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.
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