Relationships between Deficits in Tissue Mass and Transcriptional Programs after Partial Hepatectomy in Mice
2009; Elsevier BV; Volume: 175; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.090043
ISSN1525-2191
AutoresJiangning Li, Jean S. Campbell, Claudia Mitchell, Ryan S. McMahan, Xuesong Yu, Kimberly J. Riehle, Roger E. Bumgarner, Nelson Fausto,
Tópico(s)Organ Transplantation Techniques and Outcomes
ResumoLiver regeneration after two-thirds partial hepatectomy (2/3 PH) results in synchronized proliferation of hepatocytes and rapid restoration of liver mass. Understanding the mechanisms that regulate this process has both biological and clinical importance. Using cDNA microarray analysis, we investigated whether gene activation after 2/3 PH is specifically related to liver growth and hepatocyte proliferation. We generated gene expression profiles at 4, 12, 20, and 30 hours after 2/3 PH and compared them with profiles obtained at the same time points after 1/3 PH, a procedure that causes minimal DNA replication. Surprisingly, a significant number of genes whose expression is altered after 2/3 PH are similarly up- or down-regulated after 1/3 PH, particularly at 4 hours. We identified a number of genes and transcription factors that are more highly expressed (“preferential expression”) after 2/3 PH and show that a shift in transcriptional programs in the regenerating liver occurs between 4 and 12 hours after 2/3 PH, a time at which the decision to replicate appears to be made. These results show that the liver responds to PH with massive changes of gene expression, even in the absence of DNA replication. We suggest that the changes in gene expression during the first 4 to 6 hours after 2/3 PH may induce chromatin remodeling and facilitate the binding of new sets of transcription factors required for DNA replication. Liver regeneration after two-thirds partial hepatectomy (2/3 PH) results in synchronized proliferation of hepatocytes and rapid restoration of liver mass. Understanding the mechanisms that regulate this process has both biological and clinical importance. Using cDNA microarray analysis, we investigated whether gene activation after 2/3 PH is specifically related to liver growth and hepatocyte proliferation. We generated gene expression profiles at 4, 12, 20, and 30 hours after 2/3 PH and compared them with profiles obtained at the same time points after 1/3 PH, a procedure that causes minimal DNA replication. Surprisingly, a significant number of genes whose expression is altered after 2/3 PH are similarly up- or down-regulated after 1/3 PH, particularly at 4 hours. We identified a number of genes and transcription factors that are more highly expressed (“preferential expression”) after 2/3 PH and show that a shift in transcriptional programs in the regenerating liver occurs between 4 and 12 hours after 2/3 PH, a time at which the decision to replicate appears to be made. These results show that the liver responds to PH with massive changes of gene expression, even in the absence of DNA replication. We suggest that the changes in gene expression during the first 4 to 6 hours after 2/3 PH may induce chromatin remodeling and facilitate the binding of new sets of transcription factors required for DNA replication. Resection of hepatic tissue triggers a proliferative response known as liver regeneration in which quiescent hepatocytes enter the cell cycle and replicate. After 70% partial hepatectomy (2/3 PH) in mice, liver mass is fully restored during the second week after the operation. Restoration of mass is a consequence of a process of compensatory hyperplasia of cells of the liver remnant, as the liver lobes removed at the time of the operation do not re-grow.1Fausto N Campbell JS Riehle KJ Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1280) Google Scholar, 2Michalopoulos GK Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1172) Google Scholar, 3Taub R Liver regeneration: from myth to mechanism.Nat Rev Mol Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1277) Google Scholar, 4Fausto N Liver regeneration and repair: hepatocytes, progenitor cells, and stem cells.Hepatology. 2004; 39: 1477-1487Crossref PubMed Scopus (635) Google Scholar The human liver also has a high regenerative capacity, as shown by its growth in donors of right lobe grafts in living donor transplantation.5Haga J Shimazu M Wakabayashi G Tanabe M Kawachi S Fuchimoto Y Hoshino K Morikawa Y Kitajima M Kitagawa Y Liver regeneration in donors and adult recipients after living donor liver transplantation.Liver Transpl. 2008; 14: 1718-1724Crossref PubMed Scopus (92) Google Scholar, 6Pomfret EA Pomposelli JJ Gordon FD Erbay N Lyn Price L Lewis WD Jenkins RL Liver regeneration and surgical outcome in donors of right-lobe liver grafts.Transplantation. 2003; 76: 5-10Crossref PubMed Scopus (144) Google Scholar Thus, the investigation of the cellular and molecular mechanisms of liver regeneration has biological and clinical implications, and is of fundamental importance. Liver regeneration after 2/3 PH is a stepwise process that starts with an initiation phase, corresponding to the G0 to G1 transition, which primes hepatocytes to respond to growth signals. Primed hepatocytes enter the cell cycle, undergo one or two rounds of synchronous DNA replication followed by mitosis, and then return to a quiescent state. The regenerative process involves the activity of hundreds of genes and the activation of multiple pathways. However, despite the great progress achieved by the analyses of gene expression patterns in the regenerating liver,7White P Brestelli JE Kaestner KH Greenbaum LE Identification of transcriptional networks during liver regeneration.J Biol Chem. 2004; Google Scholar, 8Su AI Guidotti LG Pezacki JP Chisari FV Schultz PG Gene expression during the priming phase of liver regeneration after partial hepatectomy in mice.Proc Natl Acad Sci USA. 2002; 99: 11181-11186Crossref PubMed Scopus (170) Google Scholar, 9Otu HH Naxerova K Ho K Can H Nesbitt N Libermann TA Karp SJ Restoration of liver mass after injury requires proliferative and not embryonic transcriptional patterns.J Biol Chem. 2007; 282: 11197-11204Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 10Fukuhara Y Hirasawa A Li XK Kawasaki M Fujino M Funeshima N Katsuma S Shiojima S Yamada M Okuyama T Suzuki S Tsujimoto G Gene expression profile in the regenerating rat liver after partial hepatectomy.J Hepatol. 2003; 38: 784-792Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 11Kelley-Loughnane N Sabla GE Ley-Ebert C Aronow BJ Bezerra JA Independent and overlapping transcriptional activation during liver development and regeneration in mice.Hepatology. 2002; 35: 525-534Crossref PubMed Scopus (66) Google Scholar more information is still needed for a full understanding of the molecular mechanisms of liver regeneration. An important question that has not been explored in detail is the extent to which the widespread changes in gene expression that occur during liver regeneration after 2/3 PH are linked to hepatocyte DNA replication. Designing studies to answer this question is made difficult for various reasons, particularly, the confounding factors created by surgical stress, the problems in choosing adequate controls (whether normal livers or liver of sham-operated mice) to measure relative changes in gene expression, and the variability of the data obtained from different animals. Our approach to determine whether changes of gene expression in the regenerating liver are directly or indirectly linked to hepatocyte DNA replication has been to compare gene expression after 2/3 PH, the standard surgical procedure that produces robust DNA replication, with gene expression after 1/3 PH, a procedure that causes minimal replication. Previously, we showed that many proto-oncogenes and cytokines that are expressed early after 2/3 PH are also expressed after 1/3 PH, suggesting that some components of the immediate early gene response after 2/3 PH do not appear to be directly linked to the amount of tissue resected, and do not determine the magnitude of DNA replication after PH.12Mitchell C Nivison M Jackson LF Fox R Lee DC Campbell JS Fausto N Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.J Biol Chem. 2005; 280: 2562-2568Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar This conclusion is similar to that presented by Lambotte et al in their studies of gene expression after a “temporary hepatectomy,” who indicated that the extent of DNA replication after PH is not determined at the initiating phase of liver regeneration, but may occur several hours later, possibly at a time when most hepatocytes reach the late G1 stage.13Lambotte L Saliez A Triest S Tagliaferri EM Barker AP Baranski AG Control of rate and extent of the proliferative response after partial hepatectomy.Am J Physiol. 1997; 273: G905-G912PubMed Google Scholar To determine whether changes in gene expression after 2/3 PH occur even with a minimal replicative response, we have expanded our previous work, and performed a detailed analysis of global patterns of gene expression after 1/3 and 2/3 PH. Using an experimental design in which each mouse had its own normal liver as a control, thus reducing animal to animal variation, we analyzed gene expression profiles at 4, 12, 20 and 30 hours after both 1/3 and 2/3 PH, and identified transcription factors that may regulate genes that are preferentially expressed after 2/3 PH relative to 1/3 PH. We found that there are widespread changes of gene expression after 1/3 PH, a procedure that causes only minimal hepatocyte DNA replication, and that the expression of a large number of genes is similarly up- or down-regulated relative to normal liver after 1/3 or 2/3 PH. However, a group of genes showed a higher expression magnitude, up or down, after 2/3 PH relative to 1/3 PH (preferential expression). These genes contain binding sites for a small number of transcription factors whose profiles change drastically between 4 and 12 hours after 2/3 PH. We suggest that the change in the transcriptional program that occurs during this time may be associated with chromatin remodeling. Ten-week-old, male, wild-type C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were kept on a 12 hours light/dark cycle with free access to food and water; 1/3 PH and 2/3 PH were performed as described.12Mitchell C Nivison M Jackson LF Fox R Lee DC Campbell JS Fausto N Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.J Biol Chem. 2005; 280: 2562-2568Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar At the time of surgery, the resected left lobes were cut into approximately 5 mm3 cubes and stored in liquid nitrogen until use. Mice were sacrificed 4, 12, 20, or 30 hours later (6 mice/time point/type of surgery), and the regenerating lobes (right and caudate) were harvested as described.12Mitchell C Nivison M Jackson LF Fox R Lee DC Campbell JS Fausto N Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.J Biol Chem. 2005; 280: 2562-2568Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar The experimental design is shown in Figure 1. All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee at the University of Washington. Total RNA was extracted from a matched pair of frozen liver tissues using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s directions. RNA obtained from the left lobe (L) of each mouse was used as a control for the regenerating right lobe (R). All RNA was quantified, assessed for quality, and amplified with a single round of Eberwine amplification as previously described.14Li J Adams L Schwartz SM Bumgamer RE RNA amplification, fidelity and reproducibility of expression profiling.C R Biol. 2003; 326: 1021-1030Crossref PubMed Scopus (20) Google Scholar cDNA microarrays containing 13,425 mouse cDNAs from the NIA 15K collection (http://lgsun.grc.nia.nih.gov/cDNA/15k.html) were made at the Center for Expression Arrays at the University of Washington (http://www.expression.washington.edu) following established protocols that are described in detail elsewhere.14Li J Adams L Schwartz SM Bumgamer RE RNA amplification, fidelity and reproducibility of expression profiling.C R Biol. 2003; 326: 1021-1030Crossref PubMed Scopus (20) Google Scholar We used two arrays for each animal—one array had the Cy3/Cy5 labels associated with L/R probes while the other was a dye reversal (ie, Cy3/Cy5 for R/L). There were two identical gene sets on each slide, which generated 2 × 2 = 4 technical replicates for each gene comparison per mouse. Array data have been deposited to Array Express, (http://www.ebi.ac.uk/microarray-as/ae, accession number E-MTAB-119). We analyzed the normalized data using a custom version of TIGR’s MEV software15Saeed AI Sharov V White J Li J Liang W Bhagabati N Braisted J Klapa M Currier T Thiagarajan M Sturn A Snuffin M Rezantsev A Popov D Ryltsov A Kostukovich E Borisovsky I Liu Z Vinsavich A Trush V Quackenbush J TM4: a free, open-source system for microarray data management and analysis.Biotechniques. 2003; 34: 374-378Crossref PubMed Scopus (4019) Google Scholar that we have modified to connect to the Gene Traffic database. Several types of analyses were performed, as outlined in supplemental Table S1 (available at http://ajp.amjpathol.org): a) selection of genes that are differentially expressed (up or down) after 1/3 or 2/3 PH relative to normal liver, using a one-class significance analysis of microarray (SAM)16Tusher VG Tibshirani R Chu G Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001; 98: 5116-5121Crossref PubMed Scopus (9797) Google Scholar with a false discovery rate of 5% for each type of surgery at each time point; b) selection of genes that are differentially expressed between 1/3 and 2/3 PH at all time points, using a two-class SAM with a false discovery rate of 20% at each time point; c) selection of genes that are differentially expressed after both 1/3 and 2/3 PH in relationship to normal liver in at least one time point; d) classification of genes preferentially expressed after 2/3 PH dependent on the type of surgery, the time after surgery, or dependent on both the type of surgery and the time after PH, using a two-factor analysis of variance using the type of surgery (1/3 or 2/3 PH) as one variable, and time (4, 12, 20, and 30 hours after PH) as the other. For this analysis a P value of <0.01 calculated by permutation testing was used, without further correction for multiple testing errors. After hybridization, washing and scanning, all of the data were transferred to a local GeneTraffic database (Iobion Software, http://www.iobion.com). All data were normalized using global LOWESS normalization.17Cleveland W Robust locally weighted regression and smoothing scatter plots.J Am Stat Assoc. 1979; 74: 829-836Crossref Scopus (7460) Google Scholar All gene annotations were updated in March 2006 using the SOURCE database.18Diehn M Sherlock G Binkley G Jin H Matese JC Hernandez-Boussard T Rees CA Cherry JM Botstein D Brown PO Alizadeh AA SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data.Nucleic Acids Res. 2003; 31: 219-223Crossref PubMed Scopus (354) Google Scholar Out of 13,426 clones on the array, 10,240 have a UniGene cluster ID, and 7464 of these are unique. Duplicate UniGene IDs may not produce identical array data due to a variety of reasons, including the possible presence of alternative spliced versions of the same gene on the array.19Okazaki Y Furuno M Kasukawa T Adachi J Bono H Kondo S Nikaido I Osato N Saito R Suzuki H Yamanaka I Kiyosawa H Yagi K Tomaru Y Hasegawa Y Nogami A Schonbach C Gojobori T Baldarelli R Hill DP Bult C Hume DA Quackenbush J Schriml LM Kanapin A Matsuda H Batalov S Beisel KW Blake JA Bradt D Brusic V Chothia C Corbani LE Cousins S Dalla E Dragani TA Fletcher CF Forrest A Frazer KS Gaasterland T Gariboldi M Gissi C Godzik A Gough J Grimmond S Gustincich S Hirokawa N Jackson IJ Jarvis ED Kanai A Kawaji H Kawasawa Y Kedzierski RM King BL Konagaya A Kurochkin IV Lee Y Lenhard B Lyons PA Maglott DR Maltais L Marchionni L McKenzie L Miki H Nagashima T Numata K Okido T Pavan WJ Pertea G Pesole G Petrovsky N Pillai R Pontius JU Qi D Ramachandran S Ravasi T Reed JC Reed DJ Reid J Ring BZ Ringwald M Sandelin A Schneider C Semple CA Setou M Shimada K Sultana R Takenaka Y Taylor MS Teasdale RD Tomita M Verardo R Wagner L Wahlestedt C Wang Y Watanabe Y Wells C Wilming LG Wynshaw-Boris A Yanagisawa M Yang I Yang L Yuan Z Zavolan M Zhu Y Zimmer A Carninci P Hayatsu N Hirozane-Kishikawa T Konno H Nakamura M Sakazume N Sato K Shiraki T Waki K Kawai J Aizawa K Arakawa T Fukuda S Hara A Hashizume W Imotani K Ishii Y Itoh M Kagawa I Miyazaki A Sakai K Sasaki D Shibata K Shinagawa A Yasunishi A Yoshino M Waterston R Lander ES Rogers J Birney E Hayashizaki Y Analysis of the mouse transcriptome based on functional annotation of 60,770 full-length cDNAs.Nature. 2002; 420: 563-573Crossref PubMed Scopus (1406) Google Scholar Duplicate UniGene IDs were treated the following way: we calculated the correlation coefficient of the expression values between duplicates using a tool built into the “R” package (http://www.r-project.org/), and treated the duplicates as one gene if the correlation coefficient was above 0.8 by averaging the ratios of those duplicates. For those duplicates having a correlation coefficient less than 0.8, we treated each individual clone as an independent gene associated with its own expression ratio. This approach generates a total of 10,143 clones used in the subsequent statistical analysis. Data from all technical replicates were averaged for subsequent analyses. All of the gene functions are based on gene ontology (GO) terminology20Zeeberg BR Feng W Wang G Wang MD Fojo AT Sunshine M Narasimhan S Kane DW Reinhold WC Lababidi S Bussey KJ Riss J Barrett JC Weinstein JN GoMiner: a resource for biological interpretation of genomic and proteomic data.Genome Biol. 2003; 4: R28Crossref PubMed Google Scholar except for those specifically referenced. To identify biological schemes that are over-represented in each gene list, we used Expression Analysis Systematic Explorer (EASE).21Hosack DA Dennis Jr, G Sherman BT Lane HC Lempicki RA Identifying biological themes within lists of genes with EASE.Genome Biol. 2003; 4: R70Crossref PubMed Google Scholar, 22Ashburner M Ball CA Blake JA Botstein D Butler H Cherry JM Davis AP Dolinski K Dwight SS Eppig JT Harris MA Hill DP Issel-Tarver L Kasarskis A Lewis S Matese JC Richardson JE Ringwald M Rubin GM Sherlock G Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.Nat Genet. 2000; 25: 25-29Crossref PubMed Scopus (27616) Google Scholar We used the unique UniGene cluster IDs as identifiers. All of the over-represented schemes are determined by an EASE score of less than 0.05. Pathway analyses were performed with GenMAPP 2 β.23Doniger SW Salomonis N Dahlquist KD Vranizan K Lawlor SC Conklin BR MAPPFinder: using Gene Ontology and GenMAPP to create a global gene-expression profile from microarray data.Genome Biol. 2003; 4: R7Crossref PubMed Google Scholar, 24Dahlquist KD Salomonis N Vranizan K Lawlor SC Conklin BR GenMAPP, a new tool for viewing and analyzing microarray data on biological pathways.Nat Genet. 2002; 31: 19-20Crossref PubMed Scopus (808) Google Scholar For gene selection, the threshold of ‘fold-change’ was set at 1.5 in creating the following 3 categories: 1) up-regulated (above 1.5), 2) down-regulated (below −1.5); and 3) criteria not met (values between −1.5 and +1.5). This criterion was chosen based on statistical calculations as described.25Wei C Li J Bumgarner RE Sample size for detecting differentially expressed genes in microarray experiments.BMC Genomics. 2004; 5: 87Crossref PubMed Scopus (113) Google Scholar The Promoter Analysis and Interaction Network Toolset (PAINT) is a computational tool that can integrate functional genomics data with genomic sequence data to perform transcriptional regulatory network analysis.26Vadigepalli R Chakravarthula P Zak DE Schwaber JS Gonye GE PAINT: a promoter analysis and interaction network generation tool for gene regulatory network identification.Omics. 2003; 7: 235-252Crossref PubMed Scopus (114) Google Scholar The differentially expressed genes at each time point were analyzed using this program to identify potential transcriptional regulatory elements (TREs) by the presence of transcription factor binding sites. The desired upstream sequences were set to 2000 bp, and the TRANSFAC public match program was applied. The Core similarity threshold was set to 1 and the P value was set to 0.05. The numbers of identified TREs present at each of the four time points were combined and re-scaled, with the total number of differentially expressed genes retrieved from the TRANSFAC database. For validation of microarray results, 2 μg of RNA from 1/3 and 2/3 PH samples were reverse transcribed using the Invitrogen Retroscript kit, and real-time RT-PCR performed on the resultant cDNA amplicon using FAM-labeled primers for murine proliferating cell nuclear antigen (Pcna) and cyclin B1 (Ccnb1) (Applied Biosystems, Foster City, CA). Analyses were performed as previously described.12Mitchell C Nivison M Jackson LF Fox R Lee DC Campbell JS Fausto N Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.J Biol Chem. 2005; 280: 2562-2568Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar For immunohistochemistry, the right lobes of each liver after 1/3 or 2/3 PH at 30 hours were fixed in 10% buffered formalin and embedded in paraffin. Specimens were stained with antibodies to PCNA (Transduction Laboratories, BD Biosciences, San Jose, CA) using the ABC method with diaminobenzidine as the chromogen. Hepatocytes with nuclear staining were considered PCNA-positive cells. For each sample, three high-power (×400) fields were counted, and differences between 1/3 and 2/3 PH were calculated using an unpaired t-test with Welch’s correction using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). We examined gene expression profiles after 1/3 and 2/3 PH to study the similarities and differences in gene expression patterns after 2/3 PH, which leads to robust DNA replication, and 1/3 PH, in which the replicative response is minimal.12Mitchell C Nivison M Jackson LF Fox R Lee DC Campbell JS Fausto N Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.J Biol Chem. 2005; 280: 2562-2568Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 13Lambotte L Saliez A Triest S Tagliaferri EM Barker AP Baranski AG Control of rate and extent of the proliferative response after partial hepatectomy.Am J Physiol. 1997; 273: G905-G912PubMed Google Scholar, 27Bucher NL Swaffield MN The rate of incorporation of labeled thymidine into the deoxyribonucleic acid of regenerating rat liver in relation to the amount of liver excised.Cancer Res. 1964; 24: 1611-1625PubMed Google Scholar, 28Inderbitzin D Studer P Sidler D Beldi G Djonov V Keogh A Candinas D Regenerative capacity of individual liver lobes in the microsurgical mouse model.Microsurgery. 2006; 26: 465-469Crossref PubMed Scopus (31) Google Scholar For each animal, the left lobe resected at the time of the PH served as an internal control for its own right lobe, which was harvested 4 to 30 hours after the surgery (Figure 1, A–C). This design minimizes the variation inherent in comparing pre- and postoperative livers of different animals, and increases the statistical power for detecting differences in gene expression.29Li J Pritchard DK Wang X Park DR Bumgarner RE Schwartz SM Liles WC cDNA microarray analysis reveals fundamental differences in the expression profiles of primary human monocytes, monocyte-derived macrophages, and alveolar macrophages.J Leukoc Biol. 2007; 81: 328-335Crossref PubMed Scopus (35) Google Scholar Using a one-class SAM analysis, we first investigated the patterns of gene expression at 4, 12, 20, and 30 hours after 1/3 and 2/3 PH (see supplemental Tables S2 and S3 available at http://ajp.amjpathol.org) to select genes whose expression was either increased or decreased after PH, as compared with the normal liver from the same mouse (left lobe resected at the time of PH). Both 1/3 and 2/3 PH induce a large number of genes, ranging in number from 625 to 2877 at different time points (Figure 2A). Overall, the number of differentially expressed genes, after 2/3 PH relative to normal liver, increases from 1673 at 4 hours to 2877 at 30 hours. After 1/3 PH the largest number of differentially expressed genes relative to normal liver (ie, 2278) is detected at 4 hours. This number decreases to 625 at 12 hours, and then increases to 1945 from 20 to 30 hours. Genes that were differentially regulated after 1/3 or 2/3 PH compared with normal liver (ie, the left lobe from the same mouse) are listed in supplemental Tables S2 and S3 available at http://ajp.amjpathol.org, respectively. In summary, the results show that the expression of a large number of genes is altered, either up- or down-regulated, after 1/3 and 2/3 PH, and that between 4 and 12 hours, the number of differentially expressed genes relative to normal liver increases after 2/3 PH but decreases after 1/3 PH. After compiling a group of genes that are up- or down-regulated after 1/3 or 2/3 PH relative to normal liver shown in Figure 2A, we selected genes that are preferentially expressed after 2/3 PH, as compared with 1/3 PH, using a two-class SAM with a false discovery rate of 20%. At 4 and 12 hours, only a few genes are preferentially expressed after 2/3 PH compared with 1/3 PH but the overall number of genes whose expression is preferentially modified after 2/3 PH increases significantly after 12 hours (Figure 2B). Supplemental Table S4 available at http://ajp.amjpathol.org lists genes that are preferentially regulated after 2/3 PH compared with 1/3 PH at all 4 time points (see supplemental Table S4 available at http://ajp.amjpathol.org). Given the importance of the hepatocyte growth factor/c-met system in liver regeneration,2Michalopoulos GK Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1172) Google Scholar, 30Borowiak M Garratt AN Wustefeld T Strehle M Trautwein C Birchmeier C Met provides essential signals for liver regeneration.Proc Natl Acad Sci USA. 2004; 101: 10608-10613Crossref PubMed Scopus (407) Google Scholar, 31Huh CG Factor VM Sanchez A Uchida K Conner EA Thorgeirsson SS Hepatocyte growth factor/c-met signaling pathway is required for efficient liver regeneration and repair.Proc Natl Acad Sci USA. 2004; 101: 4477-4482Crossref PubMed Scopus (627) Google Scholar, 32Paranjpe S Bowen WC Bell AW Nejak-Bowen K Luo JH Michalopoulos GK Cell cycle effects resulting from inhibition of hepatocyte growth factor and its receptor c-Met in regenerating rat livers by RNA interference.Hepatology. 2007; 45: 1471-1477Crossref PubMed Scopus (79) Google Scholar we examined the expression of c-met by real-time PCR because this gene was not among the genes in the microarray. There were no differences in the expression of c-met mRNA between 1/3 and 2/3 PH at 4, 12, and 30 hours. Previously we reported that hepatocyte growth factor mRNA levels did not differ between 1/3 and 2/3 PH.12Mitchell C Nivison M Jackson LF Fox R Lee DC Campbell JS Fausto N Heparin-binding epidermal growth factor-like growth factor links hepatocyte priming with cell cycle progression during liver regeneration.J Biol Chem. 2005; 280: 2562-2568Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar To learn more about gene expression differences between 1/3 and 2/3 PH, we plotted the log2 ratios of preferentially expressed genes in the regenerating liver relative to normal liver tissue at each time point after 1/3 or 2/3 PH (Figure 3). The data show that at each time point examined, the magnitude of expression of most genes is higher, either up or down, after 2/3 PH compared with 1/3 PH. Functional analysis by EASE of genes that are preferentially expressed after 2/3 PH (Table 1 and supplemental Table S5 available at http://ajp.amjpathol.org) shows an enrichment of transcriptional profiles at 12 hours after 2/3 PH of genes associated with “cell adhesion” and “blood vessel development.” At 20 hours the enrichment profiles shift to genes associated with “amine metabolism” and “amino acid metabolism,” and at 30 hours, there is enrichment of genes associated with “cell cycle,” “DNA replication,” and “S-phase of the cell cycle.” Surprisingly, our functional analysis of preferentially regulated genes at 4 hours after 2/3 PH relative to 1/3 PH did not reveal any enriched categories. We conclude that differences in gene expression between 1/3 and 2/3 PH are mostly due to the magnitude of gene expression, that is, changes in expression of individual genes, either up or down, are larger after 2/3 PH compared with 1/3 PH. Furthermore, enrichment for functional categories of expressed genes in 2/3 PH relative to 1/3 PH does not occur until 12 hours after the operation.Table 1EASE Analysis of Preferentially Expressed Genes after 2/3 PH Relative to 1/3 PHSystemTime (h)Gene categoryEASE scoreGO_BP12Cell adhesion1.71E-02GO_BP12Angiogenesis2.48E-02GO_BP12Blood vessel development3.42E-02GO_BP20Amino acid and derivative metabolism1.89E-03GO_BP20Amino acid catabolism7.94E-03GO_BP20Amine metabolism9.81E-03GO_BP20Amine catabolism1.91E-02GO_BP20Amino acid metabolism2.22E-02GO_BP20Amino acid derivative metabolism4.81E-02GO_BP30Cell proliferation1.35E-03GO_BP30Cell cycle1.84E-03GO_BP30DNA replication and chromosome c
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