Proteomics Of Tree Shrew's Hepatocarcinogenesis

Hepatocellular carcinoma (HCC), of which the morbidity and mortality are in the forefront among the overall tumors worldwide, is one of the malignancies with difficult in early diagnosis and extremely poor prognosis. Hepatitis B virus (HBV) infection and aflatoxin B₁ (AFB₁) exposure are recognized as the main factors for HCC. The formation of HCC is a multistep, multistage and multifactor process like most other tumors. The conventional strategy of research on single gene has been challenged by the rapidly progress of proteomics, which can be utilized to observe the dynamical changes of nature and quantity of total protein during progression of disease. Proteomics can be used to find new protein markers and key molecules and therefore is an effective and high-throughout method for exploring mechanism of disease and for searching targets of diagnosis or therapy. By optimizing the technical platform of two-dimensional electrophoresis (2-DE), in the present study we observed dynamically the differentially expressed proteins of liver tissue that were taken at different time points during the hepatocarcinogenesis of tree shrew (Tupaia belangeri chinensis), a small animal species belonged to low Primates evolutionarily. By taking the advantages that the animal can survive repeated liver biopsies during the experiment and can be infected with HBV, the present study aimed at screening differential expressed proteins in various stages of hepatocarcinogenesis induced by AFB₁ or AFB₁+HBV. Subsequently, one group of the screened proteins, peroxiredoxin family (Prxs), was further confirmed by Western blot and RT-PCR on the tissues samples form tree shrews as well as from HCC patients. The entire study included four parts as following. Part 1. Normal physiological laboratory value of tree shrewTo further confirm the feasibility and superiority of utilizing tree shrew as animal model for studying human disease, this part is to detect the physiological laboratory values of adult tree shrew bred artificially, and to comparer the values with human normal reference.Whole blood sample and separated serum were collected from twenty two adult tree shrews bred artificially in this lab, and then all the laboratory values were determined. Blood routine examination showed that RBC count was 8.19×10¹²/L, which was higher than human normal reference value. Tree shrew WBC count was 2.2×10⁹/L, which was lower than human normal reference. Microscopic examination of whole blood smear from tree shrew showed the morphology of blood cells and platelet were similar to that of human. WBC differential count showed that each cell percentage was near to that of human. Assay of ten biochemical values of serum samples showed that seven values including urea, globulin, serum total protein, serum albumin, serumγ-GT, C-reactive protein and rheumatoid factor accorded with the range of human reference. While serum creatinine of tree shrew as 16.63μmol/L was lower than 44μmol/L of human reference, serums GPT as 77.92 U/L and GOT as 179.9 U/L were higher than the upper limit of human reference of 40 U/L and 45 U/L respectively. The result of HBsAg examination showed all of the animals were negative, with the value of 0.022 ng/ml±0.005.Analysis of these laboratory values mentioned above suggests that tree shrew, as a low grade primate related closely to humankind, is suitable as animal model for studying human disease. Part 2. Establish and optimize 2-DE technique for proteomic study on tree shrew liver samplesFor achieving a high quality on resolution and a steady result, effort was made to establish and optimize the 2-DE technique that should be adaptive for proteomic analysis on tree shrew liver samples. The conventional protocol of 2-DE was modified, especially on some critical steps such as sample processing (grinding and homogenating), quantitative methods of protein (Lowery method and modified Bradford method), loading modes (cup-loading and swelling-loading), IEF procedure, gel stain methods (Coomassie brilliant blue R-250 stain, traditional silver stain, MS compatible silver stain), and etc.The protein spots acquired from 2-DE gel by the modified method increased greatly (1228 vs 856), along with the enhanced spot-resolution, as well as with the decreased horizontal band and vertical tail. The protein spots from same sample that run repeatedly three times of 2-DE were 1241, 1192 and 1216 respectively, with the mean shiftings as 2.0±0.56mm at IEF direction and 2.3±0.61mm at SDS-PAGE direction. Establishment of this technical platform provided the foundation for following research.Part 3. Proteomic study on differential expression during tree shrew's hepatocarcinogenesisTo search the critical protein molecules responsible for tree shrew hepatocarcinogenesis, and to explore the mechanism of HCC. This part compared and analyzed protein profiles of tree shrew HCC induced by AFB₁ and AFB₁+HBV, by 2-DE and MALD1-TDF MS proteomic techniques.Twelve categories of liver samples were collected from differently treated animals at different time points during the experiment. Samples from the animals in group A that were treated with AFB₁ and developed HCC at late stage of the experiment, named A45w, A75w, ACa and ACp respectively, referring the samples were biopsies at 45th week (A45w) or 75th week (A75w) of the experiment before HCC appeared, and the HCC samples (ACa) or HCC-surrounding samples (ACp) were from the identical animals. Similarly, the samples from the animals in group C that were not only treated with AFB₁ but also infected with HBV and developed HCC at late stage of the experiment were named C45w, CCa and CCp respectively. The samples from the animals in group D that were only infected with HBV, had no HCC developed until the experiment ended at 165th week, were named D45w and D105w respectively. The samples from the control animals in group E that treated nothing else but only breed normally were named E45w, E75w and E105w respectively. The mixed total protein from samples in each category was assayed repeatedly with 2-DE method. Thirteen centimeters non-linear IPG gel strip with pH 3-10 was adopted. The IEF electrophoresis was carried out by applying the swelling sample, and pictures from 36 blocks of 2-DE gels were acquired.Profile of proteins was similar among the different categories of sample as most protein spots distributed inside the field of pI 4-8. ImageMaster 2-DE Platimum 5.0 software was utilized to carry out analysis for each well-matched gel dot between the comparable categories. The mean detected numbers of protein spot in each categories were as follows: 1255±43 in A45w, 1197±46 in A75w, 1198±50 in ACa, 1205±89 in ACp; 1233±30 in C45w, 1257±35 in CCa, 1244±39 in CCp; 1100±79 in D45w, 1115±61 in D105w; 1249±51 in E45w, 1179±62 in E75w, 1208±36 in E105w. Two hundred and six protein points which had double or more density and appeared in more than fifty percent gels were screened out.One hundred sixty protein spots with different expression were dug out from the corresponding prepared Coomassie brilliant blue stain gels and one hundred forty-two spots were identified by MALDI-TOF MS. To assure the accuracy of blots sampling, each spot dug out from three blocks of gel was identified and only identical data in MS was selected. The identified differential proteins included the members of Peroxiredoxin family (Prxâ… , Prxâ…¡, Prxâ…¢, and Prxâ…£), cytochrome C oxidase, glutathione peroxidaseâ… , thioredoxin, cytochrome B5, ketohexose kinase, heterogeneous ribose nucleoprotein, phosphotriose isomerase, nicotinamide N-methyl transferase, metastasis suppressed gene NM23-H1, translation controlled tumor protein, and others.Part 4. Verify tests on differential expression of peroxiredoxins among liver samples from tree shrews and from HCC patientsThe members in peroxiredoxin family (Prxâ… , Prxâ…¡, Prxâ…¢and Prxâ…£), which were among the differentially expressed protein screened from Part 3 and were known had widespread biological functions concerning many other tumors, were studied further to confirm the 2-DE results that showed the expressions of Prxâ… , Prxâ…¡, and Prxâ…¢in HCC tissue were up-regulated greatly compared to precancerous tissue, and the expression of Prxâ…£was up-regulated prominently in HCC tissue compared to HCC-surrounding liver tissue. By Western blot and RT-PCR assays, the expression levels of Prxâ… , Prxâ…¡, Prxâ…¢and Prxâ…£were examined on the samples from tree shrew as well as from HCC patients.Results confirmed that the expressions of Prxâ… , Prxâ…¡, Prxâ…¢, and Prxâ…£were not only up-regulated in tree shrew HCC but also in human HCC tissue in both protein level and mRNA level, which indicated that the proteins in Prx family might play important role in hepatocarcinogenesis and could be hopefully applied as a biomarker for HCC diagnosis and/or treatment. ConclusionTree shrew, a lower grade primate related closely with humankind evolutionarily, is proved suitable as animal model to research human disease because most of its laboratory values were found similar to that of human beings.The modified 2-DE technical platform for proteomic analysis on liver tissues of tree shrews provides the foundation for research. Profiles of differentially expressed protein screened from the tissues taken at different time points during tree shrew hepatocarcinogenesis induced by AFB₁ or AFB₁+HBV suggests the progression of HCC is concerned with many factors. At least some of the proteins among the 142 identified ones might play important roles in the HCC initiation, development and/or progression. Particularly, proteins in Prx family might be applied as a biomarker for HCC diagnosis, treatment and/or prognosis-evaluation.