Computational prediction of MicroRNAs targeting GABA receptors and experimental verification of miR-181, miR-216 and miR-203 targets in GABA-A receptor

Background 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. Results 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. Conclusions 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.


Background
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][4][5][6]. The functions of GABA receptors in peripheral tissues are less studied. They may be involved in ion homeostasis [7], cell proliferation and differentiation [8], development [1], 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 [10]. The expression pattern of the GABA receptor π-subunit is regulated by various culture conditions and is consistent with the type II cell phenotypes [11]. We have further identified 19 subunits of the ionotropic GABA receptors in alveolar epithelial cells [6]. Their expression is dynamically changed during lung development [12]. 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: GABA A and GABA C as ligand-gated Clchannels, and GABA B receptor as a metabotropic receptor coupled to a heterotrimeric G-protein. GABA A and GABA C 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][15][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 [17].

Construction of microRNA expression vectors
Human microRNA expression vectors were constructed as previously described [22]. Mature microRNAs with the flanking sequences (~200 base pairs at each end) were PCR-amplified from human genomic DNA. The primers used for PCR amplification are listed in Table 1. The PCR products were inserted into a modified pLVX-Puro lenti-viral vector (Clontech) between CMV-driven enhanced green fluorescent protein (EGFP) and SV40 polyA terminal sequences.

Construction of 3'-UTR reporter vectors
The full length 3'-UTR or the microRNA binding sites in the 3'-UTR of rat GABA receptors were PCR-amplified and inserted into the pRL-TK vector containing a Renilla luciferase (Promega). The primers used for PCR amplification are listed in Table 2. 3'-UTR reporter assay HEK 293T cells (2 × 10 4 /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.
However, GABA C receptors are exclusively composed of ρ subunits in the form of homo-or hetero-pentamers.
To identify the microRNA that may potentially regulate GABA A and GABA C receptors, we used the online computer software, TargetScan (v 5.2) to predict the binding sites of microRNAs on the 3'UTR of rat GABA receptors. We chose rat GABA receptors because we use rats as most of our animal or cell models. Since our microRNA expression vectors use human sequences, we only queried conserved microRNA target sites among mammals based on conserved 8 mer and 7 mer sites that match the seed sequence of a microRNA. The results are listed in Table 3. Among α-subunits, we found that α1 had most of the microRNA binding sites. There were 6 binding sites for 6 microRNAs on α1 3'-UTR. For other α-subunits, we found miR-128, miR-27ab, let-7/miR-98 for α6. We did not find any micro-RNAs for α2, α4, and α5. There was no information available for α3.
We further utilized a recently developed software, miRWalk [33], to predict the miRNAs targeting 3'-UTR and open reading frame (ORF) of GABA receptors. The results are presented in Table 4. Obviously, this method is less stringent compared to TargetScan and miRanda, since the miRWalk query yielded 79 miRNAs for GABA receptor α1 subunit in comparison with only 6 by Tar-getScan and 29 by miRanda.
We selected two subunits, α1 and γ2 for experimental verification of the predictions. For α1-subunit, we constructed a 3'-UTR reporter vector, in which the 3'-UTR of α1-subunit was placed after a Renilla luciferase reporter gene (Table 2). For γ2-subunit, we cloned the predicted binding site of miR-103/107 into the downstream of a Renilla luciferase reporter gene. We then co-transfected a microRNA expression vector with the reporter into HEK293T cells to see whether the micro-RNA depressed the reporter activity. The firefly luciferase pGL3 vector was used for normalization. As shown in Figure 1, among the predicted microRNAs tested, miR-181, miR-216, and miR-203 inhibited the reporter activity. Four miR-181 isoforms, a-2, b-1, c and d-1 generated the same mature miR-181 and all of them depressed the reporter activity. All other microRNAs tested had no effects. The results suggest that miR-181, miR-216, and miR-203 are the micoRNAs that regulates GABA receptor α1-subunit. It is noted that miR-15, miR-16, miR-146, miR-221, miR-424 and miR-497 were predicated to target the GABA receptor α1-subunit by an earlier version (v.4.2) of TargetScan. Some of the miRNAs such as miR-181 are expressed in astrocytes naturally expressing GABA receptors [34]. For γ2-subunit, we did not find major effects of miR-103-1, miR-103-2, and miR-107 on the reporter activity ( Figure 2).
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 overexpressed 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   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 [35]. 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 [13]. 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.

Conclusions
In summary, computational approaches predict many microRNA binding sites on the 3'-UTR of GABA A receptors, but not on these of GABA C receptors. 3'-UTR reporter assays only verified miR-181, miR-216, Luciferase Activity GABRA1 Figure 1 Effect of the predicted microRNAs on the 3'-UTR reporter activity of GABA A receptor a1-subunit. HEK 293T cells were transfected with the reporter and microRNA expression vectors and dual luciferase activities were assayed. The results were expressed as a ratio to the control microRNA vector. Data shown are means ± S.D. *P < 0.05 v.s. control. n = 3. Student t-Test. 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.