Altered expression of microRNAs in the myocardium of rats with acute myocardial infarction
© Shi et al; licensee BioMed Central Ltd. 2010
Received: 30 October 2009
Accepted: 1 March 2010
Published: 1 March 2010
MicroRNAs(miRNAs) are important cellular components and their dysfunction is associated with various diseases. Acute myocardial infarction (AMI) is one of the most serious cardiovascular diseases. Although several miRNAs are reported to be associated with AMI, more novel miRNAs are needed to further investigate and improve certainty
We applied a well-established acute myocardial infarction rat model and performed miRNAs microarray experiments upon the myocardium tissue of rats with AMI and under sham control. We identified the differentially expressed miRNAs and analyzed the function of miRNA targets, transcription factors, and host genes based on bioinformatics.
As a result, the levels of expression of seventeen miRNAs significantly deregulated, of which four miRNAs were further validated by qRT-PCR. In addition, we observed that the transcription factors, targets, and host genes of these deregulated miRNAs are enriched in cardiovascular-related functions.
We found that the miRNAs expression level altered in rats with AMI and differentially expressed miRNAs may be novel biomarkers of AMI.
MicroRNAs (miRNAs) are small non-coding RNAs that represent one of the most important cellular components. Increasing evidence indicates that miRNAs have distinct expression profiles and play crucial roles in the regulation of gene expression during development stages such as cell growth, differentiation, and apoptosis. Deregulation of miRNAs is associated with various diseases including cardiovascular diseases [1–4]. In recent years, efforts have been made to identify the miRNAs associated with cardiovascular diseases . For example, miRNAs involved in cardiac hypertrophy and heart failure such as miR-208, miR-133, miR-195, miR-21, and miR-126 have been reported in several studies [5–8].
Acute myocardial infarction (AMI) is one of the most serious cardiovascular diseases . Recently, some miRNAs for example miR-21, miR-1, miR-216, and miR-29 family, were reported to be deregulated in myocardial infarction. However, considering the complex genetic variants in AMI, further investigation is needed to ascertain if more novel miRNAs are associated with AMI.
In this study, to explore the expression of miRNA in ischemic myocardium in rat with AMI, we employed microarray technique on the border zone myocardium of rats with AMI and sham-operated controls. As a result, we found that the levels of expression of seventeen miRNAs significantly changed in the progression of AMI. Furthermore, some of the deregulated miRNAs were confirmed by qRT-PCR, and the functions of miRNA targets, transcription factors, and host genes were also analyzed.
A rat model of AMI
We utilized an in vivo rat model to study altered expression of miRNA in the border zone myocardium of rats with AMI. Experimental acute myocardial infarction models were induced by the ligation of anterior descending coronary artery in adult Spraque-Dawley rats (180 to 200 gram), as described by Shyu et al. . On the first postoperative day, the survivals were randomly divided into three groups: 2 days after AMI, 7 days after AMI, 14 days after AMI. In the sham-control group, rats were exposed to all surgical procedures except the ligation of the anterior descending coronary artery. Each group has five individual samples. This study was approved by the Institutional Laboratory Animal Care and Use Committee of Peking University Health Science Center (Peking, China). The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the China National Institute of Health.
For miRNAs microarray analysis, total RNA from the myocardial tissues in the border zone of infracted myocardium was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Low-molecular-weight RNA (<200 nt) was isolated from the 30 μg total RNA using a mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). Profiling of miRNAs expression was analyzed by CapitalBio (CapitalBio Corp, Peking, China) using CapitalBio Mammalian miRNA Array V3.0.(Which includes 924 known miRNAs for human, mouse and rat). The miRNA microarray from CapitalBio Corporation was a double-channel fluorescent chip. Experimental procedures were performed as described in detail on the website of CapitalBio http://www.capitalbio.com. The T4 RNA ligase labeling method according to Thomson's protocol was adopted. Briefly, 4 ug of low-molecular-weight RNA was labeled with 500 ng 5'-phosphate-cytidyl-uridyl-cy3/5-3' (Dharmacon) with 2 units T4 RNA ligase (NEB). The labeling reaction was performed at 4°C for 2 hours. Labeled RNA was precipitated with 0.3 M sodium acetate, 2.5 volumes ethanol and resuspended in 15 μl of hybridization buffer containing 3× SSC, 0.2% SDS and 15% formamide. The array was hybridized at 42°C overnight and washed with two consecutive solutions (0.2% SDS, 2× SSC at 42°C for 5 min, and 0.2% SSC for 5 min at room temperature). The miRNAs Arrays were scanned with a confocal LuxScan™ scanner (CapitalBio Corp, Peking, China).
Intensity data was extracted from the TIFF images using LuxScan™ 3.0 software (CapitalBio Corp, Peking, China). The low-intensity spots in which fewer than 30% of the signal pixels exceeded the median background plus two times its standard deviation were removed. Then normalization was performed based on mean array intensity for inter-array comparison. To determine the significant differentially expressed miRNAs, Significance Analysis of Microarrays (SAM, version 3.0, from Stanford University) was performed using a two class unpaired comparison in the SAM procedure . miRNAs with adjusted P values less than 0.05 and with either a greater than 2.0-fold change or less than a 0.5-fold fold change in expression compared with controls were identified as differentially regulated miRNAs[15, 16]. The entire datasets (for miRNAs) described here are available from the Gene Expression Omnibus(GEO, http://www.ncbi.nlm.nih.gov/geo/) through series accession number GSE19695.
Quantitative reverse trancriptase-polymerase chain reaction (qRT-PCR)
To validate the microarray results in this study, four of the de-regulated miRNAs were randomly selected for further validation by qRT-PCR.
Since miRNAs are ~ 22 nucleotides in length, it could not be reverse transcribed according to traditional method. So we designed a stem-loop RT primer to induce a reverse transcribe reaction. For PCR reaction, Gene-specific anti-sense primers and a universal sense primer were designed according to miRNAs sequences. All primers used in this study were synthesized at Invitrogen (Invitrogen, Peking, China). All the primer sequences were listed in an additional file (see Additional file 1).
Each reaction mixture of RT contained RNA samples including about 100 ng of purified total RNA, 50 nM stem-loop RT primer, 1× RT buffer, 0.25 mM each of dNTPs(Takara), 0.1 mM DTT, 200 U Superscript II reverse transcriptase (Invitrogen), and 20 U RNase inhibitor(Takara). The 20 uL reactions were incubated for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then held at 4°C.
Quantitative Real-Time PCR was performed using the Roche 1.2 real-time PCR instrument (Roche). The expression of the U6 small nucleolar RNA gene was used as an internal control.
The PCR was performed in triplicate, the 20 uL PCR reaction contained: 1 μL of cDNA solution, 2 uL LightCycler-FastStart DNA Master SYBR Green I (Roche), and 0.3 uM of each primer. The reactions were incubated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s. Samples were also run on a 1.5% agarose gel to confirm specificity.
The relative expression level for each miRNA was calculated using the comparative CT method [18, 19]. To account for possible differences in the amount of starting RNA, miRNA expression was normalized to small nucleolar RNA U6.
Transcription factors (TFs) and targets of miRNAs
The transcription of miRNAs is regulated by TFs and miRNAs exert their functions by regulating target mRNAs. Therefore, the TFs and targets of miRNAs may provide clues to the functions of miRNAs. We obtained experimentally validated miRNA targets from TarBase . As a result, 9 of the 17 deregulated miRNAs having one or more targets were found. In total, 195 miRNA-target pairs were identified (see Additional File 2). We obtained the TFs that regulate the 17 deregulated miRNAs from TransmiR. As a result, we identified 14 experimentally validated TF-miRNA pairs, in which 14 unique TFs regulate 6 of the 17 deregulated miRNAs (see Additional File 3).
Gene function enrichment analysis
We carried out gene function enrichment analysis for miRNA transcription factors, miRNA targets, and miRNA host genes using EASE. We chose all human genes as the background gene list. Bonferroni corrections to P values were used in the analysis.
Altered expression of miRNAs in the myocardium of rats with AMI
miRNAs differentially expressed in the myocardial tissues of rats with acute myocardial infarction.
Tissues in which miRNA is most highly expressed tissue
Confirmation of the altered expression of miRNAs by qRT-PCR
Enriched functions of TFs and targets of miRNAs
We analyzed using EASE the enriched functions of TFs that were regulating the 17 deregulated miRNAs and the enriched functions of the targets of the 17 deregulated miRNAs . As a result, both sets of genes were enriched in cardiovascular-associated functions (see Additional File 5 and File 6). For example, the significant gene functions for miRNA TFs include "myogenesis" (Bonferroni P = 1.59 × 10-4) and "muscle development" (Bonferroni P = 9.21 × 10-4); the significant gene functions for miRNA targets include "muscle thin filament tropomyosin" (Bonferroni P = 5.93 × 10-3) and "striated muscle thin filament" (Bonferroni P = 5.85 × 10-2). This result indicates that the deregulated miRNAs tend to have roles in cardiovascular disease by coupling with cardiovascular function-associated TFs and target genes.
Deregulated miRNAs that are located in the introns of protein-coding genes and host genes as well as the functions of host genes.
Function of host gene
UNC45B plays a role in myoblast fusion and sarcomere organization
blood vessel development; angiogenesis; and vasculogenesis
filopodium formation; centronuclear myopathy; growth and development of megakaryocytes
filopodium formation; centronuclear myopathy; growth and development of megakaryocytes
cardiac muscle, striated muscle contraction, striated muscle thick filament
miRNA regulates gene expression by binding and modulating the translation of specific miRNAs. In this study, we identified 17 deregulated miRNAs in a rat model of AMI. Our data shows that the change of expression levels of miRNAs is in accordance with the progression of AMI. In the early stage of myocardial infarction, the pathological change in the myocardium occurs with inflammation and intracellular and interstitial myocardial edema. Other pathological changes such as hypoxia, myocardial necrosis, fibrosis are secondary. Some deregulated miRNAs that respond to AMI are reported to be associated with hypoxia, cell differentiation, inflammation, development, and fibrosis [1, 5, 7, 23, 25]. These results suggest that miRNAs have a critical role in cardiac remodeling after AMI. Furthermore, miRNAs highly expressed in cardiovascular tissues tend to be down-regulated after AMI, indicating that a high level of expression is needed for these miRNAs to maintain the normal functions of the cardiovascular system. This suggests that miRNAs may have important roles not only in the normal development of the cardiovascular system but also in cardiovascular diseases.
Finally, function enrichment analyses was carried out upon TFs, targets, and host genes of the deregulated miRNAs. All these gene sets are enriched in cardiovascular functions, providing more solid evidence of the associations between these deregulated miRNAs and acute myocardial infarction. Although it is still unclear how these deregulated miRNAs exert their effects in the progression of myocardial infarction.
In conclusion, we identified some miRNAs that showed altered expressions in rat myocardium with acute myocardial infarction. These miRNAs may be potential biomarkers for the diagnosis and treatment of acute myocardial infarction. Furthermore, investigating how these miRNAs exert their effects in acute myocardial infarction may become increasingly important.
This work was supported by State Basic Research Development Program of People's Republic of China (No. 2007CB512100).
- Latronico MV, Catalucci D, Condorelli G: Emerging role of microRNAs in cardiovascular biology. Circ Res. 2007, 101 (12): 1225-1236. 10.1161/CIRCRESAHA.107.163147.View ArticlePubMedGoogle Scholar
- Calin GA, Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer. 2006, 6 (11): 857-866. 10.1038/nrc1997.View ArticlePubMedGoogle Scholar
- Esquela-Kerscher A, Slack FJ: Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer. 2006, 6 (4): 259-269. 10.1038/nrc1840.View ArticlePubMedGoogle Scholar
- Lu M, Zhang Q, Deng M, Miao J, Guo Y, Gao W, Cui Q: An analysis of human microRNA and disease associations. PLoS ONE. 2008, 3 (10): e3420-10.1371/journal.pone.0003420.View ArticlePubMedPubMed CentralGoogle Scholar
- Harris TA, Yamakuchi M, Ferlito M, Mendell JT, Lowenstein CJ: MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1. Proc Natl Acad Sci USA. 2008, 105 (5): 1516-1521. 10.1073/pnas.0707493105.View ArticlePubMedPubMed CentralGoogle Scholar
- van Rooij E, Sutherland LB, Liu N, Williams AH, McAnally J, Gerard RD, Richardson JA, Olson EN: A signature pattern of stress-responsive microRNAs that can evoke cardiac hypertrophy and heart failure. Proc Natl Acad Sci USA. 2006, 103 (48): 18255-18260. 10.1073/pnas.0608791103.View ArticlePubMedPubMed CentralGoogle Scholar
- van Rooij E, Sutherland LB, Qi X, Richardson JA, Hill J, Olson EN: Control of stress-dependent cardiac growth and gene expression by a microRNA. Science. 2007, 316 (5824): 575-579. 10.1126/science.1139089.View ArticlePubMedGoogle Scholar
- Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, Muth AN, Tsuchihashi T, McManus MT, Schwartz RJ, Srivastava D: Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell. 2007, 129 (2): 303-317. 10.1016/j.cell.2007.03.030.View ArticlePubMedGoogle Scholar
- Ozaki K, Sato H, Inoue K, Tsunoda T, Sakata Y, Mizuno H, Lin TH, Miyamoto Y, Aoki A, Onouchi Y, et al: SNPs in BRAP associated with risk of myocardial infarction in Asian populations. Nat Genet. 2009, 41 (3): 329-333. 10.1038/ng.326.View ArticlePubMedGoogle Scholar
- Shan ZX, Lin QX, Fu YH, Deng CY, Zhou ZL, Zhu JN, Liu XY, Zhang YY, Li Y, Lin SG, et al: Upregulated expression of miR-1/miR-206 in a rat model of myocardial infarction. Biochem Biophys Res Commun. 2009, 381 (4): 597-601. 10.1016/j.bbrc.2009.02.097.View ArticlePubMedGoogle Scholar
- van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA, Olson EN: Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci USA. 2008, 105 (35): 13027-13032. 10.1073/pnas.0805038105.View ArticlePubMedPubMed CentralGoogle Scholar
- Shyu KG, Wang MT, Wang BW, Chang CC, Leu JG, Kuan P, Chang H: Intramyocardial injection of naked DNA encoding HIF-1alpha/VP16 hybrid to enhance angiogenesis in an acute myocardial infarction model in the rat. Cardiovasc Res. 2002, 54 (3): 576-583. 10.1016/S0008-6363(02)00259-6.View ArticlePubMedGoogle Scholar
- Thomson JM, Parker J, Perou CM, Hammond SM: A custom microarray platform for analysis of microRNA gene expression. Nat Methods. 2004, 1 (1): 47-53. 10.1038/nmeth704.View ArticlePubMedGoogle Scholar
- Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA. 2001, 98 (9): 5116-5121. 10.1073/pnas.091062498.View ArticlePubMedPubMed CentralGoogle Scholar
- Tan KS, Armugam A, Sepramaniam S, Lim KY, Setyowati KD, Wang CW, Jeyaseelan K: Expression profile of microRNAs in young stroke patients. PLos One. 2009, 4 (11): e7689-10.1371/journal.pone.0007689.View ArticlePubMedPubMed CentralGoogle Scholar
- Deng X, Nguyen DK, Hansen RS, Van Dyke DL, Gartler SM, Disteche CM: Dosage regulation of the active x chromosome in human triploid cells. PLos Genet. 2009, 5 (12): e1000751-10.1371/journal.pgen.1000751.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ: Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005, 33 (20): e179-10.1093/nar/gni178.View ArticlePubMedPubMed CentralGoogle Scholar
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29 (9): e45-10.1093/nar/29.9.e45.View ArticlePubMedPubMed CentralGoogle Scholar
- Tichopad A, Dilger M, Schwarz G, Pfaffl MW: Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res. 2003, 31 (20): e122-10.1093/nar/gng122.View ArticlePubMedPubMed CentralGoogle Scholar
- Papadopoulos GL, Reczko M, Simossis VA, Sethupathy P, Hatzigeorgiou AG: The database of experimentally supported targets: a functional update of TarBase. Nucleic Acids Res. 2009, D155-158. 10.1093/nar/gkn809. 37 Database
- Wang J, Lu M, Qiu C, Cui Q: TransmiR: a transcription factor-microRNA regulation database. Nucleic Acids Res. 2010, D119-D122. 10.1093/nar/gkp803. 38 Database
- Hosack DA, Dennis G, Sherman BT, Lane HC, Lempicki RA: Identifying biological themes within lists of genes with EASE. Genome Biol. 2003, 4 (10): R70-10.1186/gb-2003-4-10-r70.View ArticlePubMedPubMed CentralGoogle Scholar
- Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, Bozzoni I: A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis. Cell. 2005, 123 (5): 819-831. 10.1016/j.cell.2005.09.023.View ArticlePubMedGoogle Scholar
- Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, Brummelkamp TR, Fleming MD, Camargo FD: Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature. 2008, 451 (7182): 1125-1129. 10.1038/nature06607.View ArticlePubMedGoogle Scholar
- Joost P Sluijter AVM, Piet van Vliet, Corina H Metz, Pieter A Doevendans, Marie-Jose Goumans: Microrna-499 Regulates (cardio)myocyte Differentiation in Human-Derived Cardiomyocyte Progenitor Cells. Circulation. 2008, 118 (S_239):
- Liang Y, Ridzon D, Wong L, Chen C: Characterization of microRNA expression profiles in normal human tissues. BMC Genomics. 2007, 8: 166-10.1186/1471-2164-8-166.View ArticlePubMedPubMed CentralGoogle Scholar
- Baskerville S, Bartel DP: Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA. 2005, 11 (3): 241-247. 10.1261/rna.7240905.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/10/11/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.