in human cancers. This approach is named as SEREX (Serological analysis of recombinant cDNA expression libraries), which defining antigens by the reaction with autologous patient sera. Many antigens were defined by this approach, SEREX makes a great progress in defining human tumor antigens. Morbidity and mortality rate of liver cancer, especially hepatocellular carcinoma (HCC) were very high in our country. The majority of people with HCC will die within 1 year of its detection. This high case-fatality rate can in part be attributed to lack of diagnostic methods that allow early detection. So how to find more valuable tumor associated antigens (TAAs) and establish a methodology to identify the high-risk individuals for HCC remains to be badly in need of investigation. At the present study, 81 genes encoding HCC-associated antigen were identified by SEREX method in our Lab. By bioinformatics analysis, 9 genes (clone number: X7,X21,X64,X28,X41,X37,X87,X33,X53) which were closely related to HCC in literature reports were found to be involved in cell differentiation, aging, apoptosis, metabolism, signal transduction of hepatocyte. The proteins encoded by these genes were DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), pyruvate dehydrogenase kinase, isoenzyme 2(PDK2), keratin 23 (histone deacetylase inducible) (KRT23), transcript variant 1, alpha-2-HS-glycoprotein( AHSG), activator of heat shock 90kDa protein ATPase (AHSA1) , selenoprotein P, plasma, 1 (SEPP1) , Human osteopontin (SPP1) , ribosomal protein L17 (RPL17) , ferritin, light polypeptide (FTL) . This study also was designed to investigate the autoantibody responses in heterologous serum samples from HCC, chronic hepatitis and normal human individuals to 9 HCC-associated antigens which were identified using SEREX, and further evaluate whether or not these identified antigens can be used as markers in the detection of HCC. We hope the identified antigens can not only provide new potential markers for the diagnosis and therapy of HCC, but also highlight a new direction for investigating HCC vaccines and monitoring immune impacts. The results from this study may also contribute to the understanding of HCC tumorigenesis.Methods Bioinformatics analysis: By using bioinformatics analysis, 9 genes encoding HCC-associated antigen were anticipated from the structure and function, cellular location, protein products and so on. These genes were associated with development,infiltration and metastasis of tumor more or less. Serological analysis: 1.E.coli transfected with recombinant phages identified by SEREX were plated onto LB-agar plates. Plagues were transferred onto the nitrocellulose membranes. 2. IPTG was used to induce the expression of recombinant proteins and the membranes were blocked with 3% non-fat milk. 3. Serum was preabsorbed with E.coli lasyte. 4. The nitrocellulose membranes were incubated with preabsorbed patient's serum and then incubated with a 1: 2000 dilution of the Goat anti-Human IgG(H+L)- labeled antibody specific for human IgG. Reactive dots were visualized by staining with DAB. 5. The results were analyzed by SPSS statistic analysis software.Results By using bioinformatics analysis, the proteins encoded by these genes(clone number:X7,X21,X64,X28,X41,X37,X87,X33,X53) were DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41), pyruvate dehydrogenase kinase, isoenzyme 2(PDK2), keratin. 23 (histone deacetylase inducible) (KRT23), transcript variant 1, alpha-2-HS-glycoprotein (AHSG) , activator of heat shock 90kDa protein ATPase (AHSA1), selenoprotein P, plasma, 1 (SEPP1), Human osteopontin (SPP1), ribosomal protein L17(RPL17), ferritin, light polypeptide (FTL). The frequency of autoantibody against 5 HCC-associated antigens keratin23(KRT23), transcript variant 1, a2-HS glycoprotein (AHSG) , ribosomal protein L17 (RPL17), ferritin, light polypeptide (FTL) , DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 (DDX41) were 76.4% (42/55) ,81.8% (45/55) ,78.2% (43/55) ,78.2% (43/55) ,91.0% (50/55) in HCC;60.0% (18/30) ,53.3% (16/30) ,76.7% (23/30) ,56.7% (17/30) ,96.7%(29/30) in Chronic Hepatitis; 43.3% (13/30) , 46.7% (14/30) , 33.3% (10/30) , 43.3% (13/30) , 76.7% (23/30) in Normal Human sera, respectively. There was significant difference in HCC,chronic hepatitis and normal individuals at KRT23(x~2=9.27, P=0.0097) , AHSG ( x~2 =12.99, P=0.0015) , RPL17 ( x~2 =19.28, P<0.0001) , FTL (x~2=10.89, P=0.0043) , DDX41 (x~2=6.44, P=0.0399) . The significant difference existed between HCC and Normal Human sera in KRT23( x~2=9.27, P=0.002), AHSG ( x~2=11.30,P=0.001), RPL17(x~2=16.63,P<0.0001), FTL (x~2=10.49, P=0.001) . After analyzed the other antigens such as human osteopontin ( SPP1 ) , pyruvate dehydrogenase kinase(PDK2), isoenzyme 2, activator of heat shock 90kDa protein ATPase (AHSA1), selenoprotein P, plasma, 1 (SEPP1), no significant difference was found in HCC, chronic hepatitis and normal human. When 4 antigens(KRT23,AHSG,RPL17,FTL) were used by in parallel the sensibility and specificity were 98.2%, 65.3%; in series, the sensibility and specificity were 62.7%, 90.0%.Conclusions Bioinformatics analysis is an effective method to recognize an unknown gene. It can help us to make a good anticipation for a new gene on its structure and function, cellular location, encoding proteins and so on. There was significant difference in HCC, chronic hepatitis and normal individuals at 5 HCC-associated antigens such as KRT23, AHSG, RPL17, FTL, DDX41. The 4 HCC-associated antigens in which the significant difference existed between HCC and Normal Human sera such as KRT23, AHSG, RPL17, FTL may be considered as potential markers in HCC early detection. The preliminary results from our study suggest that the combinational detection of different HCC-associated antigens may be potentially useful for early diagnosis of HCC. |
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