The Dynamic Alteration Of Genome And Proteome During Neoplastic Transformation Of Rat Hepatic Oval-like Cells WB-F344

The cellular origin of hepatocellular carcinoma (HCC) remains controversial. To investigate the molecular mechanism of hepatocarcinogenesis from cellular origin aspect may provide us some new clues for explaining further development and progression of hepatocarcinoma and for finding the valuable biomarkers of diagnosis, prevention and treatment. Hepatic oval cell (HOC) may be one of the cellular origins of HCC due to its relationship to many liver diseases. Here, the rat oval like cells WB-F344 were treated by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H₂O₂), and that resulted in inducible neoplastic transformation. Then, the changes of gene expression profile and protein expression profile during transformation respectively were obtained through oligo microarray detection and two-dimensional electrophoresis (2-DE), followed by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS/MS), respectively. Bioinformatics analysis suggested that the imbalance between proliferation and apoptosis, mRNA metabolism, the regulation of transcription and translation, the process of metabolism, etc may play an important role in neoplastic transformation of oval cells. Genes or proteins, such as phosphatase and tensin homolog (pten), cyclin dependent kinase inhibitor 1A (cdkn1a), cyclin dependent kinase 2 (cdk2), caspase-8 (casp-8), heterogeneous nuclear ribonucleoprotein A2/B1 isoform (HnRPA2B1), heterogeneous nuclear ribonucleoprotein A3 (HnRPA3), heterogeneous nuclear ribonucleoprotein D (HnRPD), Non-POU domain-containing octamer-binding protein (NONO),glucose transporter type 4 (GLUT4), etc, may be some crucial molecules, and may also play an important role in hepatocarcinogenesis. Part OneThe neoplastic transformation and identification of rat oval-like cells WB-F344For inducing neoplastic transformation of WB-F344 cells (Abbreviated as "WB cells"), the cells firstly were treated by MNNG, and then these MNNG-initiated cells were exposed to H₂O₂ once a week, repeated 21 times. Morphological change and transformed foci were observed under light microscope or transmission electron microscope. The change of chromosome in transformed cells was detected by karyotype analysis. Meanwhile, anchoring-independent growth, the characteristic of transformed cells or tumor cells, also was determined through the soft agar assay. The proliferative ability of transformed cells was detected by MTT analysis. The flow cytometry (FCM) was used for analyzing cell cycle of transformed cells.The morphologic analysis displayed that transformed foci were observed at weeks 2 of H₂O₂-treatment. Under light microscope, the cells in transformed foci were multilayer, overlapping and confused arrangement. Through transmission electron microscope detection, compared to control, transformed cells were polygon or cube, including more mitochondria and endoplasmic reticulum organelles, and microvillus and gap junctions occurred. Furthermore, karyotype analysis showed transformed cells possessed heteroploid karyotype, the chromosome number in half of transformed cells was abnormal, ranged from 27 to 103. In the soft agar assay, transformed cells have the characteristic of anchoring-independent growth, and the clonal forming ability gradually increased. The frequency of clonal forming respectively was 0, 0.1%, 0.3%, 0.8% and 2% in WB-3, -5, -7,-11 and -21 cells, however, WB cells could not grow in soft agar. WB-5 cells (at weeks 5 of H₂O₂-treatment) firstly formed clones in soft agar, but the frequency of clones forming was low (0.1%) and the clones were small. However the clonal forming ability of WB-21 cells (at weeks 21of H₂O₂-treatment) significantly increased (2%). MTT assay revealed that the proliferation of transformed cells were active. Transformed cells had more cells in S phase and G2-M phase as compared to WB cells.These results suggested that WB cells treated by MNNG and H₂O₂ could have neoplastic transformation, and their proliferation was more active. The transformed cells provided us basis for further the analysis of gene expression profile and protein expression profile.Part TwoThe analysis of gene expression profile during neoplastic transformation of rat oval-like cells WB-F344The objective of this work was to analyze the alteration of gene expression of transformed cells at different time points during the process of neoplastic transformation, and to investigate the biological significance of differential genes, and reveal some biological pathways or some genes those may associated with the neoplastic transformed of WB cells.Total RNA was extracted from WB cells (control), WB-5 and WB-21 cells and then applied to oligo microarray detection (rat Cancer PathwayFinder oligo microarray, SuperArray). We found 21 significantly up- or down-regulated genes in WB-5, WB-21 cells as compared to WB control. We validated the expression of pten, casp-8, cdkn1a and tumor necrosis factor receptor superfamily, member 10b (tnfrsf10b) in those three groups of cell by real time PCR. All q-PCR confirmed the data obtained by oligo microarray analysis. Most differential genes could classified as protein or nucleic acid binding and signal transducer related genes, which were involved in cell cycle control, apoptosis, cell adhesion or motility based on the DIVID database and the GENE ONTOLOGY database. Among these genes, up-regulation genes included cdk2, neural cell adhesion molecule 1 (ncam1), etc, down-regulation genes had interferon, beta 1 (ifnb1), interferon-alpha 1 (ifna1), collagen, type XVIII, alpha 1 (col18a1), etc. The expression of pten was increased gradually, while cdkn1a was continuously down-regulated during the stepwise transformation of WB cells. Otherwise, myc,fos and casp-8 were first up-regulation in WB-5 cells and then down in WB-21 cells. The analysis of interaction networks about differential genes through STRING database indicated the imbalance between proliferation and apoptosis may be part of reason for neoplastic transformation of WB cells, and these of genes, including cdk2, cdkn1a, pten, casp-8, etc, may be associated with the transformation. Part ThreeThe dynamic alteration of proteome during neoplastic transformation of rat oval-like cells WB-F344In this part, we further analyzed the change of protein expression profile of transformed cells at different time points during the transforming process and the functions of differential proteins to reveal some important proteins these may associate with the neoplastic transformation of WB cells.Total proteins were extracted from WB cells (control), WB-5 and WB-21 cells. Proteins were separated by 2-DE using IPG stripe (13cm, pH 3-11) and 12% SDS-PAGE in triplicate under identical condition. Analytical gels were stained with Coomassie brilliant blue. The stained gels were then scanned using ImageScanner and analyzed with ImageMaster 2D Platinum 5.0 software. Averagel279±13, 1141±6 and1503±8 protein spots had been detected in protein profile of WB, WB-5 and WB-21 cells, among of them, 629±29 (WB/WB-5), 752±21 (WB/WB-21) and 643±31 (WB-5/ WB-21) protein spots were well matched each other, and 148 protein spots were considered as differential protein spots (2 or more than 2 folds difference)132 differential protein spots were cut out and digested into peptides. Theses PMF or amino acid sequences of peptides were analyzed by MALDI-TOF-MS/MS and 81 protein spots were finally identified. Among of them, WB compared to WB-5 cells contained 29 differential protein spots, WB compared to WB-21 cells had 41 differential protein spots, and 27 differential protein spots were detected between WB-5 and WB-21 cells. Compared to between WB cells and transformed cells (WB-5 and WB-21), most of differential proteins were involved in metabolism, oxidation-reduction/stress reaction, such as aldehyde dehydrogenase A1 (ALDH3A1) and malate dehydrogenase 2 (MDH2) involved in the regulation of metabolism were up-regulated in transformed cells, however, adenylate kinase 1 (AK1) and phosphomannomutase 2 (PMM2) were down-regulated. Serine proteinase inhibitor, clade H, member 1 (SERPINH1) and heat shock protein 8 (HSP7C) were up, whereas peroxiredoxin 4 (PRDX4), PDZ and LIM domain protein 1 (ELFIN) were down in transformed cells, those of proteins were associated with oxidation-reduction/stress reaction. Most of differential proteins between transformed cells contributed to the regulation of transcription /translation or mRNA metabolism, cell adhesion or motility, oxidation-reduction/stress reaction and cell signal transduction. Especially, a group of RNA binding proteins, including HnRPA2Bl, HnRPA3 and HnRPD, had similar expressional trend in three groups of cells. Their level of expression was up-regulation in WB-5 cells, and then was down-regulation in WB-21 cells. The other RNA binding protein was NONO, it may interact with 40S ribosomal protein SA (RPSA), HSP7C, PRDX3 and ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5b) through HnRPK according to the analysis of proteins interaction network. Interestingly, glucose transporter type 4 (GLUT4) was common nodal point in all of interaction networks, and may be associate with many of differential proteins, which suggested it may relate to the neoplastic transformation of WB cells. In addition, the expression of CK8 and RPSA in three groups of cells was verified by immunoblotting.Above of these results indicated the change of these biological processes may contribute to neoplastic transformation of WB cells, including transcription /translation or mRNA metabolism, cell adhesion or motility, oxidation-reduction/stress reaction and metabolism. HnRPA2Bl, HnRPA3, HnRPD and NONO, etc, may be some crucial factors during the transformation of WB cells. GLUT4, interacted with many proteins, may also associate with the transformation or hepatocarcinogenesis.Conclusions1. Rat oval-like cells WB-F344 were transformed after treating with MNNG and H₂O₂.2. To establish the expression profile of proteins in WB, WB-5 and WB-21 cells; to analyze the profile of gene expression and protein expression.3. Most differential genes during transformation were involved in the regulation of proliferation, apoptosis and cell adhesion or motility. And differential expressed proteins contributed to the metabolic process, the regulation of transcription and translation/mRNA metabolism, cell adhesion or motility and oxidation-reduction/stress reaction, etc. The results suggested these biological processes may play an important role in the neoplastic transformation of WB cells. resulting in transformation. Among of these differential genes, cdk2, cdkn1a, pten, and casp-8, etc, may be some crucial factors involving in the transformation. A group of RNA binding proteins such as HnRPA2B1, HnRPA3, HnRPD, NONO and GLUT4, etc, may associate with WB cells transformation or hepatocarcinogenensis.The potential application of this work1. To provide some new evidences for further understanding the molecular mechanism of hepatocarcinogenesis.2. Some identified differential genes or proteins may become some new biomarkers for diagnosis or prevention, meanwhile, they may provide some new clues for exploring new targets of treatment.Novelty of project1. To establish firstly the expression profile of proteins during neoplastic transformation of WB cells (oval cells).2. To investigate hepatocarcinogenesis from cellular origin and further to understand the relationship between oval cells and hepatocarcinoma.3. The change of gene expression and protein expression profile was analyzed using microarray and proteomics technology. The imbalance between proliferation and apoptosis, metabolism, transcription/translation or mRNA metabolism, cell adhesion or motility and oxidation-reduction/stress reaction may be involved in neoplastic transformation of WB cells. Some proteins and some genes, such as HnRPA2B1, HnRPA3, HnRPD, NONO, GLUT4, cdk2, cdkn1a, pten, casp-8, etc, may associate with transformation or hepatocarcinogenesis, and they provided some new clues for exploring new targets of treatment.4. The combination of gene expression profile and protein expression profile will help us to preferably understand the molecular mechanism of WB cells neoplastic transformation and hepatocarcinogenesis.