Overexpression of miR-128 specifically inhibits the truncated isoform of NTRK3 and upregulates BCL2 in SH-SY5Y neuroblastoma cells
© Guidi et al; licensee BioMed Central Ltd. 2010
Received: 29 March 2010
Accepted: 10 December 2010
Published: 10 December 2010
Neurotrophins and their receptors are key molecules in the regulation of neuronal differentiation and survival. They mediate the survival of neurons during development and adulthood and are implicated in synaptic plasticity. The human neurotrophin-3 receptor gene NTRK3 yields two major isoforms, a full-length kinase-active form and a truncated non-catalytic form, which activates a specific pathway affecting membrane remodeling and cytoskeletal reorganization. The two variants present non-overlapping 3'UTRs, indicating that they might be differentially regulated at the post-transcriptional level. Here, we provide evidence that the two isoforms of NTRK3 are targeted by different sets of microRNAs, small non-coding RNAs that play an important regulatory role in the nervous system.
We identify one microRNA (miR-151-3p) that represses the full-length isoform of NTRK3 and four microRNAs (miR-128, miR-485-3p, miR-765 and miR-768-5p) that repress the truncated isoform. In particular, we show that the overexpression of miR-128 - a brain enriched miRNA - causes morphological changes in SH-SY5Y neuroblastoma cells similar to those observed using an siRNA specifically directed against truncated NTRK3, as well as a significant increase in cell number. Accordingly, transcriptome analysis of cells transfected with miR-128 revealed an alteration of the expression of genes implicated in cytoskeletal organization as well as genes involved in apoptosis, cell survival and proliferation, including the anti-apoptotic factor BCL2.
Our results show that the regulation of NTRK3 by microRNAs is isoform-specific and suggest that neurotrophin-mediated processes are strongly linked to microRNA-dependent mechanisms. In addition, these findings open new perspectives for the study of the physiological role of miR-128 and its possible involvement in cell death/survival processes.
Neurotrophins are a family of growth factors that play important roles in the nervous system. They exert multiple functions, being crucial for the survival and maintenance of the central and peripheral nervous system as well as in axon and dendrite patterning. Recent evidence has shown that neurotrophins also act as modulators in synaptic plasticity and are consequently involved in cognitive processes, learning and memory formation .
In mammals, the neurotrophin family is composed of four members: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4/5). Each member binds with high affinity to a specific neurotrophic tyrosine kinase (NTRK) receptor: NGF is the preferred ligand for NTRK1 (TrkA), BDNF and NT4/5 for NTRK2 (TrkB), and NT3 for NTRK3 (TrkC) . Upon neurotrophin-induced stimulation, NTRK receptors can activate the Ras/MAPK pathway, the PI3K (phosphoinositide 3-kinase) pathway, and/or PLC-γ1 (phospholipase C gamma 1)-dependent signaling, respectively promoting cell survival, differentiation and activity-dependent plasticity.
Neurotrophins exert neuroprotective activity against different paradigms of neuronal cell death [3–5], linking neurotrophic factors, in particular NGF, to neurodegenerative disorders . Neurotrophins and their receptors have also been implicated in the etiology of psychiatric and mood disorders, often in a dosage-dependent manner . As for many other growth factors, the deregulation of neurotrophin signal transduction is involved in several types of cancers, where NTRK receptor activation can either support or suppress tumor growth. This is the case for example of NTRK3, which is highly expressed in neuroblastomas with good prognosis and highly correlates with patient survival .
The human NTRK3 gene is located on chromosome 15q25 and spans ~380 kb of genomic DNA (chr15: 86,220,992-86,600,665, March 2006, hg18). It contains 19 introns and undergoes alternative splicing. In humans, three transcript variants have been well characterized: a full-length catalytic form (NM_002530) containing a tyrosine kinase (TK) domain, a full-length isoform (NM_001012338) with an insertion of 14 amino acids in the TK domain - which is less abundant and shows reduced signaling potential  - and a single non-catalytic truncated form (NM_001007156) that completely lacks the TK domain . Two major protein isoforms have correspondingly been detected in human brain samples: the full-length, kinase-active 150-kDa receptor (FL-NTRK3) and a truncated non-catalytic isoform (TR-NTRK3) of 50 kDa . The main function that has long been attributed to the truncated isoform is the inhibition of catalytic receptors, which is achieved through a dominant-negative or a ligand-sequestering mechanism. However, a recent work has identified a new signaling pathway activated by the truncated isoform, which links NT3 to downstream molecules affecting membrane remodeling, cytoskeletal reorganization and cell movement, such as the scaffold protein tamalin, the Rac1 GTPase and the adenosine diphosphate-ribosylation factor Arf6 .
In humans, the full-length receptor is expressed at low but relatively constant levels throughout development, while the truncated receptor is expressed at moderate levels early in development and increases to reach mature levels by adolescence. In contrast, both full-length and truncated transcripts are uniformly expressed throughout postnatal life and decline in ageing . This discrepancy between changes in protein and mRNA levels of NTRK3 suggests that post-transcriptional regulation may play a role in controlling the expression of the two isoforms, in addition to alternative splicing.
MicroRNAs (miRNAs) are post-transcriptional regulators that have been shown to play a key role in the nervous system. The distribution of miRNAs in the developing and adult nervous system is spatially and/or temporally restricted , indicating that they may contribute to the fine-tuning of neuronal gene expression. Structurally, mature miRNAs are small RNAs of approximately 22 nucleotides in length, and regulate gene expression by means of partial complementarity to miRNA binding sites located in the 3' untranslated regions (3'UTRs) of target mRNAs . In particular, perfect complementarity between nucleotide #2 through #7 or #8 at the 5' end of the mature miRNA - the so-called seed region - and the 3'UTR of genes is considered determinant for successful binding . Genes with long 3'UTRs are more prone to miRNA-mediated regulation compared with genes with short 3'UTRs, which tend to be specifically deprived of miRNA target sites . Interestingly, full-length and truncated NTRK3 transcripts show different 3'UTR regions that do not overlap, supporting the hypothesis that the two isoforms may be differentially regulated by miRNAs. Furthermore, it has recently been shown that miRNAs miR-9 and miR-125a/b are able to specifically regulate the expression of the truncated isoform of the NTRK3 receptor .
Only one study has so far analyzed the contribution of miRNAs to determining the balance between different variants of the same gene . Here, we show that the full-length and truncated isoforms of NTRK3 are regulated by different sets of miRNAs. In addition, we demonstrate that, among the miRNAs that inhibit the truncated isoform, the overexpression of miR-128 - a brain-enriched miRNA - in SH-SY5Y neuroblastoma cells alters the expression profile of genes involved in cytoskeletal organization and of genes related with apoptosis and cell cycle regulation, including the anti-apoptotic factor BCL2.
Firefly luciferase constructs
The 3'UTRs of the full-length and truncated isoforms of NTRK3 were PCR-amplified from BAC CTD-2508H23 with PfuTurbo® DNA polymerase (Stratagene, La Jolla, CA), using primers containing an Xba I restriction site at the 5' end: forward 5'-acacactctagagtctgccccaaagaggtgta-3' and reverse 5'-acacactctagaccaaactgccttacagggttt-3' for the full-length isoform and forward 5'-acacactctagaaataagccttcccggacatt-3' and reverse 5'-acacactctagatgcaaaatttccaaataagagg-3' for the truncated isoform. PCR fragments of 334 and 2110 bp respectively were purified, Xba I-digested and cloned into an Xba I site located downstream of the firefly luciferase reporter gene in the pGL4.13 vector (Promega Corporation, Madison, WI). Constructs were propagated in E. coli One Shot® TOP 10 cells (Invitrogen). Mutant reporter plasmids were generated as previously described  using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), with the wild-type pGL4.13 construct as a template and primers carrying the desired point mutations. The authenticity and orientation of the inserts as well as the presence of the mutations were confirmed by direct sequencing.
Cell culture and transfection
HeLa cells and SH-SY5Y neuroblastoma cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml Streptomycin (GIBCO™, Invitrogen). In the case of SH-SY5Y cells, fetal bovine serum was heat inactivated for 45 min at 56°C prior to use. Transfection with small RNAs was performed with Lipofectamine 2000 (Invitrogen), using either small RNAs that mimic endogenous mature miRNAs and the related negative controls (miRIDIAN™miRNA Mimics and miRIDIAN™miRNA Mimics Negative Controls #2 and #4, Dharmacon Inc.) or miRNA inhibitors and the corresponding negative control (miRCURY LNA™ microRNA Inhibitors and miRCURY LNA™ microRNA Inhibitor Negative Control A, Exiqon). The cotransfection of plasmid DNA and small RNAs into HeLa cells was optimized using a GFP-containing plasmid (pEGFP-C1, Clontech) and a commercially available GFP-specific siRNA (Ambion), obtaining a transfection efficiency of over 80%. Transfection of SH-SY5Y cells with miRNA mimics was optimized and monitored in each experiment using a fluorescein-labeled double-stranded RNA oligomer (siGLO Green Transfection Indicator, Dharmacon). Optimal efficiency was obtained at a concentration of 50-200 nM and miRNA mimics as well as miRNA inhibitors were ultimately used at 100 nM.
Luciferase activity assay
HeLa cells were seeded at 1.3 × 104 cells/well in 96-well plates and cotransfected 24 h later with the Firefly reporter constructs described above or the empty pGL4.13 vector (10-24 ng), the Renilla reporter plasmid pGL4.75 (3 ng) and the appropriate miRNA mimic (30 nM). The activity of Firefly and Renilla luciferases was determined 24 h after transfection using the Dual-Glo™ Luciferase Assay System (Promega). Relative reporter activity was obtained by normalization to the Renilla luciferase activity. In order to correct for vector-dependent unspecific effects, each relative reporter activity was normalized to the empty vector cotransfected with the corresponding miRNA. Results were then compared to the mean of the two negative controls. Each experiment was done in triplicate and at least three independent experiments were performed for each miRNA. Statistical significance was determined using Student's t test (p < 0.05).
For the analysis of NTRK3 expression during RA-induced differentiation, SH-SY5Y cells were treated with 10 μM all-trans-RA and harvested at time 0 (untreated), day 3, day 6 and day 10 of RA treatment. For TR-NTRK3, undifferentiated SH-SY5Y cells were plated at 2 × 105 cells per well in 6-well plates and transfected 24 h later with 100 nM miRNA mimic; 72 h after transfection cells were lysed and analyzed by western blotting. For FL-NTRK3, SH-SY5Y cells were differentiated with 10 μM all-trans-RA, plated in 6-well plates at a concentration of 3 × 105 cells per well at day 3 of RA treatment, transfected with 100 nM miRNA mimic at day 4 of RA treatment and analyzed 72 h after transfection (day 7 of RA treatment).
For protein extraction, cells were rapidly rinsed with ice-cold PBS and solubilized with RIPA buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Nonidet P 40, 1% sodium deoxycholate, 1 mM Na3VO4, 1 mM PMSF, 50 mM NaF and 1× protease inhibitors (Complete Mini, EDTA-free, Roche). Cells were then scraped off, incubated on ice for 15 min and centrifuged at 12000 rpm for 15 min. Samples were resolved in NuPAGE® 4-12% Bis-Tris polycrylamide gels using the NuPAGE® MES SDS Running Buffer and transferred to nitrocellulose membranes using the iBlot™Dry Blotting System (Invitogen). Before blotting, gels were equilibrated in 100 ml equilibration buffer (2× NuPAGE® Transfer Buffer, 10% methanol and 1:1000 NuPAGE® Antioxidant) for 20 min at room temperature (RT; all NuPAGE® products are from Invitrogen).
Immunodetection was performed using the ODYSSEY® infrared imaging system (LI-COR® Biosciences), following the manufacturer's instructions for two-color western blotting. This detection system allows the precise quantification of low-abundance proteins (as is the case of TR-NTRK3) for which chemiluminescence is not enough sensitive and accurate, and has the advantage that the signal generated by the proteins on the membrane is measured in a static state. Membranes were blocked in ODYSSEY® blocking buffer for 1 h at RT and incubated with the appropriate primary antibody (Ab) for 1 h at RT (anti-TrkC goat polyclonal IgG, Upstate, catalog number 07-226; anti-Bcl-2 (N-19), Santa Cruz, catalog number sc-492; anti-Caspase-3, Millipore, catalog number 06-735; anti-Caspase-9, Cell Signaling, catalog number 9508). As a loading control, membranes were simultaneously incubated with an anti-GAPDH Ab (anti-GAPDH mouse monoclonal Ab, Chemicon, catalog number MAB374). Blots were subsequently probed with the appropriate fluorophore-labeled secondary antibodies (LI-COR® Biosciences) and finally scanned on an ODYSSEY® infrared scanner. Fluorescent bands were quantified using the ODYSSEY® software. Each experiment was repeated at least four times; data are reported as means ± SE and statistical significance was determined using Student's t test.
For the analysis of ERK phosphorylation, differentiated cells were seeded and transfected as explained above; 72 h after transfection cells were rinsed twice with serum-free medium, incubated with serum-free medium for 5 h and then acutely stimulated for 10 min with 30 ng/mL neurotrophin-3 (Alomone Labs). Cells were then lysed in 50 mM Tris-HCl pH 7.4, 50 mM NaF, 10 mM sodium pyrophosphate and 0.005% Triton X-100. After transfer, membranes were incubated with an anti-phopsho-ERK (Anti-MAP kinase, activated mouse monoclonal Ab, Sigma, catalog #M9692) and an anti-pan-ERK Ab (Anti-p44/42 MAP kinase rabbit polyclonal Ab, Cell Signaling, catalog # 9102). To make sure that the quantification was accurate in each WB, a gradient (5, 10 and 15 μg) of one of the control samples was loaded on each gel. The gradients of FL-NTRK3, TR-NTRK3 and GAPDH were quantified by densitometry, and the corresponding standard curve equations were calculated for each WB; blots were considered reliable only if standard curves showed a correlation coefficient (R2) > 0.9 (Additional file 1). Standard curve equations were then applied to the densitometry counts of each sample for quantification.
miRNA expression analysis using custom microarrays (Agilent, 11k)
Total RNA, including short RNAs, was extracted from HeLa and SH-SY5Y cells using the miRNeasy® Mini Kit (QIAGEN). miRNA expression in HeLa and SH-SY5Y cells was analyzed using custom 11k oligonucleotide microarrays (Agilent Technologies), including probes for the 325 known human miRNAs according to Sanger miRBase release 7.1. The expression values obtained were compared with results reported in other studies [20, 21], and the general correlation was good; however, to avoid possible false results due to a failure in the hybridization of specific individual probes, we eliminated from subsequent analysis those miRNAs whose expression values were discordant with the other studies. Negative controls included probes for 20 Bacillus subtilis-specific sequences and 20 rare non-human sequences. Two μg of total RNA were labeled with Hy5™or Hy3™fluorescent labels using the miRCURY™LNA microRNA Labelling kit (Exiqon), following the manufacturer's instructions. Pairs of labeled samples were hybridized to dual-channel microarrays for 40 h at 55°C using Agilent hybridization reagents. Microarray images were quantified using the GenePix 6.0 (Axon) software; only spots with signal intensities twice above the local background and not saturated were considered reliable and used for subsequent analysis. Extracted intensities were subtracted from the local background and the log2 ratios were normalized in an intensity-dependent fashion. Statistical analyses were performed using the MMARGE tool.
Whole-genome expression analysis using beadchip microarrays
Total RNA samples obtained from four independent experiments (SH-SY5Y cells transfected with miR-128 and the related negative controls) were analyzed on HumanRef-8 BeadChips from Illumina, which target 24,500 well-annotated RefSeq transcripts. Starting from 200 ng of total RNA, biotin-labeled cRNA was synthesized and hybridized according to the manufacturer's instructions. Data were analyzed using the Array File Maker (AFM) 4.0 software package. The whole genome microarray data obtained here are part of a larger project that is still ongoing. These results will be deposited in MIAME format in the ArrayExpress public repository once the experiment is completed.
Real-time quantitative RT-PCR
DNase-treated RNA extracted from transfected cells was retrotranscribed with the SuperScript™III First-Strand Synthesis System for RT-PCR (Invitrogen). RNA levels of the full-length and truncated isoforms of NTRK3 were analyzed by real-time quantitative RT-PCR using SYBR Green I (Roche), following the manufacturer's instructions. Reactions were performed with the LightCycler® 480 Instrument (Roche) in 384-multiwell plates. Specific primers were designed for the two NTRK3 isoforms (FL-NTRK3 forward 5'-cagccatggttccaactctc-3', FL-NTRK3 reverse 5'-ccagcatgacatcgtacacc-3', TR-NTRK3 forward 5'-tccagagtggggaagtgtct-3', TR-NTRK3 reverse 5'-ccatggttaagaggcttgga-3') and for GAPDH (forward 5'-ctgacttcaacagcgacacc-3', reverse 5'-ccctgttgctgtagccaaat-3'), which was used as housekeeping gene. All amplicons were ~100 bp in length, and forward and reverse primers were located in different exons. Thermal cycling was performed as follows: one pre-incubation cycle at 95°C for 10 min (ramp rate: 4.8°C/s); 45 amplification cycles at 95°C for 10 sec, 57°C for 30 sec and 72°C for 5 sec (ramp rates: 4.8, 2.2 and 4.8°C/s). The expression levels of miR-128 in different human tissues and in SH-SY5Y cells were analyzed using the TaqMan® MicroRNA Assays, following the manufacturer's instructions.
Cells cultured on 6-well plates and transfected with miR-128, the TR-NTRK3-specific siRNA (sense 5'-gagucuaugccuuuggcaatt-3', antisense 5'-uugccaaaggcauagacuctt-3', Gene Link) and the related negative controls were trypsinized and resuspended in 1 mL DMEM; 100 μL of each sample were then diluted in 10 mL of Coulter Isoton II diluent (Beclman Coulter) and counted using a Z2™ Series Coulter Particle Count and Size Analyzer (Beckman Coulter).
SH-SY5Y cells transfected with miR-128 and the related negative controls were trypsinized and resuspended in PBS 72 h after transfection cells. Samples were examined using a BD FACSCanto Flow Cytometer and the BD FACSDiva software (BD Biosciences). A two-parameter forward/side scatter (FSC/SSC) analysis was performed recording 5000 events in each analysis.
Three web-based miRNA target prediction methods were used: miRanda (http://www.microrna.org, 2005 release; ), TargetScan (http://www.targetscan.org, releases 2.0, 2.1, 3.0 and 3.1; ) and PicTar (http://pictar.mdc-berlin.de; ). Genomic coordinates were according to the human assembly release of March 2006 (H. sapiens hg18). Sequences were obtained from the University of California Santa Cruz (UCSC) Genome Browser http://www.genome.ucsc.edu. Sequence analysis was performed using the 4peaks software http://mekentosj.com/4peaks/ and Multalin http://multalin.toulouse.inra.fr/multalin/. Primers were designed using the Primer3 software http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi and PCR annealing temperatures were calculated with the Optimase ProtocolWriter™ http://mutationdiscovery.com. Pathway analysis from microarray expression data was performed with the Ingenuity Pathway Analysis Software (IPA) version 6.3 http://www.ingenuity.com.
Functional screening of miRNA target sites in NTRK3
Tested miRNAs and predicted target
The entire 3'UTRs of the full-length and truncated isoforms of NTRK3 (178 and 1981 bp, respectively) were cloned immediately downstream of the firefly luciferase open reading frame in the pGL4.13 plasmid, and the resulting constructs were designated pGL4.13-FL and pGL4.13-TR. The renilla reporter plasmid pGL4.75 was used to control for transfection efficiency. A total of 55 miRNAs and 2 non-targeting controls were tested on both pGL4.13-FL and pGL4.13-TR. Tested miRNAs (Table 1) included the 32 mentioned above; 6 additional miRNAs that appeared in more recent versions of the miRbase database and had putative target sites in the truncated isoform (miR-485-3p, miR-509, miR-617, miR-625, miR-765 and miR-768-5p) were also included because allelic variants were found in their target sites after re-sequencing of patients with anxiety disorders . Eleven out of the 35 miRNAs were predicted to regulate the truncated isoform by more than one method, and at least 7 were conserved target sites for miRNA families broadly conserved among vertebrates (Table 1). Finally, 17 miRNAs with no predicted target sites according to the interrogated methods were also analyzed for comparison.
Since multiple target sites in the same 3'UTR are thought to be cooperative and to increase the efficacy of miRNA inhibition, we analyzed whether the cotransfection of different miRNAs repressing pGL4-13-TR could lead to a synergistic effect and a more dramatic reduction in luciferase activity. Thirteen combinations of two miRNAs were tested, but we could not detect a significant decrease in luciferase expression compared with the corresponding miRNAs taken individually; a combination of the three most effective miRNAs - miR-128, 509 and 625 - was also analyzed and no synergistic effect was observed (Additional file 2).
miRNA-mediated regulation of endogenous NTRK3 in neuroblastoma cells
SH-SY5Y neuroblastoma cells were used to investigate whether miRNAs causing a significant decrease in luciferase activity were also able to downregulate endogenous NTRK3. Upon exposure to retinoic acid (RA), SH-SY5Y cells reduce their growth rate and differentiate by extending neurites into cells that are biochemically, ultrastructurally and electrophysiologically similar to neurons . Furthermore, RA treatment has been shown to induce the appearance of functional FL-NTRK3 receptors in this cell line .
miRNA expression levels in HeLa and SH-SY5Y cells
Differentiated SH-SY5Y (RA 6 days)
Finally, full-length and truncated NTRK3 transcripts were quantified by real-time quantitative RT-PCR after miRNA overexpression, using isoform-specific primers. mRNA levels were not affected by any of the regulating miRNAs, indicating that they do not act by destabilizing NTRK3 transcripts and suggesting that the observed downregulation is achieved through translational repression.
miR-128 overexpression affects the morphology and number of SH-SY5Y cells
Transcriptome analysis of SH-SY5Y cells transfected with miR-128
Top 10 upregulated genes upon miR-128 overexpression in SH-SY5Y cells
Microtubule-based vesicular trafficking;retrograde
axonal transport of signaling endosomes containing
neurotrophins and associated downstream kinases
Lipogenesis and biosynthesis of cholesterol
Regulation of cell death and
caspase activity; antiapoptotic
Transcription factor of the CREB3 family;
astrocyte proliferation in response
to inflammation and trauma (gliosis)
Activation of the transcription factor NFkB;
implicated in malignant processes and in the
regulation of the apoptotic response; mediates the
antiapoptotic effect of TGF-beta and TNF-alpha
Integral membrane protein
Zinc finger transcription factor; adipogenesis
and inhibition of cellular growth
May regulate neurite formation
during brain development
Component of the energy homeostatic mechanism;
increases fatty acid oxidation and glucose uptake;
may participate in the control higher brain functions
Encodes mesotrypsin, a brain-specific
trypsin form thought to protect neural
cells from apoptotic cell death
Top 10 downregulated genes upon miR-128
Organization of the cytoskeleton, cell motility and spreading:
binds to actin monomers and inhibits actin polymerization;
anti-apoptotic action in chickembryo motoneurons
Vesicular transport from endoplasmic reticulum to golgi;
possible role in postsynaptic membrane trafficking
Regulation of glucose homeostasis: catalyzes
hydrolysis of G6P to glucose and phosphate
Chaperone protein; maturation of the cytoskeleton
and morphogenesis; possible role in the
transport and correct localization of the tumor
suppressor von Hippel-Lindau (VHL) protein
p75NTR-associated protein; mediates
apoptosis in response to NGF
Member of the brain-abundant 14-3-3 protein
family; may play a role in linking
mitogenic signaling and the cell cycle machinery
Translational repressor; acts on VEGF
(Vascular Endothelial Growth Factor)
Secretory protein expressed in neuronal cells;
guides and sorts the secretion of neuropeptides
RNA-binding protein; major autoantigen
in patients suffering from autoimmune diseases;
binds small RNAs of unknown function (Y RNAs)
and misfolded noncoding RNAs; possible function
in the degradation of defective RNAs
Target predictions and genes deregulated by miR-128
Overexpression of miR-128 upregulates BCL2
In this study we explore how miRNAs take part in the regulation of two isoforms of the neurotrophin-3 receptor NTRK3, which present non-overlapping 3'UTRs. We demonstrate that the two isoforms are targeted by different sets of miRNAs, providing new evidence for a role of miRNAs in determining the balance among different splice variants of a gene. In fact, we identify one miRNAs regulating the full-length isoform of NTRK3 (miR-151-3p) and 4 miRNAs regulating the truncated isoform (miR-128, miR-485-3p, miR-765 and miR-768-5p).
Our approach followed two steps: a first screening of miRNAs with predicted targets in the 3'UTR of either isoform, performed in HeLa cells using luciferase constructs, and a subsequent validation of the ability of luciferase-positive miRNAs to repress endogenous NTRK3 receptors in SH-SY5Y neuroblastoma cells. Upon exposure to RA, SH-SY5Y differentiate into cells that are similar to neurons ; the presence of FL-NTRK3 receptors after RA stimulation had previously been demonstrated in this cell system , where we could well characterize the expression of both NTRK3 isoforms.
If we compare the results obtained with the two strategies, 5 out of 10 (50%) luciferase-positive miRNAs were validated on endogenous NTRK3. Given that the constitutive expression of the remaining 5 miRNAs is similarly low in both HeLa and SH-SY5Y cells, we could rule out the possibility that high levels of endogenous miRNAs in SH-SY5Y cells affected the results of the validation, making the overexpression of miRNA mimics less effective; such rate of false-positives is probably related with the artificial context in which the 3'UTRs are inserted in the luciferase assay strategy.
One limitation of this study is that, due to the generally low expression levels of the analyzed miRNAs in SH-SY5Y cells, the experiments were based on miRNA overexpression, rather than inhibition of endogenous miRNAs. It would be certainly interesting to analyze the effect of miRNA inhibition in a different cell system where the miRNAs of interest are highly expressed, including cell lines where NTRK3 is not present and where the inhibition of the regulating miRNAs might indeed induce its expression. In addition, the levels of the truncated isoform of NTRK3 were also low in SH-SY5Y, and standard WB quantification by enhanced chemiluminescence detection proved to be not enough sensitive. We therefore used fluorescent detection, employing fluorescently-labelled secondary antibodies and an infrared imaging system, which allows the precise quantification of low-abundance proteins , and has the advantage that the signal generated by the proteins on the membrane is measured in a static state, as opposed to the enzymatic reaction of chemiluminescence, which is dynamic and changes over time . Given that the signal with this detection system is linear and not exponential as in chemiluminescence, the differences between bands are less apparent but the quantification by densitometry becomes much more accurate. This allowed us to detect the subtle changes originated by the overexpression of miRNAs.
Interestingly, miR-151-3p and miR-185 have partially overlapping target sequences in the full-length isoform and, in a similar way, miR-128, miR-509 and miR-768-5p target the same segment of the 3'UTR of the truncated isoform (Figure 1B). It is worth of notice that while both miR-128 and miR-509 cause a strong reduction in luciferase activity (30-50%), only miR-128 seems to repress the corresponding protein isoform in SH-SY5Y cells. Intriguingly, miR-128 is expressed in brain, whereas miR-509 is not present in brain but shows a strong expression in kidney and testis. This suggests that the binding of these miRNAs to their target sequences might be mutually exclusive, and that the regulation of NTRK3 by miRNAs could be coordinated in a tissue specific fashion.
The second part of this study focused on miR-128, one of the miRNAs repressing the truncated isoform. miR-128 is a brain-enriched miRNA, whose expression has been shown to positively correlate with neuronal differentiation [27, 28]. Furthermore, accumulation of miR-128 has been detected in the hippocampus of Alzheimer's disease brains . Here, we show that the overexpression of miR-128 causes morphological changes in SH-SY5Y cells, which resemble those observed using an siRNA specifically directed against TR-NTRK3. In accordance with the morphological changes, as revealed by microarray analysis, miR-128 alters the expression of genes involved in cytoskeletal organization, a process with which the truncated isoform has been related. These results suggest an involvement of miR-128 in the organization of the cytoskeleton through the regulation of NTRK3, possibly in cooperation with other miR-128 targets.
Another consequence of the overexpression of miR-128 in SH-SY5Y cells is an increase in cell number, which is accompanied by the deregulation of genes involved in apoptosis, cell death/survival and proliferation, with a remarkable upregulation of BCL2. BCL2 codes for an outer mitochondrial membrane protein that blocks cytochrome c release from mitochondria and inhibits caspase activity, suppressing apoptosis. However, the analysis of caspase-3 and caspase-9 activation revealed that in normal culture conditions there is virtually no apoptosis in SH-SY5Y cells, making the hypothesis of an increased inhibition of this process unsuitable for our results. It would be therefore interesting to analyze the effects of miR-128 in the presence of apoptosis-promoting agents.
Besides apoptosis, BCL2 also plays a role in the regulation of the cell cycle, having an antiproliferative effect that drives cells into enhanced G0 arrest. This is thought to promote cell survival, especially in unfavorable environments, since quiescent cells are more resistant to killing than proliferating cells . Although this could be could be compatible with the increase in cell number and with the general reduction of metabolic activity observed in our analyses, further investigation is needed to support this hypothesis and to exhaustively evaluate the contribution of BCL2 to the observed phenotype, as well as that of other proliferation-regulating genes whose expression is altered by miR-128.
The full-length and truncated isoforms of NTRK3 are regulated by different sets of miRNAs, demonstrating that the regulation of NTRK3 by microRNAs is isoform-specific and indicating that neurotrophin-mediated processes are strongly linked to microRNA-dependent mechanisms. In addition, overexpression of the brain-enriched miRNA miR-128 - one of the miRNAs that regulates the truncated isoform of NTRK3 - causes morphological changes in neuroblastoma cells and alters the expression profile of genes involved in cytoskeletal organization, apoptosis and cell proliferation, including the anti-apoptotic factor BCL2. These findings open new perspectives for the study of the physiological role of miR-128 and its possible involvement in cell death/survival processes.
We would like to thank S. Porta and M. Bañez for their technical support and advice. This work was supported by the European Union Sixth Framework Programme Integrated Project SIROCCO (Grant LSHG-CT-2006-037900), the "Instituto Carlos III and Fondo de Investigaciones Sanitarias" (CIBER-CB06/02/0058, FIS/ISCIII:P1052565, ISCIII:GO3/184), the "Departament d'Universitats Innovació i Empresa, Generalitat de Catalunya" (2005SGR00008) and the Spanish Ministry of Science and Innovation (MICINN-SAF2008-00357). M.G. was funded by the Spanish Ministry of Education and Science (FPU fellowship). M.M.G. is a recipient of a FIS fellowship (FI05/0006). Y.E.P. was supported by the "Ramón y Cajal" Program (Spanish Ministry of Science and Innovation). E.M. is supported by the Spanish Ministry of Health. This publication includes part of the work performed by M.G.'s and M.M.G.'s for their doctoral theses, presented at Pompeu Fabra University (UPF), Barcelona.
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