- Research article
- Open Access
Computational prediction of MicroRNAs targeting GABA receptors and experimental verification of miR-181, miR-216 and miR-203 targets in GABA-A receptor
© Zhao et al; licensee BioMed Central Ltd. 2012
Received: 13 July 2011
Accepted: 9 February 2012
Published: 9 February 2012
GABA receptors are well known as the inhibitory receptors in the central nervous system and are also found in peripheral tissues. We have previously shown that GABA receptors are involved in lung development and fluid homeostasis. However, the microRNAs that regulate GABA receptors have not yet been identified.
In this study, we used the online software, TargetScan and miRanda, to query the microRNAs that directly target GABA receptors and then selected some of them to verify experimentally using 3'-UTR reporter assays. Computational approaches predict many microRNA binding sites on the 3'-UTR of GABAA receptors, but not on GABAC receptors. 3'-UTR reporter assays only verified miR-181, miR-216, and miR-203 as the microRNAs that target GABA receptor α1-subunit among 10 microRNAs tested.
Our studies reinforce that microRNA target prediction needs to be verified experimentally. The identification of microRNAs that target GABA receptors provides a basis for further studies of post-transcriptional regulation of GABA receptors.
GABA receptors are well known as the inhibitory receptors in the central nervous system [1, 2]. However, GABA receptors are also found in several peripheral tissues [3–6]. The functions of GABA receptors in peripheral tissues are less studied. They may be involved in ion homeostasis , cell proliferation and differentiation , development , and hormone secretion [5, 9].
We have initially identified GABA receptor π-subunit as a specific alveolar epithelial type II cell marker through DNA microarray analysis . The expression pattern of the GABA receptor π-subunit is regulated by various culture conditions and is consistent with the type II cell phenotypes . We have further identified 19 subunits of the ionotropic GABA receptors in alveolar epithelial cells . Their expression is dynamically changed during lung development . Functionally, GABA receptors play important roles in fluid homeostasis in the adult lung and fetal lung development [6, 13].
GABA receptors can be classified into two major types: GABAA and GABAC as ligand-gated Cl- channels, and GABAB receptor as a metabotropic receptor coupled to a heterotrimeric G-protein. GABAA and GABAC receptors share a conserved structure that contains a long extracellular N-terminal region, 4 transmembrane domains (TM1-TM4), a large intracellular loop between TM3 and TM4, and a short extracellular C-terminus [1, 2, 14–16]. The N-terminal segment is responsible for ligand binding and subunit assembly. The TM2 domain forms the lining of the ion pore. The intracellular loop is the site for post-translational modifications and binding with other proteins. This loop harbors a number of consensus phosphorylation sites for protein kinase A and C (PKA and PKC) and tyrosine kinases .
MicroRNAs are small non-coding RNAs. They form a ribonucleoprotein complex, termed RISC that cleaves mRNA or represses protein translation. MicroRNAs regulate various biological processes [18, 19]. Several microRNAs such as miR-17-92 cluster and miR-127 are involved in lung development [20–22]. MicoRNAs have also been implicated in many lung diseases including lung inflammation, Chronic Obstructive Pulmonary Disease, Asthma and Idiopathic Pulmonary Fibrosis [23–30]. Nevertheless, microRNAs that regulate GABA receptors have not yet been reported. In this study, we used online software, TargetScan (http://www.targetscan.org)  and mRanda (http://www.microrna.org)  to predict the microRNAs that possibly target to GABA receptors and then selected some of them to verify experimentally using 3'-UTR reporter assays. We found that miR-181, miR-23 and miR-216 target the GABA receptor α1-subunit.
Construction of microRNA expression vectors
Primers for miRNA expression vectors
Construction of 3'-UTR reporter vectors
Primers used for constructing 3'-UTR reporter vectors
3'-UTR or binding Sites
3'-UTR reporter assay
HEK 293T cells (2 × 104/well) were seeded in each well of a 96-well plate. After one day of culture, the cells were transfected with100 ng microRNA expression vector or control vector without miRNA insert, 2.5 ng 3'-UTR Renillia luciferase reporter vector and 15 ng pGL3 control vector (firefly luciferase reporter) using Lipofectamine. After a 2 day transfection, the cells were lyzed and dual luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The Renilla luciferase activities were normalized with firefly luciferase activity. Data was expressed as a ratio to the control vector without miRNA insert.
Results and discussion
Both GABAA and GABAC receptors are ligand-gated Cl- channels. However GABAC receptors have very unique ligand binding characteristics in comparison with GABAA and GABAB receptors, including a high sensitivity to the physiological ligand, GABA, insensitivity to bicuculline, barbiturates, and bacofen, very weak de-sensitization, a smaller single-channel conductance, and a longer open time [14–16]. Eight different subunits of GABAA and GABAC receptors (α1-6, β1-3, γ1-3, δ, θ, ε, π, and ρ1-3) have been identified. The assembly of a heteropentamer, with at least one α-, one β-, and one γ-subunit, forms functional GABAA receptor channels. δ-, θ-, ε-, and π-subunits can substitute for the γ-subunit. However, GABAC receptors are exclusively composed of ρ subunits in the form of homo- or hetero-pentamers.
Predicted microRNAs targeting rat GABA receptor subunits by TargetScan and miRanda
GABA receptor subunits
Entrez Gene symbol
Lengths of 3'-UTR in TargetScan (v5.2)
Conserved microRNAs targeting to GABA receptors predicted by TargetScan (v5.2)
Lengths of 3'-UTR in miRanda
Conserved microRNAs targeting to GABA receptors predicted by miRanda
miR-129 (2), miR-130b, miR-136, miR-137, miR-148b-3p,
miR-150, miR-152, miR-181a (2) bc (3) d, miR-182,
miR-186, miR-203 (2), miR-210, miR-216a, miR-26ab,
miR-30acde, miR-30b-5p, miR-320, miR-340-5p, miR-361,
miR-374, miR-375, miR-376c, miR-377, miR-384-5p,
miR-410, miR-433, miR-488 (2), miR-539, miR-874
miR-186, miR-200bc, miR-203, miR-429, miR-495
miR-124, miR-132, miR-133ab, miR-195, miR-212, miR-223,
miR-30acde, miR-30b-5p, miR-322, miR-346, miR-376c,
miR-378, miR-384-5p, miR-494 (2), miR-495, miR-539
miR-103, miR-107, miR-128, miR-143, miR-148b-3p,
30/384-5p (2), miR-103/107
miR-152, miR-30a (2) c (2) d (2) e (2), miR-30b-5p (2),
miR-384-5p (2), miR-411
miR-128, miR-199a-5p, miR-203, miR-33, miR-411, miR-485
miR-101, miR-19, miR-144,
miR-9 (2), miR-455/455-5p,
miR-122, miR-186, miR-199a-5p, miR-204, miR-210, miR-211,
miR-23ab, miR-27ab, miR-218
miR-23ab, miR-26ab, miR-320, miR-324-5p, miR-329,
miR-203, miR-218, miR-379, miR-410, miR-455, miR-488
miR-15b, miR-16, miR-195, miR-26ab, miR-322, miR-497
miR-145, miR-19ab, miR-24, miR-328, miR-365
For β-subunits, we found two binding sites for miR-30a/30a-5p/30b/30b-5p/30/384-5p and one binding site for miR-103/107. There were 15 binding sites for 15 microRNAs on β2 and 5 binding sites for 7 microRNAs on β3. For γ-subunits, we found two binding sites on γ2 and no binding sites on γ1 and γ3, There was only one binding site for ε and no binding sites on π-, δ-, θ-, ρ1- and ρ2-subunits. In general, the "common" subunits (α, β, and γ) had more miRNA target sites than the "rare" subunits (δ, θ, ε, π, and ρ). This is probably because these subunits had shorter 3'-UTRs, in particular for ρ-subunits.
We also used another software, miRanda to predict the microRNA that target to GABA receptors (Table 3). In general, miRanda predicted more microRNAs than TargetScan. There were some common microRNAs that were predicted by both software. For example, miR-137, miR-181, miR-203, and miR-216a for α1; miR-103, miR-107, miR-30, and miR-384-5p for β1; and miR-204, miR-211, miR-23, and miR-26 for β3.
Predicted microRNAs targeting 5'-UTR, ORF and 3'-UTR region using miRWalk software
GABA receptor subunits
Entrez Gene symbol
MicroRNAs targeting 5'-UTR
MicroRNAs targeting ORF
Numbers of microRNA targeting 3'-UTR with p-value < 0.05
miR-326, miR-28*, miR-29b-1*,
miR-341, miR-503, miR-150, miR-378
miR-539, miR-542-5p, miR-147,
miR-27b, miR-27a, miR-185,
miR-350, miR-431, miR-542-3p, miR-322, miR-323*,
miR-343, miR-346, miR-17-5p,
miR-140, miR-148b-3p, miR-29a*, miR-152, miR-497
miR-93, miR-128, miR-143,
miR-345, miR-22, miR-451,
miR-24-1*, miR-24-2*, let-7d, miR-346, miR-153,
miR-296, miR-376c, miR-466c
miR-126*, miR-743b, miR-323*, miR-330*, miR-21*,
miR-140, miR-351, miR-324-5p, miR-325-3p, miR-7a*,
miR-10a-5p, miR-125a-5p, miR-125b-5p, miR-376b-5p,
miR-20a*, miR-150, miR-297, miR-541
miR-300-5p, miR-350, miR-433, miR-881, miR-672,
miR-497, miR-322, miR-103-2,
miR-182, miR-216a, miR-483, miR-327, miR-338,
miR-205, miR-296, miR-320, miR-880
miR-182, miR-483, miR-382, miR-505
miR-142-3p, miR-298, miR-15b, miR-16, miR-28,
miR-34a, miR-195, miR-214, miR-290, miR-449a,
miR-345-5p, miR-199a-3p, miR-873
miR-322, miR-338, miR-193, miR-370, miR-497, miR-873
miR-485, miR-484, miR-342-3p, miR-344-5p, miR-223,
miR-671, miR-322, miR-24, miR-139-3p, miR-199a-5p,
miR-298, miR-483, miR-497, miR-743b, miR-672
miR-350, miR-34c, miR-92a, miR-92a, miR-300-5p,
miR-92b, miR-7a*, miR-32
miR-338, let-7d, miR-204*, miR-421, miR-672,
miR-873, miR-134, miR-210,
miR-218*, let-7d, miR-34a, miR-204*, miR-421,
miR-449a, miR-431, miR-381, miR-674-3p
miR-350, miR-30c-1*, miR-30c-
miR-338, miR-341, miR-23a*, miR-143, miR-384-5p,
2*, miR-148b-3p, miR-152,
miR-324-3p, miR-30c, miR-30e, miR-30b-5p, miR-30d,
miR-872*, miR-874, miR-672
miR-30a, miR-204*, miR-539, miR-742, miR-873
It should be noted that we did not measure miRNA levels in the miRNA-overexpressed cells. Thus, there are possibilities that some of miRNAs may not be over-expressed in the experimental set-up; particularly for these miRNAs that had no effect on 3'-UTR reporter activity. However, the transfection efficiency is 90-100% under our experimental conditions based on the GFP reporter expression encoded in the same miRNA expression vector. Additionally, the effect of a miRNA on the luciferase activity does not necessarily mean that it was a direct effect on the binding of a miRNA to the 3'-UTR reporter construct. A miRNA could have indirect effects. The mutations of seed sequences in the miRNA binding sites are needed to exclude indirect effects. Further studies are also needed to see whether the overexpression of miR-181, miR-203, and miR-216 in a physiologically relevant cell type modifies GABA receptor expression, and whether these miRNAs are differentially regulated in diseased states.
It is also interesting to note that miR-15b and miR-146a/b actually increased the 3'-UTR reporter activity. It has been reported that miRNA increases translation . However, it is also possible that this is a result of indirect effects.
We have previously shown that the activation of GABA receptors promotes fetal lung development . The inhibition of miRNAs that target GABA receptors may increase receptor density and thus sensitivity of GABA receptors, which may benefit the development of therapy in treating diseases related to developmental anomalies.
In summary, computational approaches predict many microRNA binding sites on the 3'-UTR of GABAA receptors, but not on these of GABAC receptors. 3'-UTR reporter assays only verified miR-181, miR-216, and miR-203 as the microRNAs that target GABA receptor α1-subunit among 10 microRNAs tested. These studies reinforce that micoRNA target prediction needs to be verified experimentally. The identification of microRNAs that target to GABA receptors provides a basis for further studies of post-transcriptional regulation of GABA receptors.
We thank Ms. Tazia Cook for editorial assistance. This work was supported by the National Institutes of Health, HL-087884 and HL-095383 and OCAST, AR101-037.
- Ben Ari Y: Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci. 2002, 3: 728-739.PubMedView ArticleGoogle Scholar
- Owens DF, Kriegstein AR: Is there more to GABA than synaptic inhibition?. Nat Rev Neurosci. 2002, 3: 715-727. 10.1038/nrn919.PubMedView ArticleGoogle Scholar
- Akinci MK, Schofield PR: Widespread expression of GABA(A) receptor subunits in peripheral tissues. Neurosci Res. 1999, 35: 145-153. 10.1016/S0168-0102(99)00078-4.PubMedView ArticleGoogle Scholar
- Glassmeier G, Hopfner M, Buhr H, Lemmer K, Riecken EO, Stein H, Quabbe HJ, Rancso C, Wiedenmann B, Scherubl H: Expression of functional GABAA receptors in isolated human insulinoma cells. Ann N Y Acad Sci. 1998, 859: 241-248. 10.1111/j.1749-6632.1998.tb11138.x.PubMedView ArticleGoogle Scholar
- Park HS, Park HJ: Effects of gamma-aminobutyric acid on secretagogue-induced exocrine secretion of isolated, perfused rat pancreas. Am J Physiol Gastrointest Liver Physiol. 2000, 279: G677-G682.PubMedGoogle Scholar
- Jin N, Kolliputi N, Gou D, Weng T, Liu L: A novel function of ionotropic gamma-aminobutyric acid receptors involving alveolar fluid homeostasis. J Biol Chem. 2006, 281: 36012-36020. 10.1074/jbc.M606895200.PubMedView ArticleGoogle Scholar
- Limmroth V, Lee WS, Moskowitz MA: GABAA-receptor-mediated effects of progesterone, its ring-A-reduced metabolites and synthetic neuroactive steroids on neurogenic oedema in the rat meninges. Br J Pharmacol. 1996, 117: 99-104.PubMedPubMed CentralView ArticleGoogle Scholar
- Mong JA, Nunez JL, McCarthy MM: GABA mediates steroid-induced astrocyte differentiation in the neonatal rat hypothalamus. J Neuroendocrinol. 2002, 14: 45-55. 10.1046/j.1365-2826.2002.00737.x.PubMedView ArticleGoogle Scholar
- Mayerhofer A, Hohne-Zell B, Gamel-Didelon K, Jung H, Redecker P, Grube D, Urbanski HF, Gasnier B, Fritschy JM, Gratzl M: Gamma-aminobutyric acid (GABA): a para- and/or autocrine hormone in the pituitary. FASEB J. 2001, 15: 1089-1091.PubMedGoogle Scholar
- Chen Z, Jin N, Narasaraju T, Chen J, McFarland LR, Scott M, Liu L: Identification of two novel markers for alveolar epithelial type I and II cells. Biochem Biophys Res Commun. 2004, 319: 774-780. 10.1016/j.bbrc.2004.05.048.PubMedView ArticleGoogle Scholar
- Jin N, Narasaraju T, Kolliputi N, Chen J, Liu L: Differential expression of GABA(A) receptor pi subunit in cultured rat alveolar epithelial cells. Cell Tissue Res. 2005, 321: 173-183. 10.1007/s00441-005-1130-8.PubMedView ArticleGoogle Scholar
- Jin N, Guo Y, Sun P, Bell A, Chintagari NR, Bhaskaran M, Rains K, Baviskar P, Chen Z, Weng T, Liu L: Ionotropic GABA receptor expression in the lung during development. Gene Expr Patterns. 2008, 8: 397-403. 10.1016/j.gep.2008.04.008.PubMedPubMed CentralView ArticleGoogle Scholar
- Chintagari NR, Jin N, Gao L, Wang Y, Xi D, Liu L: Role of GABA receptors in fetal lung development in rats. PLoS One. 2010, 5: e14171-10.1371/journal.pone.0014171.PubMedPubMed CentralView ArticleGoogle Scholar
- Bormann J: The 'ABC' of GABA receptors. Trends Pharmacol Sci. 2000, 21: 16-19. 10.1016/S0165-6147(99)01413-3.PubMedView ArticleGoogle Scholar
- Enz R, Cutting GR: Molecular composition of GABAC receptors. Vision Res. 1998, 38: 1431-1441. 10.1016/S0042-6989(97)00277-0.PubMedView ArticleGoogle Scholar
- Zhang D, Pan ZH, Awobuluyi M, Lipton SA: Structure and function of GABA(C) receptors: a comparison of native versus recombinant receptors. Trends Pharmacol Sci. 2001, 22: 121-132. 10.1016/S0165-6147(00)01625-4.PubMedView ArticleGoogle Scholar
- Moss SJ, Smart TG: Modulation of amino acid-gated ion channels by protein phosphorylation. Int Rev Neurobiol. 1996, 39: 1-52.PubMedView ArticleGoogle Scholar
- Bushati N, Cohen SM: microRNA functions. Annu Rev Cell Dev Biol. 2007, 23: 175-205. 10.1146/annurev.cellbio.23.090506.123406.PubMedView ArticleGoogle Scholar
- Croce CM: Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009, 10: 704-714. 10.1038/nrg2634.PubMedPubMed CentralView ArticleGoogle Scholar
- Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL: Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol. 2007, 310: 442-453. 10.1016/j.ydbio.2007.08.007.PubMedPubMed CentralView ArticleGoogle Scholar
- Carraro G, El-Hashash A, Guidolin D, Tiozzo C, Turcatel G, Young BM, De Langhe SP, Bellusci S, Shi W, Parnigotto PP, Warburton D: miR-17 family of microRNAs controls FGF10-mediated embryonic lung epithelial branching morphogenesis through MAPK14 and STAT3 regulation of E-Cadherin distribution. Dev Biol. 2009, 333: 238-250. 10.1016/j.ydbio.2009.06.020.PubMedPubMed CentralView ArticleGoogle Scholar
- Bhaskaran M, Wang Y, Zhang H, Weng T, Baviskar P, Guo Y, Gou D, Liu L: MicroRNA-127 modulates fetal lung development. Physiol Genomics. 2009, 37: 268-278. 10.1152/physiolgenomics.90268.2008.PubMedPubMed CentralView ArticleGoogle Scholar
- Kumar MS, Erkeland SJ, Pester RE, Chen CY, Ebert MS, Sharp PA, Jacks T: Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc Natl Acad Sci USA. 2008, 105: 3903-3908. 10.1073/pnas.0712321105.PubMedPubMed CentralView ArticleGoogle Scholar
- Izzotti A, Calin GA, Arrigo P, Steele VE, Croce CM, De FS: Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke. FASEB J. 2009, 23: 806-812. 10.1096/fj.08-121384.PubMedPubMed CentralView ArticleGoogle Scholar
- Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E: miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proc Natl Acad Sci USA. 2009, 106: 15819-15824. 10.1073/pnas.0901216106.PubMedPubMed CentralView ArticleGoogle Scholar
- Sato T, Liu X, Nelson A, Nakanishi M, Kanaji N, Wang X, Kim M, Li Y, Sun J, Michalski J, Patil A, Basma H, Holz O, Magnussen H, Rennard SI: Reduced miR-146a increases prostaglandin Ein chronic obstructive pulmonary disease fibroblasts. Am J Respir Crit Care Med. 2010, 182: 1020-1029. 10.1164/rccm.201001-0055OC.PubMedPubMed CentralView ArticleGoogle Scholar
- Polikepahad S, Knight JM, Naghavi AO, Oplt T, Creighton CJ, Shaw C, Benham AL, Kim J, Soibam B, Harris RA, Coarfa C, Zariff A, Milosavljevic A, Batts LM, Kheradmand F, Gunaratne PH, Corry DB: Proinflammatory role for let-7 microRNAS in experimental asthma. J Biol Chem. 2010, 285: 30139-30149. 10.1074/jbc.M110.145698.PubMedPubMed CentralView ArticleGoogle Scholar
- Pandit KV, Corcoran D, Yousef H, Yarlagadda M, Tzouvelekis A, Gibson KF, Konishi K, Yousem SA, Singh M, Handley D, Richards T, Selman M, Watkins SC, Pardo A, Ben-Yehudah A, Bouros D, Eickelberg O, Ray P, Benos PV, Kaminski N: Inhibition and role of let-7d in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 2010, 182: 220-229. 10.1164/rccm.200911-1698OC.PubMedPubMed CentralView ArticleGoogle Scholar
- Cushing L, Kuang PP, Qian J, Shao F, Wu J, Little F, Thannickal VJ, Cardoso WV, Lu J: MIR-29 is a major regulator of genes associated with pulmonary fibrosis. Am J Respir Cell Mol Biol. 2010, 45: 287-294.PubMedPubMed CentralView ArticleGoogle Scholar
- Liu G, Friggeri A, Yang Y, Milosevic J, Ding Q, Thannickal VJ, Kaminski N, Abraham E: miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis. J Exp Med. 2010, 207: 1589-1597. 10.1084/jem.20100035.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhang B, Pan X, Anderson TA: MicroRNA: a new player in stem cells. J Cell Physiol. 2006, 209: 266-269. 10.1002/jcp.20713.PubMedView ArticleGoogle Scholar
- Betel D, Wilson M, Gabow A, Marks DS, Sander C: The microRNA.org resource: targets and expression. Nucleic Acids Res. 2008, 36: D149-D153.PubMedPubMed CentralView ArticleGoogle Scholar
- Dweep H, Sticht C, Pandey P, Gretz N: miRWalk-Database: prediction of possible miRNA binding sites by "walking" the genes of three genomes. J Biomed Inform. 2011, 44: 839-847. 10.1016/j.jbi.2011.05.002.PubMedView ArticleGoogle Scholar
- Ouyang YB, Lu Y, Yue S, Giffard RG: miR-181 targets multiple Bcl-2 family members and influences apoptosis and mitochondrial function in astrocytes. Mitochondrion. 2011, [http://dx.doi.org/10.1016/j.mito.2011.09.001]Google Scholar
- Vasudevan S, Tong Y, Steitz JA: Switching from repression to activation: microRNAs can up-regulate translation. Science. 2007, 318: 1931-1934. 10.1126/science.1149460.PubMedView ArticleGoogle Scholar
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