Proteomics Research In The Gender Disparity Of Hepatitis B Virus-Related Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancy in the world, more than500,000new patients come out around the world each year. Hepatitis B virus infection is the most important carcinogenic factors in China. The epidemiological investigation indicated that the incidence of HCC is higher in male than female. The male incidence of HCC ranks fifth for common tumor worldwide while women’s ranks seventh, with the male:female ratio usually averaging between2:1and4:1. It is few that people suffer from HCC before the age of forty, as the peak of incidence age is probably sixty. According to the distribution worldwide, the peak age of HCC incidence for male is about five years earlier than female. So is the chronic liver disease. Chronic liver disease caused by HBV progresses more rapidly to HCC in males than females. The vitro and vivo research about the mechanism of gender disparity of incidence and prognosis in HCC has begun in the1970s. In recent years the rapid development of proteomics has became an important field of research. Quantitative proteomics with large scale, high throughput and sensitivity provide a new platform to comprehensively observe the type and quantity changes of protein in the disease process, to investigate the pathogenesis of the disease and to find specific markers. According to this research strategy, our study used the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic approach to identify differentially expressed proteins derived from HCC of male and female tissue and normal liver tissue, then establish the protein database of gender disparity in HBV-related HCC, and verify some potential value proteins in both mRNA and protein level.PART1PROTEOMIC ANALYSIS OF GENDER DISPARITY IN HEPATITIS B VIRUS-RELATED HEPATOCELLULAR CARCINOMAThe main purpose of this part is to quantitative analysis the proteins of early stage HBV-related hepatocellular carcinoma, screen out proteins of gender disparity expression.Firstly, screen out26samples, including surgical resection tissue of early stage HCC in male and female and normal control liver tissue, then label with iTRAQ reagents113,114,115,116and117in the group of male HCC l,male HCC2, female HCC, male normal, female normal respectively, then analysis by2D-LC-MS/MS quantitative proteomics. After analysis by strong cation column and reversed phase column chromatography, mass spectrometry and searching SWISS-PROT database, a total of652distinct proteins are identified (95%confidence). These were subsequently filtered with manually selected filter inclusion parameters (P<0.05and proteins were considered up or down regulated when their ratios were>1.2or <0.8). Then73proteins were screened out as differential expressed proteins in gender diaparity of HCC (113:115,114:115) and122proteins were screened out as differential expressed proteins between normal and cancer (113:116,114:116,115:117). Using bioinformatics analysis of differentially expressed proteins, most of them localized in intracellular organelles, involved in the biosynthesis, metabolism, biological regulation and stress and other biological processes. According to the signaling pathway analysis, the majority of differentially expressed proteins involved in energy metabolism pathways.These results showed that we have created the differential expressed protein database of HCC gender disparity with the iTRAQ technology. The proteins as potential molecular markers would help to reveal the potential molecular mechanism of high HCC incidence in male.PART2VALIDATION OF SOME DIFFERENTIALLY EXPRESSED PROTEINSIn this section, we verify some differentially expressed proteins in the first part of iTRAQ data to confirm the reliability of the mass spectrometry. And the proteins are to analyze what roles do they play in the development of gender disparity in HCC.According to bioinformatics analysis results,38cases of male and14cases of female HCC tissue,4cases of male normal liver tissues, and7cases of female normal liver tissue samples, are selected to validate the mass spectrometry results by RT-PCR and Western-Blot experiments, while β-actin as internal control. The results showed that the gene expression trends of five differentially expressed proteins are consistent with mass spectrometry results. The TSPY1protein express highly in male HCC group comparing with the female HCC group and normal group, and it elevated with the high expression of androgen receptor. The verification results show that the mRNA and protein expression trends of TSPY1are consistent with the mass spectrometry results.CONCLUSION1. Proteomics is the high-throughput technology to screen differentially expressed proteins in HBV-related male and female HCC, and the proteins could be in-depth study as the potential molecular markers.2. Compared to female HCC, male HCC are found some differently expression in the proteins, indicating that the male HCC have special different factors involved in the carcinogenic process. A total of73differentially expressed proteins may be involved in the occurrence of liver cancer and development.3. Of HBV-related hepatocellular carcinoma compared with normal liver tissues, it is shown that a few differentially expressed proteins are detected in the occurrence and development of HCC process. A total of122differentially expressed proteins were identified, may be significantly associated with HCC.4. Our study has explored the relation between TSPY1and male HCC to help clarifying the mechanism of HCC pathology. It is likely to be an important factor resulted in gender disparity of HBV-related HCC. It may be a new target for the diagnosis and treatment of HCC. NOVELTY OF THIS PROJECTIt is first time to filter out73differentially expressed proteins of HBV-related HCC in gender disparity with the application of iTRAQ proteomics technology, some of which are the new report in hepatocellular carcinoma.THE POTENTIAL APPLICATION OF THIS WORK1. TSPY1can be used as a molecular marker for the potential value of diagnosis and prognosis of HCC.2. It is rich the choice of the diagnosis and treatment indicators for HCC by the differentially expressed proteins.