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Microrna Profiling in Murine Liver After Partial Hepatectomy

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Microrna Profiling in Murine Liver After Partial Hepatectomy INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 29: 747-755, 2012 MicroRNA profiling in murine liver after partial hepatectomy IOANNIS CHAVELES1, APOSTOLOS ZARAVINOS2, IOANNIS G. HABEOS3, DIMITRIOS D. KARAVIAS1, IOANNIS MAROULIS1, DEMETRIOS A. SPANDIDOS2 and DIONISIOS KARAVIAS1 1Surgical Department, Medical School, University of Patras, 26504 Patras; 2Laboratory of Virology, Medical School, University of Crete, 71110 Heraklion, Crete; 3Medical Department, Medical School, University of Patras, 26504 Patras, Greece Received January 9, 2012; Accepted February 3, 2012 DOI: 10.3892/ijmm.2012.902 Abstract. Liver is uniquely capable to repair itself after injury. early up-regulation of mmu-miR-21 during the process of liver Multiple molecular and biochemical processes initiated after regeneration suggests a regulatory role in liver regeneration partial hepatectomy, lead to proliferation of all cells within the in vivo. liver. MicroRNAs (miRNAs) are a class of highly abundant non-coding RNA molecules that cause post-transcriptional Introduction gene repression and are involved in several biological processes including cell cycle regulation and differentiation. In this study, Partial hepatectomy (PH) represents the most commonly used we examined the expression levels of miRNAs in liver tissue model for the study of liver regeneration. It is a very complex and received from control mice (L0) and compared them with the well-orchestrated phenomenon that is carried out by the partici- corresponding levels in liver tissue 12 h after liver regeneration pation of all mature liver cell types. The extent of hepatocyte induced by 2/3 partial hepatectomy (L12). MiRNA expression proliferation is directly proportional to the amount of resected was investigated using microRNA profiling. Further qPCR liver tissue and 2/3 partial hepatectomy (2/3 PH) leads to a analysis was used for validation of the differentially expressed highly synchronized hepatocyte cell-cycle entry and progres- miRNAs at an early stage of liver regeneration, induced by sion. The first phase, known as the ‘priming phase’, occurs in 2/3 partial hepatectomy. TargetScan and Gene Ontology (GO) the first hours after PH and poises the hepatocytes to enter the analyses were performed in order to identify the possible G1 phase and to become receptive to growth factors. The second miRNA target genes and their ontology, respectively. A subset phase corresponds to an increased metabolic demand imposed of miRNAs was found to be differentially expressed during on the remnant liver. During this phase, among other metabolic liver regeneration. Mmu-miR-21 and mmu-miR-30b* showed changes, transient hypoglycemia is suggested to induce systemic the higher levels of up-regulation in liver tissue from the hepa- lipolysis followed by a lipid droplets accumulation in the hepa- tectomized mice at the end of the experiment (L12) compared tocytes. Between 36-42 h after 2/3 PH, most hepatocytes are to the sham operated mice (L0). Mmu-miR-21 up-regulation in the S phase of the cell cycle. The remnant liver lobes will was further confirmed by qPCR. In situ hybridization (ISH) compensate for lost tissue and recover the initial liver mass in revealed that mmu-miR-21 exhibited the higher levels of expres- less than 2 weeks. The last and much less studied step is the sion at 12 h post hepatectomy. On the contrary, mmu-miR-34c*, termination of liver regeneration. A large number of genes are mmu-miR-144, mmu-miR-207, mmu-miR-207, mmu-miR-451, involved in liver regeneration, but the essential circuitry required mmu-miR-582-3p and mmu-miR-290-5p exhibited <0.5 down- for the process may be categorized into 3 networks: cytokine, regulation in liver tissue after partial hepatectomy in L12 vs. L0 growth factor and metabolic. mice. The results from microarray and qPCR analyses were in MicroRNAs (miRNAs) are short regulatory RNAs of good agreement. In conclusion, our results provide important approximately 22 nucleotides in length (1). They bind to the information regarding the differentially expressed miRNAs in 3'-untranslated region (3'UTR) of target mRNA based on murine liver tissue before and after partial hepatectomy. The sequence complementarity and result in the degradation or suppression of translation of the target miRNA (2). Nevertheless, new findings reveal a new mode of action of miRNAs by which they may regulate gene expression, by binding not only to regions confined to the 3'UTR, but to the complete sequence Correspondence to: Dr Ioannis G. Habeos, Department of Internal (promoter, 5'UTR, CDS and 3'UTR) of a gene (3-5). It is thought Medicine, Medical School, University of Patras, 26504 Patras, that more than 30% of human genes are post-transcriptionally Greece regulated by miRNAs (6,7). There has been growing interest E-mail: [email protected] in understanding the specific role of miRNAs in regulating cellular processes, such as cell growth, proliferation, apoptosis, Key words: liver regeneration, mmu-miR-21, microRNA profiling, qPCR, in situ hybridization stress response and metabolism (8-10). There are previous studies implicating miRNAs in the regeneration of zebrafish fins (11), regeneration in planarian worms (12) and wound healing (13). Mice with hepatocyte- 748 CHAVELES et al: MicroRNA IMPLICATION IN MURINE LIVER REGENERATION specific inactivation of DGCR8, which is required for miRNA image analysis was carried out using the ImaGene 8.0 software biogenesis, have also been generated. They develop normally (BioDiscovery, Inc., El Segundo, CA). after adulthood but their hepatocytes fail to transit into the S phase after PH (7). In a recent study miR-21 was found to Microarray data filtering, background correction and normali- be up-regulated during the proliferative phase of liver regenera- zation. Filtering was performed based on the signal intensity. tion. MiR-21 has been shown to target Pellino-1 and inhibit the Spots with no signal above the background (flags 1 and 2) were NF-κB signalling (14). detected and removed. Background correction was performed Since recent studies have identified miRNAs as important to remove non-biological contributions (‘background’) to the regulators of gene expression, modulating critical cellular measured signal, by subtracting the median global background functions of hepatocytes, such as proliferation, apoptosis, lipid from the median local background from the signal intensity. metabolism and liver regeneration, we aimed to identify the A threshold of 1.5 (L12 vs. L0) was set as the cut-off value. differentially expressed miRNAs during an early phase of liver Normalization was performed using the Lowess regression regeneration. To this effect, the expression of liver miRNAs algorithm, in order to remove certain systematic biases from was investigated using miRNA profiling and qPCR, in total microarray data, such as dye effects or intensity dependence. RNA extracted from murine liver tissue 12 h after 2/3 PH. The positive effect from normalization is illustrated on each slide sheet with an M-A plot before and after normalization Materials and methods (Fig. 1). After normalization the spots appeared symmetrically scattered around the horizontal line (M=0). Normalized data Mice, PH and RNA isolation. Twenty male wild-type mice were extracted, pre-processed and sorted with Microsoft Excel®. C57BL6J (8 weeks old) fed a standard diet were used for the liver regeneration experiment. Mice were housed in the Slide quality check using spike-in controls. Spike-in co-efficient animal facility of the University of Patras Medical School in of variation (CV) values were calculated between 48 replicates temperature-, light- and humidity-controlled rooms with a 12-h for each of the spike-in controls on each slide. CVs did not light/dark cycle. All animal procedures were approved by the exceed 30%, thus reflecting lack of spatial effects on the array Institutional Review Board of the University of Patras Medical (smear across the slide) or misplacement of the grid for anno- School and were in accordance with EC Directive 86/609/EEC. tating the spots. A 2/3 PH was performed on 10 mice according to a previously published standardized protocol (15) whereby the median and Clustering. We clustered the miRNAs between the L0 and left lobes of the liver were removed and another 10 sham-oper- L12 murine livers, using hierarchical clustering (HCL) and ated mice were used as the control group. The liver was allowed k-means clustering, as previously reported (16,17). to regenerate and mice were sacrificed 12 h after operation. Liver samples were collected from the sham-operated mice (L0) Gene Expression Omnibus (GEO) accession numbers. and the hepatectomized mice at the end of the experiment (L12). Array data were deposited at the GEO (National Center for Biotechnology Information) with accession numbers Liver tissue RNA extraction. Liver tissue was excised from mice GSM862374 through GSM862375 (GSE35128). and was immediately submerged in RNAlater solution (Ambion, Foster City, CA). For miRNA profiling, total RNA was isolated Reverse transcription (RT) and qPCR validation. The miRNA from pooled livers from the L0 and L12 mice, whereas for qPCR in the total RNA sample was converted to cDNA by reverse experimentation, total RNA was prepared from individual liver transcription using the miRCURY LNA miRNA PCR system samples, using the TRIzol® reagent (Invitrogen, Carlsbad, CA) First-strand cDNA synthesis kit and a miRNA-specific primer. as the manufacturer instructed. The quality of the
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