Analysis Of Differentially Expressed Proteome In Rat Hepatocarcinogenesis Induced By Diethylnitrosamine And Study On The Candidate Protein AKR1B10

Hepatocellular carcinoma (HCC) is a type of highly malignant tumor with extremely poor prognosis, and its overall 5-year survival rate is only about 7%. HCC is widespread all over the world, accounting for 5.6% of human malignant tumor, and with an increasing incidence in recent years. The pathogenesis of HCC has not yet been fully explained, although significant progress has been made in the decades-long research. As the formation of HCC is a multi-factor, multi-step and multi-gene process, the study on HCC with the methods that only focus on single factor or single gene can not accurately reflect the complexity of its pathological mechanism. In recent years, the traditional research strategy has been challenged by the rapid progress of proteomics, a technology that is helpful for studying the quantitative and dynamic changes of total proteins during the initiation and progression of a disease.This study applied two-dimensional electrophoresis (2-DE) and mass spectrometry (MALDI-TOF-MS/MS) to compare the differentially expressed proteins among the normal tissue, preneoplastic tissue and HCC tissue during rat hepatocarcinogenesis induced by diethylnitrosamine (DEN). The preneoplastic tissue was distinguished byγ-glutamyl transpeptidase (γ-GT) staining which marks the foci of liver cell proliferation positive. Among the differentially expressed proteins, aldo-keto reductase family 1 member B10 (AKR1B10) and VIMENTIN were confirmed by Western blot and RT-PCR. The biological functions and mechanism of AKR1B10 were further studied with RNA interference (RNAi) technique. Its expression in a larger scale of human HCC samples and the clinical significance were studied by tissue microarray (TMA) and immunohistochemical techniques. The results indicated that AKR1B10 probably affects cell proliferation and apoptosis by regulating the expression of tumor-associated genes, and thereby plays a role in the occurrence and development of HCC. The expression level of AKR1B10 in HCC tissue was related to the degree of HCC's differentiation. AKR1B10 up-expressed in 75% (21/28) HCC tissues of which the preoperative serum AFP were negative. AKR1B10 therefore may be helpful to predict the occurrence and prognosis of HCC, and it potentially could be a molecular marker for early diagnosis of HCC.The entire study includes four parts.Part One Study on differentially expressed proteome in the rat hepatocarcinogenesis induced by DENThe purpose of this study was to screen the protein molecules that were crucial for HCC, by comparing the protein expression profiles during the rat hepatocarcinogenesis induced by DEN.Male Wistar rats, 6 weeks old, were divided into DEN group and control group. Rats in DEN group were injected DEN in order to induce HCC, and the rats in control group were raised normally. The animal experiment lasted 30 weeks, during which a few animals from each group were sacrificed periodically for collecting liver tissues samples. The samples were stained withγ-GT and HE to distinguish the preneoplastic lesion from HCC and the normal. Total proteins were extracted respectively from six tissue samples out of the three categories such as HCC, preneoplastic and normal. The 6 protein samples in each category were mixed equally. The mixed proteins were then separated by 2-DE, and the 2-DE gels were scanned by Image Scanner and analyzed with ImageMaster 2D Platinum 6.0 software. The differentially expressed protein sports were screened out and excised for identifying by MALDI-TOF-MS/MS. The data were then analyzed with the search engine MASCOT (Matrix Science) against a NCBI nonredundant protein sequence database. The cellular location, the molecular function and the biological process of these differentially expressed proteins were analyzed through databases such as Gene Ontology, SWISSPROT and NCBI. The analysis of protein interaction networks was carried on by IntACT database.A total of nine rats in DEN group developed HCC until the end of animal experiment. The earliest HCC occurred at the 21st week in the animal in DEN group, while none in control group. With the extension of time by DEN-induced HCC, the number and size ofγ-GT foci in the liver of animals in DEN group increased, and the totalγ-GT positive area reached 29%～43% of the whole liver tissue area. HCC tissues wereγ-GT positive entirely. There was noγ-GT focus in control group. 127 differentially expressed proteins, which were with the expression level equal to or more than 2 fold comparing to the corresponding tissues, were screened by 2-DE. 82 proteins including AKR1B10, VIMENTIN and laminin receptor 1 (LAMR1) were identified by MALDI-TOF-MS/MS. Among them, 75 differentially expressed proteins were detected from HCC comparing the normal, 38 were detected from HCC comparing preneoplastic tissue, 14 were detected from preneoplastic tissue comparing the normal, and 5 were detected from the three categories. The proteins were mainly located in cytoplasm and mitochondria, and played roles of enzyme activity, oxidoreductase activity and binding activity. The main biological processes that they involved themselves in were metabolism, transport, biosynthesis, oxidative stress and so on. According to the analysis of proteins interaction network, glucose transporter type 4 (GLUT4) was a common point in all of interaction networks.The results showed that the methods thatγ-GT staining combined with HE were able to distinguish the preneoplastic lesion from HCC and the normal. The protein expression profiles at different stages during rat hepatocarcinogenesis were significant differences. The occurrence and development of HCC was related to the change in the expression level of multiple proteins.Part 2 Validation on differential expression of AKR1B10 and VIMENTIN in rat and human HCCTo ensure the reliability of the results from 2-DE and MALDI-TOF-MS/MS, some of the differentially expressed proteins, such as AKR1B10 and VIMENTIN, were validated by immunohistochemical staining, Western blot and RT-PCR.Immunohistochemical staining showed that AKR1B10 was located in cytoplasm of hepatocyte. AKR1B10 was up-regulated at both mRNA and protein level in rat HCC compared to preneoplastic tissue, and in preneoplastic tissue compared to normal liver tissue. Also, the protein level of AKR1B10 was up-regulated in human HCC tissues compared to the corresponding adjacent liver tissues, and in adjacent liver tissues compared to normal liver tissue. The protein level of VIMENTIN was up-regulated, from rat normal liver tissue to preneoplastic tissue and then to HCC tissue, while its mRNA level was only obviously up-regulated in HCC tissue. The protein level of VIMENTIN was up-regulated from human normal liver tissue to adjacent liver tissue and then to HCC tissue.It was confirmed that the expression trends of AKR1B10 and VIMENTIN were consistent with the results from 2-DE, indicating the proteomics technology platform used in this study has considerable stability and reliability. Further results showed that AKR1B10 and VIMENTIN were up-regulated, from human normal liver tissue to adjacent liver and then to HCC tissue, suggesting these two proteins may be involved in the occurrence and development of human HCC.Part 3 Biological function and mechanism study of AKR1B10 in hepatocarcinogenesisTo explore the role of AKR1B10 in hepatocarcinogenesis, further study on its biological function and mechanism was carried on by RNAi technique on the HCC cell line of MHCC97H.At first, AKR1B10 mRNA and protein levels in HCC cell lines MHCC97L, MHCC97H, SMMC-7721 and BEL-7402 and normal liver cell line Changliver were examinated by RT- PCR and Western blot. Then two pairs of chemically synthesized small interfering RNA (siRNA) targeted on AKR1B10 were transfected into MHCC97H cells. The one pair which had the relatively obvious interfering effect was selected for the following RNA interfering (RNAi) experiments which three groups were set: RNAi group, in which the MHCC97H cells were transfected with AKR1B10 specific siRNA. Mock group, in which the MHCC97H cells were transfected with the dsRNA that had no target at all. Control group, in which the MHCC97H cells were without any transfection. The abilities about cell apoptosis, proliferation, adhesion, migration and the expressions of tumor-associated genes like c-myc were observed after AKR1B10 expression was inhibited.The results showed that AKR1B10 existed in all of the four HCC cell lines, and the highest expression was in MHCC97H, while there was no expression in the normal liver cell Changliver. The expression levels of AKR1B10 mRNA and protein were down-regulated obviously after transfecting the AKR1B10-targeted siRNA for 48h and 72h respectively. The CCK-8 assay showed that, compared to the two control groups, the cell growth inhibition rates in the RNAi group were 6.5%, 18.0%, 26.6% and 22.7% respectively after transfecting for 24h, 48h, 72h and 96h. The results from flow cytometry analysis showed the proportion of apoptotic cells in RNAi group (37.3%) was significantly higher than that in mock (5.2%) and control group (5.6%). The results from cell adhesion test showed that, after 30min, 50min and 70min, the average cell numbers in the RNAi group were not significantly different from that in the two control groups. Cell motility test showed the average cell numbers per field in the group RNAi, mock and control were 18±2,20±3 and 21±4 respectively, without significant difference from each other. The results of Real-time PCR showed that the oncogenes c-myc, c-fos, N-ras and the proliferation-associated gene ki-67 were down-regulated in MHCC97H, while the apoptosis-promoting genes caspas-3 and bax were up-regulated.The results in this part showed AKR1B10 was overexpressed in HCC cell lines. When the expression of AKR1B10 had been interfered, the cell growth was inhibited and the cell apoptosis increased, while the abilities about cell adhesion and migration had no significant change. Further assay found some tumor-associated genes changed after the expression of AKR1B10 had been interfered, which might indicate AKR1B10 caused the malignant transformation of liver cells by regulating the expression level of some related genes to promote proliferation and inhibit apoptosis.Part 4 The expression and clinical significance of AKR1B10 in a larger scale of human HCC samplesTo explore the clinical significance of AKR1B10 expression in HCC and evaluate the potential value of AKR1B10 as a new molecular marker for diagnosis early HCC, the AKR1B10 protein level was examinated in 65 cases of human HCC and the adjacent liver tissues, as well as in 14 cases of normal human liver tissue, by tissue microarray (TMA) and immunohistochemical techniques. The correlation analysis between AKR1B10 expression and some of the clinical-pathological characters of the HCC cases was then performed.The results showed the expression rate and level of AKR1B10 protein was up-regulated in human HCC samples compared to the corresponding adjacent liver tissues, and in adjacent liver tissues compared to normal liver tissue (p<0.05). The AKR1B10 level had significant correlation with Edmondson stage of the HCC cases, namely the AKR1B10 protein level in stage I～II was significantly higher than that in stage III～IV. The up-regulating AKR1B10 expression was found in 50 of 65 (76.9%) HCC cases, which was higher than that of preoperative serum AFP of the patients. Moreover, AKR1B10 protein was detected in 21 HCC tissues of 28 (75.0%) negative serum AFP HCCs. The statistical analysis showed the expression of AKR1B10 protein in HCC tissue was not correlated with the preoperative serum AFP level. AKR1B10 was up-regulated, from human normal liver tissue to adjacent liver and then to HCC tissue. Its expression rate is high in HCCs, especially in negative serum AFP HCCs. These results might imply AKR1B10 had certain value of clinical applications for early diagnosis of HCC.Conclusion1. Byγ-GT staining combined with HE, the rat normal liver tissue, preneoplastic lesion tissue and HCC tissue can be distinguished from each other and therefore obtained for further comparative proteome analysis.2. The differentially expressed proteins obtained through 2-DE and mass spectrometry in this study might serve as new prognostic biomarkers and/or therapeutic targets for HCC.3. AKR1B10 might play a role in hepatocarcinogenesis by regulating the expression of some related genes. Its gradually up-regulated pattern in both rat and human hepatocarcinogenesis and the fairly high expression rate in negative serum AFP HCCs, might imply it could serve as a new diagnostic marker for early HCC.Potential value for application1. AKR1B10 may be a potential early diagnostic molecular marker of HCC, which can enrich the combination and selection of diagnostic markers and be helpful to improve the rate of early diagnosis for HCC.2. It is helpful to elucidate the pathogenesis of HCC. Its evaluation may be helpful to explore new therapeutic targets. Novelty1. The preneoplastic tissue can be distinguished from HCC and the normal byγ-GT staining combined with HE. By this method, the accurate and valuable tissue samples can be obtained for observing the changes of protein expression profiles during hepatocarcinogenesis.2. Protein expression profiles were obtained from the preneoplastic lesion and HCC tissue in the rat hepatocarcinogenesis induced by DEN. Eighty-two proteins were screened and identified, some of which were firstly reported.3. Analysis on biological function and mechanism of AKR1B10 indicates it may be involved in the occurrence and development of HCC by regulating the expression level of some genes. The dynamic differential expression pattern of AKR1B10 in rat and human hepatocarcinogenesis will make AKR1B10 possible to serve as a new diagnosis marker for early HCC.