Screening And Validation Of Biomarkers For AFB1/HBV Double Exposure HCC | Posted on:2013-02-18 | Degree:Master | Type:Thesis | Country:China | Candidate:H Lv | Full Text:PDF | GTID:2234330371974637 | Subject:Clinical Laboratory Science | Abstract/Summary: | PDF Full Text Request | Primary hepatocellular carcinoma (HCC) is one of malignant tumors which cause serious harm to human life and health. Half of new liver cancer cases were estimated to occur in China. The occurrence of HCC was related to many factors. Chronic infections with hepatitis B virus and long-term exposure to aflatoxin B1were considered to be the two main risk factors for liver cancer. Guangxi is a high endemic area for HBV, and also is a high exposure area of AFB1. These two points are the main factors that why Guangxi have a high incidence of HCC. Previous animal experiments and prospective epidemiologic studies have shown that the coexistence of HBV and AFB1present a notable synergistic effect and have a higher risk of hepatocellular carcinoma than that HBV or AFB1alone caused of hepatocellular carcinoma. Therefore, elucidating the molecular mechanism of the interaction of AFB1and HBV will have great values in early prevention and treatment of HCC in high AFB1/HBV exposure areas. In recent years, establishment of quantitative proteomics (iTRAQ), stable isotope labeling combined with mass spectrometry, make tumor biological markers and pathogenesis research upgrade to a higher level.In this study, we applied iTRAQ-2DLC-MS/MS quantitative proteomics technology to compare the protein expression profile between HBV-HCC and AFB1/HBV-HCC for screening the candidate differential proteins that related for AFB1/HBV-HCC. And western blot analysis was applied to confirm the differential proteins.Part One SCREENING THE BIOMARKERS RELATED TO AFB1/HBV DOUBLE EXPOSURE HCCObjective:To screen the molecular markers related to AFB1/HBV double exposure HCC.Methods:HCC tissues with a complete biochemical and clinical pathological data were taken from Hepatobiliary Surgery of The First Affiliated Hospital of Guangxi Medical University. Determination of exposure level of HCC tissues was according to the expression of each sample AFB1-DNA adduct detected by Immunohistochemistry assay and Codon249mutation of the p53gene detected by PCR combined with direct sequencing.18cases of HBsAg positive HCC tissues were divided into two groups:HBV single exposure group (6cases) and AFB1/HBV double exposure group (12cases). iTRAQ/2D LC-MS/MS quantitative proteomics technology was used to screen the differentially expressed proteins, and then analyzed by bioinformatics analysis.Results:A total of387non-redundant proteins (95%confidence) were identified by mass spectrometry, and there were249proteins which had quantitative information and one or more than one peptide segment in the library peptides matching95%or more among those. Totally79proteins with quantitative information were finally identified. Among those differential proteins,44proteins were up-regulated in AFB1/HBV-HCC with the comparison of HBV-HCC (>1.2folds),while35proteins were down-regulated (<0.8folds).The differential expressed proteins were mainly localized in the cytoplasm and nucleus, mainly involved in metabolic process, response to stress, transport, organelle organization, cell death and signal transduction.Conclusion:This study screened a variety of molecular markers related to AFB1/HBV double exposure HCC, and provided new clues for further research on the pathogenesis of human HCC in the region of AFB1/HBV double exposure.Part Two VERIFICATION ON DIFFERENTIAL EXPRESSIONObjective:To verificate the mass results of identificating HBV-HCC and AFB,/HBV-HCC.Methods:Extracting the total protein from the HCC tissue samples of6patients with HBV single exposure and14patients with AFB1/HBV double exposure. We used western blot to confirm the iTRAQ results that showed the expressions of GSTA1and TGFBI. Reference protein was GAPDH.Results:The results from western blot for GSTA1and TGFBI, confirm to the iTRAQ results that showed the expressions of GSTA1and TGFBI. As to compare with HBV-HCC, both of GSTA1and TGFBI was low-expression in AFB,/HBV-HCC. Conclusion:We validated the change on GSTA1and TGFBI protein expression levels in HBV-HCC and AFB,/HBV-HCC.The results of western blot were consistent with the mass results. | Keywords/Search Tags: | hepatocellular carcinoma, aflatoxin B1, hepatitis B virus, iTRAQ | PDF Full Text Request | Related items |
| |
|