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• W2000539324 abstract "Liver regeneration after a two-thirds partial hepatectomy (PHx) is a complex process requiring interaction and cooperation of many growth factors and cytokines and cross talk between multiple pathways. Along with hepatocyte growth factor and its receptor MET (HGF-MET), the epidermal growth factor receptor (EGFR) signaling pathway is activated within 60 minutes after PHx. To investigate the role of EGFR in liver regeneration, we used two EGFR-specific short hairpin silencing RNAs to inhibit EGFR expression in regenerating normal rat liver. Suppression of EGFR mRNA and protein was evident in treated rats. There was also a demonstrable decrease but not complete elimination of bromo-deoxyuridine incorporation and mitoses at 24 hours after PHx. In addition, we observed up-regulation of MET and Src as well as activation of the ErbB-3-ErbB-2-PI3K-Akt pathway and down-regulation of STAT 3, cyclin D1, cyclin E1, p21, and C/EBP β. The decrease in the ratio of C/EBP α to C/EBP β known to occur after PHx was offset in shEGFR-treated rats. Despite suppression of hepatocyte proliferation lasting into day 3 after PHx, liver weight restoration occurred. Interestingly, hepatocytes in shEGFR-treated rats were considerably larger when compared with ScrRNA-treated controls. The data indicate that although the MET and EGFR pathways are similar, the contributions made by MET and EGFR are unique and are not compensated by each other or other cytokines. Liver regeneration after a two-thirds partial hepatectomy (PHx) is a complex process requiring interaction and cooperation of many growth factors and cytokines and cross talk between multiple pathways. Along with hepatocyte growth factor and its receptor MET (HGF-MET), the epidermal growth factor receptor (EGFR) signaling pathway is activated within 60 minutes after PHx. To investigate the role of EGFR in liver regeneration, we used two EGFR-specific short hairpin silencing RNAs to inhibit EGFR expression in regenerating normal rat liver. Suppression of EGFR mRNA and protein was evident in treated rats. There was also a demonstrable decrease but not complete elimination of bromo-deoxyuridine incorporation and mitoses at 24 hours after PHx. In addition, we observed up-regulation of MET and Src as well as activation of the ErbB-3-ErbB-2-PI3K-Akt pathway and down-regulation of STAT 3, cyclin D1, cyclin E1, p21, and C/EBP β. The decrease in the ratio of C/EBP α to C/EBP β known to occur after PHx was offset in shEGFR-treated rats. Despite suppression of hepatocyte proliferation lasting into day 3 after PHx, liver weight restoration occurred. Interestingly, hepatocytes in shEGFR-treated rats were considerably larger when compared with ScrRNA-treated controls. The data indicate that although the MET and EGFR pathways are similar, the contributions made by MET and EGFR are unique and are not compensated by each other or other cytokines. Partial hepatectomy (PHx), in which two thirds of the rat liver is surgically removed, has been extensively used to study the highly complex phenomenon of liver regeneration. Although hepatocytes in normal adult liver are quiescent and rarely divide, they do retain an astounding ability to reenter the cell cycle and regenerate on surgical insult or injury. PHx in rats/mice results in rapid induction of more than 100 genes that are not expressed in the normal resting liver.1Michalopoulos GK Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1149) Google Scholar A rapid up-regulation of genes encoding transcriptional factors like AP1 breakdown of extracellular matrix by uPA and release of pre-existing stores of HGF is observed within 60 minutes of a PHx.2Michalopoulos GK DeFrances MC Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2884) Google Scholar The hepatocytes leave the quiescent G0 phase and enter the cell cycle. Methods to identify extrahepatic signals leading to liver regeneration have included mitogenic effects on hepatocyte cultures, stimulation of DNA synthesis in the liver of normal (unoperated) animals, and decrease in regeneration-related events in animals genetically or pharmacologically depleted of the agent under study. Of the various agents implicated in liver regeneration, HGF and ligands of the epidermal growth factor receptor (EGFR) are the only ones that stimulate DNA synthesis in hepatocyte cultures maintained in chemically defined media.3Patijn GA Lieber A Schowalter DB Schwall R Kay MA Hepatocyte growth factor induces hepatocyte proliferation in vivo and allows for efficient retroviral-mediated gene transfer in mice.Hepatology. 1998; 28: 707-716Crossref PubMed Scopus (103) Google Scholar They are also the only ones that stimulate DNA synthesis in the liver of normal mice and rats.4Bucher NL Liver regeneration: an overview.J Gastroenterol Hepatol. 1991; 6: 615-624Crossref PubMed Scopus (94) Google Scholar, 5Bucher NL Patel U Cohen S Hormonal factors concerned with liver regeneration.Ciba Found Symp. 1977; 55: 95-107PubMed Google Scholar, 6Yang J Chen S Huang L Michalopoulos GK Liu Y Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.Hepatology. 2001; 33: 848-859Crossref PubMed Scopus (101) Google Scholar HGF and EGF signaling pathways are activated within 60 minutes after a PHx,7Mars WM Liu ML Kitson RP Goldfarb RH Gabauer MK Michalopoulos GK Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration.Hepatology. 1995; 21: 1695-1701PubMed Google Scholar, 8Stolz DB Mars WM Petersen BE Kim TH Michalopoulos GK Growth factor signal transduction immediately after two-thirds partial hepatectomy in the rat.Cancer Res. 1999; 59: 3954-3960PubMed Google Scholar as evidenced by tyrosine phosphorylation of MET and EGFR within 30 to 60 minutes after PHx. There is evidence of cross talk and cooperation between MET and EGFR, and it is possible that MET transactivates EGFR.9Agarwal S Zerillo C Kolmakova J Christensen JG Harris LN Rimm DL Digiovanna MP Stern DF Association of constitutively activated hepatocyte growth factor receptor (Met) with resistance to a dual EGFR/Her2 inhibitor in non-small-cell lung cancer cells.Br J Cancer. 2009; 100: 941-949Crossref PubMed Scopus (82) Google Scholar, 10Jo M Stolz DB Esplen JE Dorko K Michalopoulos GK Strom SC Cross-talk between epidermal growth factor receptor and c-Met signal pathways in transformed cells.J Biol Chem. 2000; 275: 8806-8811Crossref PubMed Scopus (279) Google Scholar, 11Presnell SC Stolz DB Mars WM Jo M Michalopoulos GK Strom SC Modifications of the hepatocyte growth factor/c-met pathway by constitutive expression of transforming growth factor-alpha in rat liver epithelial cells.Mol Carcinog. 1997; 18: 244-255Crossref PubMed Scopus (39) Google Scholar, 12Scheving LA Stevenson MC Taylormoore JM Traxler P Russell WE Integral role of the EGF receptor in HGF-mediated hepatocyte proliferation.Biochem Biophys Res Commun. 2002; 290: 197-203Crossref PubMed Scopus (59) Google Scholar, 13Xu KP Yu FS Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing.Invest Ophthalmol Vis Sci. 2007; 48: 2242-2248Crossref PubMed Scopus (77) Google Scholar, 14Seki E Kondo Y Iimuro Y Naka T Son G Kishimoto T Fujimoto J Tsutsui H Nakanishi K Demonstration of cooperative contribution of MET- and EGFR-mediated STAT3 phosphorylation to liver regeneration by exogenous suppressor of cytokine signalings.J Hepatol. 2008; 48: 237-245Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar The EGFR family contains four members: ErbB-1, ErbB-2, ErbB-3, and ErbB-4. ErbB-3 is expressed in the adult liver but has no intrinsic kinase activity and relies on ErbB-1 for activity,15Michalopoulos GK Khan Z Liver regeneration, growth factors, and amphiregulin.Gastroenterology. 2005; 128: 503-506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar whereas ErbB-4 is not expressed in liver. Recently, both ErbB-2 and ErbB-3 have been shown to play a role in appendix regeneration in zebra fish.16Rojas-Munoz A Rajadhyksha S Gilmour D van Bebber F Antos C Rodriguez Esteban C Nusslein-Volhard C Izpisua Belmonte JC ErbB2 and ErbB3 regulate amputation-induced proliferation and migration during vertebrate regeneration.Dev Biol. 2009; 327: 177-190Crossref PubMed Scopus (53) Google Scholar The four ErbB receptors recognize 11 different but structurally related growth factors that mediate diverse processes like development, cell proliferation, and cell survival.17Carver RS Stevenson MC Scheving LA Russell WE Diverse expression of ErbB receptor proteins during rat liver development and regeneration.Gastroenterology. 2002; 123: 2017-2027Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Linggi B Carpenter G ErbB receptors: new insights on mechanisms and biology.Trends Cell Biol. 2006; 16: 649-656Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar Some of the ligands of EGFR that also increase after PHx and appear to affect liver regeneration are transforming growth factor α,19Fausto N Mead JE Regulation of liver growth: protooncogenes and transforming growth factors.Lab Invest. 1989; 60: 4-13PubMed Google Scholar Heparin binding EGF (HB-EGF),20Mitchell 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: 2562-2568Crossref PubMed Scopus (125) Google Scholar and amphiregulin.21Berasain C Garcia-Trevijano ER Castillo J Erroba E Lee DC Prieto J Avila MA Amphiregulin: an early trigger of liver regeneration in mice.Gastroenterology. 2005; 128: 424-432Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar There is thus a certain redundancy built in the EGFR pathway with multiple ligands with overlapping functions. The role of EGFR in embryonic development has been demonstrated by targeted deletion of EGFR. The resulting phenotype was dependent on strain and genetic background with abnormalities in various organs like liver, brain, and kidneys.22Sibilia M Kroismayr R Lichtenberger BM Natarajan A Hecking M Holcmann M The epidermal growth factor receptor: from development to tumorigenesis.Differentiation. 2007; 75: 770-787Crossref PubMed Scopus (258) Google Scholar, 23Miettinen PJ Berger JE Meneses J Phung Y Pedersen RA Werb Z Derynck R Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor.Nature. 1995; 376: 337-341Crossref PubMed Scopus (874) Google Scholar, 24Dackor J Strunk KE Wehmeyer MM Threadgill DW Altered trophoblast proliferation is insufficient to account for placental dysfunction in Egfr null embryos.Placenta. 2007; 28: 1211-1218Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar There have been two recent studies that addressed the role of EGFR in liver regeneration after a PHx. In one study, a monoclonal antibody (mAB) targeting EGFR was used to inhibit EGFR,25Van Buren 2nd, G Yang AD Dallas NA Gray MJ Lim SJ Xia L Fan F Somcio R Wu Y Hicklin DJ Ellis LM Effect of molecular therapeutics on liver regeneration in a murine model.J Clin Oncol. 2008; 26: 1836-1842Crossref PubMed Scopus (55) Google Scholar and its impact on liver regeneration was studied. In the second study, effects on liver regeneration after liver-specific perinatal deletion of EGFR were studied.26Natarajan A Wagner B Sibilia M The EGF receptor is required for efficient liver regeneration.Proc Natl Acad Sci USA. 2007; 104: 17081-17086Crossref PubMed Scopus (235) Google Scholar In the first study by Van Buren et al,25Van Buren 2nd, G Yang AD Dallas NA Gray MJ Lim SJ Xia L Fan F Somcio R Wu Y Hicklin DJ Ellis LM Effect of molecular therapeutics on liver regeneration in a murine model.J Clin Oncol. 2008; 26: 1836-1842Crossref PubMed Scopus (55) Google Scholar inhibiting EGFR had no effect on liver regeneration, whereas in the study performed by Natarajan et al,26Natarajan A Wagner B Sibilia M The EGF receptor is required for efficient liver regeneration.Proc Natl Acad Sci USA. 2007; 104: 17081-17086Crossref PubMed Scopus (235) Google Scholar mice lacking EGFR exhibited increased mortality and impaired liver regeneration. However, a number of pitfalls such as histopathological changes of using targeted gene deletions have been recognized that can complicate interpretation of results.27Fausto N Campbell JS Riehle KJ Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1262) Google Scholar, 28Muller U Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis.Mech Dev. 1999; 82: 3-21Crossref PubMed Scopus (203) Google Scholar, 29Wu H Wade M Krall L Grisham J Xiong Y Van Dyke T Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration.Genes Dev. 1996; 10: 245-260Crossref PubMed Scopus (215) Google Scholar To avoid pitfalls resulting from adaptations to gene loss30Gkretsi V Apte U Mars WM Bowen WC Luo JH Yang Y Yu YP Orr A St-Arnaud R Dedhar S Kaestner KH Wu C Michalopoulos GK Liver-specific ablation of integrin-linked kinase in mice results in abnormal histology, enhanced cell proliferation, and hepatomegaly.Hepatology. 2008; 48: 1932-1941Crossref PubMed Scopus (69) Google Scholar and histopathological alterations, we investigated the role of EGFR in liver regeneration in rats after a two-thirds PHx by using short hairpin silencing RNA (shRNA) targeting EGFR, a method we have recently applied for the HGF receptor (c-Met).31Paranjpe 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 (77) Google Scholar Our data show severe effects on hepatocyte proliferation and compensatory increases in expression of ErbB-3, ErbB-2, MET, and Src. Liver weight was restored in part by increase in hepatocyte size. Male Fisher 344 rats (150 to 200 gram) were obtained from Charles River Laboratories (Frederick, MD). Animals were allowed access to food and water ad libitum. Metofane was used to anesthetize animals. A two-thirds PHx was performed by resecting median and left lateral lobes.32Higgins GM A. RM: Experimental pathology of the liver, 1: restoration of the liver of the white rat following partial surgical removal.Arch Pathol. 1931; 12: 186-202Google Scholar For controls, a time-matched sham operation was performed. Sham operations involved a laparotomy and resection of the xyphoid process of the sternum. At defined time points, the animals were anesthetized with Nembutal (Abbott, Chicago, IL), and the remaining lobes were removed. All liver samples were promptly frozen in liquid nitrogen and stored at −80°C. All procedures performed on these rats were approved under the Institutional Animal Care and Use Committee protocol number 0507596A and conducted according to National Institutes of Health guidelines. The following primary antibodies were obtained from Cell Signaling Technology (Danvers, MA): EGFR rabbit mAB (1:1000, clone C74B9); phosphoEGFR Tyr992 (1:1000); PhosphoEGFR Tyr1068 mouse mAB (1:1000, clone 1H12); ErbB-3 rabbit mAB (1:1000, clone 1B2); phosphoErbB-2 Tyr1248, rabbit antibody (1:1000); ErbB-2 mouse mAB (1:1000, clone 44E7); MET mouse mAB (1:1000, clone 25H2); and PI3Kinase p85 rabbit mAB (1:1000, clone 19H8). The following antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA): ErbB-3 (clone G-4, for co-immunoprecipitation studies); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mouse mAB (1:1000, clone 0411); Platelet derived growth factor receptor (PDGFR), rabbit polyclonal antibody (1:1000, C-20); MET mouse mAB (clone B-2, for co-immunoprecipitation studies); and Cyclin D1 mouse mAB (1:1000, clone CD1.1). β-Actin antibody was from Millipore (Bedford, MA). Secondary antibodies used for this project were donkey anti-rabbit and donkey anti-mouse (Jackson ImmunoResearch Laboratories, West Grove, PA), used at 1:50,000 dilution. Rat EGFR-specific silencing RNAs were designed to down-regulate EGFR expression in regenerating rat livers. Two shRNA sequences, E1 and E2, were based on published rat EGFR, NM_031517 sequence. A mismatch sequence differing from shEGFR 2 sequence was also designed: (1) shEGFR 1: 5′-AGTAACAGGCTCACCCAAC-3′; (2) shEGFR 2: 5′-CACCGTGGAGAGAATCCCT-3′; and (3) mismatch shEGFR: 5′-gACgGTGGAGtGtATCCaT-3′. As a negative control, a scrambled sequence (Invivogen, San Diego, CA) was also used. All sequences were checked against databases by using the Smith-Waterman algorithm.33Smith TF Waterman MS Identification of common molecular subsequences.J Mol Biol. 1981; 147: 195-197Crossref PubMed Scopus (7115) Google Scholar Oligonucleotides were designed to contain TCAAGAG loop sequence and were cloned in the Bbs1 site of the shRNA-Hh1-gfp ZEO vector (Invivogen). The plasmid also has a Cytomegalovirus (CMV) enhancer/promoter driven Green Fluorescent Protein (GFP): zeo fusion gene that encodes a red-shifted variant of the jellyfish GFP and resistance to zeocin. The insert was cloned downstream of a RNA polymerase III promoter, the human H1 promoter. It is transcribed into a short double stranded RNA (dsRNA) with a hairpin structure (shRNA) consisting of a 21-bp double stranded region corresponding to the target sequence and a small loop formed by the spacer region. Competent GT116 Escherichia coli (Invivogen) were transformed and selected by growing them in media lysogeny broth (LB) in the presence of 50 μg/ml of zeocin. Positive clones were confirmed by restriction digestion and sequencing. A mixture of two EGFR-specific endotoxin-free shRNA plasmids at 300 μg was diluted in 5% glucose and was complexed with linear Polyethylenimine (In vivo JET PEI; Q-Biogene, Carlsbad, CA) at an N/P ratio of six, following the manufacturer’s protocol. The gene-specific shRNA/PEI complex was injected (in separate experiments) via the superior mesenteric vein 1 day before partial PHx. A second dose of 300 μg/rat was administered at the time of PHx. The mismatch and scrambled plasmids were similarly injected. Two parameters viz. bromo-deoxyuridine (BrdU) index and mitotic index were estimated following standard protocols. The labeling index corresponds to the ratio between positive nuclei to the total hepatocyte population. Three rats per time point per treatment group were used. Results were expressed as mean (±SEM). BrdU was injected intraperitoneally within 1 hour after PHx and then every 24 hours until day of sacrifice. All of the cells that had progressed through the cell cycle in a given animal could be assessed by this cumulative approach. Anti-BrdU was obtained from Amersham Biosciences (Buckinghamshire, UK). BrdU antibody was diluted at 1:100 in a nuclease that was received with the antibody kit. For each animal, at least 10 random fields (original magnification, ×200) were observed and a total of 200 to 400 positively stained nuclei counted. Data were plotted as mean ± SEM. Liver tissues were fixed in formalin, paraffin embedded, sectioned, and stained with H&E. Mitotic index was estimated by counting cells in 10 optical fields on each slide by means of light microscopy at ×200 magnification. Results were plotted as mean ± SEM. Livers were harvested from rats at defined time points with three rats per time point (four separate experiments). Each set included animals subjected to PHx alone, PHx + shRNA, PHx + ScrRNA, PHx + mismatch injected set, or a sham operation. In each set, the time points of animal sacrifice ranged from 0 to 72 hours. Total RNA was isolated by using TRIzol (Invitrogen, Carlsbad, CA). Briefly, 5 mg of liver tissue frozen in liquid nitrogen was homogenized in 1 ml of TRIzol. RNA was purified by using RNeasy kit from Qiagen (Valencia, CA). The isolated RNA was treated with Turbo DNA-Free (Ambion, Austin, TX) according to the manufacturer’s instructions. RNA was quantified by spectrophotometry at 260 nm, and purity was assessed by optical density 260/280 ratio. The RNA was stored at −80°C. Five micrograms of total RNA isolated from liver tissue was converted to cDNA by using random hexamers and reverse transcribed by using Superscript III (Invitrogen) following the manufacturer’s protocol. A no Reverse Transcriptase (RT) control was also included. PCR Primers used for amplification of EGFR, ErbB-3, and ErbB-2 were obtained from SABiosciences (Frederick, MD). Expression levels of EGFR, ErbB-2, and ErbB-3 were determined by Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) using SYBR green, and levels were normalized relative to expression of GAPDH in each sample. Fold change in gene expression was calculated by using the 2(−ΔΔCt) method.34Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123298) Google Scholar Reverse transcribed samples were amplified in parallel on an ABI Prism 7000 SDS instrument (Applied Biosystems, Foster City, CA). Quantitative real-time PCR for each sample was performed in triplicate in a 25-μl reaction with 50 ng of cDNA, two picomoles of each primer, and 1X SYBR Green PCR Master Mix (Applied Biosystems). The standard conditions for real-time PCR were as follows: 2 minutes at 50°C, 10 minutes at 95°C followed by 40 cycles of 15 seconds denaturation at 95°C, and elongation at 60°C for 45 seconds. A dissociation curve analysis was performed at the end of every run. A no RT and a no template controls were also included in every run. Whole liver cell lysates were prepared by homogenizing 100 mg liver tissue in radioimmunoprecipitation assay buffer (1X PBS, 1% NP-40, 0.1% SDS, pH 7.4) containing protease and phosphatase inhibitor mixtures (Sigma, St. Louis, MO), and then centrifuging at 14,000 × g for 30 minutes. Protein concentration was determined by using bicinchoninic acid assay (Sigma). Supernatants were saved at −80°C. Whole liver cell lysates (75 μg in NuPAGE LDS Sample Buffer) were separated by using precast 4% to 12% NuPAGE Bis-Tris gels with 1X MOPS (3-(N-morpholino) propanesulfonic acid; Invitrogen), then transferred to Immobilon-P membranes (Millipore) for 1 hour in transfer buffer containing 10% methanol and 0.005% SDS. Membranes were stained with Ponceau-S to verify loading and transfer efficiency. Membranes were probed with primary and secondary antibodies in Tris-buffered saline Tween 20 containing 5% nonfat mild or 5% bovine serum albumin. All antibodies were used at 1:1000 dilution following the manufacturer’s recommendations. Horseradish-peroxidase conjugated secondary antibodies to mouse and rabbit immunoglobulin was used at 1:50,000 (Chemicon, Temecula, CA). Membranes were processed by using SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL) and exposed to X-ray film (Lab Product Sales, Rochester, NY). Films were scanned, and densitometry readings were measured by using ImageJ (NIH). Band density for each time point (three rats per time point) was averaged, plotted in Excel (Microsoft Corporation, Redmond, WA), and standardized to Actin or GAPDH. Normal-, ScrRNA-, and shEGFR-treated rat liver protein lysates (500 μg) were diluted to final volume of 500 μl and precleared by incubation with 1 μg of hamster IgG (Santa Cruz Biotechnology) and 20 μl of Agarose A/G plus beads (Santa Cruz Biotechnology) for 1 hour at room temperature. For ErbB-3-phosphoErbB-2-PI3K heterodimerization studies, protein complexes were immunoprecipitated from cleared lysates with anti-Erbb-3 antibody (2 μg/500 μg of total protein lysates, G-4, Santa Cruz Biotechnology) or with control hamster IgG overnight at 4°C followed by 5-hour incubation at 4°C with agarose A/G beads. Immune complexes were washed three times with radioimmunoprecipitation assay buffer followed by resuspension in gel-loading sample buffer and 1X reducing agent (Invitrogen). Seventy-five micrograms of immunoprecipitated samples from shEGFR- and ScrRNA-treated and control livers were then electrophoresed and processed as described above. Blots were probed with phosphoErbB-2 Tyr1248 antibody (Cell Signaling Technology), stripped, and reprobed with p85 antibody (clone 19H8; Cell Signaling Technology). Equal protein loading was ascertained by immunoblotting with an ErbB-3 antibody. For MET-EGFR heterodimerization studies, whole cell lysates were processed as described above, and protein complexes were immunoprecipitated by using MET mouse mAB (2 μg/500 μg of total protein lysates, clone B-2, Santa Cruz Biotechnology) or with control hamster IgG and processed as described above. Blots were probed with EGFR antibody (1:1000, clone C74B9, Cell Signaling Technology). Equal protein loading was ascertained by immunoblotting with a MET antibody. The Affymetrix oligonucleotide chip specific for the rat (U230.2 A chip) containing 10,000 expressed sequences was used for analyzing changes in global gene expression in EGFR-silenced and ScrRNA-treated rats at 3 hours, 12 hours, and 24 hours after PHx. RNA was pooled from three rats per time point/treatment. Sample preparation, data generation, and analyses were as reported previously.35Michalopoulos GK Bowen WC Mule K Luo J HGF-, EGF-, and dexamethasone-induced gene expression patterns during formation of tissue in hepatic organoid cultures.Gene Expr. 2003; 11: 55-75Crossref PubMed Scopus (101) Google Scholar Normalization and preprocessing of data were performed by using dChip software. Expression intensities were log transformed, and genes with less than 80% present calls or expression level less than 7 or SD smaller than 0.5 were filtered out. A statistical analyses microarrays package was then used to analyze the data with a false discovery rate of 5%. R software (http://www.r-project.org) was used to generate the hierarchical clustering dendrograms and the heat maps for EGFR and control data set. Clustering was based on similarity of gene expression profiles.36Eisen MB Spellman PT Brown PO Botstein D Cluster analysis and display of genome-wide expression patterns.Proc Natl Acad Sci USA. 1998; 95: 14863-14868Crossref PubMed Scopus (13201) Google Scholar shRNA/small-interfering RNA treatment can often result in unexpected silencing of unrelated genes and induction of interferon response.37Semizarov D Frost L Sarthy A Kroeger P Halbert DN Fesik SW Specificity of short interfering RNA determined through gene expression signatures.Proc Natl Acad Sci USA. 2003; 100: 6347-6352Crossref PubMed Scopus (444) Google Scholar Analysis of expression signatures generated by different shRNAs (in this case, gene specific and scrambled) by DNA microarray is a much more stringent test for investigating off target effects and assessing specificity of shRNA mediated gene silencing.37Semizarov D Frost L Sarthy A Kroeger P Halbert DN Fesik SW Specificity of short interfering RNA determined through gene expression signatures.Proc Natl Acad Sci USA. 2003; 100: 6347-6352Crossref PubMed Scopus (444) Google Scholar Expression intensities of the top 500 expressed genes 3 hours and 24 hours after PHx were plotted, and lines were fitted by statistics through the individual expression values. We also analyzed the expression pattern of seven genes known to be involved in the interferon response. These were IP10, Oligoadenylate synthetase, cox2, interferon α, interferon β, interleukin-6, and interleukin-12. Expression values derived from the oligo-array for each of these genes for the different treatment groups were plotted. We also performed pathway-specific expression profiling of genes associated with cell cycle, apoptosis, and EGFR/platelet-derived growth factor (PDGF) signaling by using pathway-specific PCR arrays (SuperArray Bioscience, Frederick, MD). These arrays are in a 96-well format and contain 84 pathway-specific genes, 6 house keeping genes, and both positive and negative controls and were used to perform pathway-specific expression profiling by qRT-PCR of genes associated with these pathways. Assays were performed as recommended by the manufacturer. Total RNA was extracted from shEGFR-treated and untreated control rat liver (n = 3) at day 1 after PHx and was converted to cDNA as described earlier. PCR was performed on ABI Prism 7000 Sequence Detector (Applied Biosystems). Data were analyzed by using the 2(−ΔΔCt) method incorporated in the Excel-based (Microsoft Corporation) PCR Array Data Analysis Template provided by the manufacturer. Fold changes were expressed as log-normalized ratios of values from shEGFR-treated/control liver tissues. A positive value indicated up-regulation and a negative value indicated down-regulation in expression as compared with control animals. Unpaired Student’s t-test was performed for evaluation of significant differences between the control and shRNA-treated animals by using the Minitab computer program (Minitab, Inc, State College, PA). Data are expressed as mean ± SEM. For real-time PCR and Western blot data, P values were determined by student’s t-test with P < 0.05 being significant. A mixture of the two shEGFR plasmids was complexed with PEI and administered 24 hours before and at the time of PHx.31Paranjpe 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 (77) Google Scholar A qRT-PCR was performed to estimate levels of EGFR mRNA. Data were normalized to GAPDH. As seen i" @default.
• W2000539324 created "2016-06-24" @default.
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• W2000539324 date "2010-06-01" @default.
• W2000539324 modified "2023-10-15" @default.
• W2000539324 title "RNA Interference Against Hepatic Epidermal Growth Factor Receptor Has Suppressive Effects on Liver Regeneration in Rats" @default.
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