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Deep Sequencing the microRNA profile in rhabdomyosarcoma reveals down-regulation of miR-378 family members
- Francesca Megiorni†1Email author,
- Samantha Cialfi†1, 2,
- Heather P McDowell1, 3, 4,
- Armando Felsani5,
- Simona Camero1,
- Alessandro Guffanti5,
- Barry Pizer3,
- Anna Clerico1,
- Alessandra De Grazia1,
- Antonio Pizzuti6,
- Anna Moles5 and
- Carlo Dominici1, 4
© Megiorni et al.; licensee BioMed Central Ltd. 2014
Received: 19 May 2014
Accepted: 4 November 2014
Published: 25 November 2014
Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. MicroRNAs (miRNAs) represent a class of short, non-coding, regulatory RNAs which play a critical role in different cellular processes. Altered miRNA levels have been reported in human cancers, including RMS.
Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Q-PCR, MTT, cytofluorimetry, migration assay, western blot and immunofluorescence experiments were carried out to determine the role of miR-378a-3p in cancer cell growth, apoptosis, migration and differentiation. Bioinformatics pipelines were used for miRNA target prediction and clustering analysis.
Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS. MiR-378a-3p over-expression in an RMS-derived cell line suppressed IGF1R expression and affected phosphorylated-Akt protein levels. Ectopic expression of miR-378a-3p caused significant changes in apoptosis, cell migration, cytoskeleton organization as well as a modulation of the muscular markers MyoD1, MyoR, desmin and MyHC. In addition, DNA demethylation by 5-aza-2′-deoxycytidine (5-aza-dC) was able to up-regulate miR-378a-3p levels with a concomitant induction of apoptosis, decrease in cell viability and cell cycle arrest in G2-phase. Cells treated with 5-aza-dC clearly changed their morphology and expressed moderate levels of MyHC.
MiR-378a-3p may function as a tumour suppressor in RMS and the restoration of its expression would be of therapeutic benefit in RMS. Furthermore, the role of epigenetic modifications in RMS deserves further investigations.
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood , representing approximately 50% of all sarcomas in children aged 0–14 years and 4-5% of malignant solid tumours in the paediatric population. The two major histological subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS), have distinct clinical features and outcomes. ERMSs are more frequent (~80% of cases) and generally affect younger children (0–4 years); they occur more commonly in the neck, head and genito-urinary tract . As the name implies, tumour cells resemble embryonal skeletal muscle cells. ARMSs (~20% of cases) usually present throughout childhood, typically originating in the limbs and trunk, often with regional or metastatic lymph node involvement already at diagnosis, and carry a significantly worse outcome [2, 3]. ARMS is so called because tumour cells form small spaces or pseudo-alveoli.
The role of genetic factors in the development of RMS has been confirmed by several recent epidemiological observations and advances in molecular genetics. Although the majority of RMS cases are sporadic, i.e. not associated with hereditary syndromes, a small proportion are linked to congenital anomalies, e.g. Beckwith-Wiedemann syndrome, or are associated with particular familial syndromes, such as neurofibromatosis type I and Li-Fraumeni syndrome [4–6]. ARMS and ERMS are both characterised by particular genetic alterations that are likely to play a decisive role in cancer pathogenesis. Eighty percent of ARMS tumours have either t(2;13)(q35,q14) or t(1;13)(p36;q14) chromosomal translocations, which generate PAX3-FOXO1 and PAX7-FOXO1 fusion proteins, respectively . However, constitutive expression of PAX3/7-FOXO1 chimeric genes is not sufficient to induce RMS development in transgenic animals [8, 9]. Loss of heterozygosity of the short arm of chromosome 11 (11p15.5), with over-expression of the insulin-like growth factor II, is often associated with ERMS . However, although several tumour causative genes have been identified, a detailed understanding of the molecular mechanisms underlying RMS development has not yet been achieved.
Recent studies have suggested that microRNAs (miRNAs) may play an essential role in RMS [11, 12]. MiRNAs are a class of conserved, short, non-coding molecules which regulate gene expression through binding to non-perfect complementary sequences at the 3′-untranslated regions (UTRs) of target messenger RNAs (mRNAs), resulting in translational repression . Several pre-miRNAs stem-loops are processed to produce two mature and functional miRNAs, designated with the suffix “-3p” or “-5p,” depending on the originating hairpin arm . It has been predicted that about one-third of all mammalian genes are targeted by miRNAs [15, 16]. Deregulation of miRNA expression is associated with various cancers and many studies indicate that miRNAs may act either as oncogenes or tumour suppressors [17–19], controlling key processes in tumorigenesis, such as tumour initiation, progression and metastatic spread. An increasing number of miRNAs, such as miR-1, miR-133a, miR-200c, miR-206, miR-214 and miR-9*, have been identified to have a role in RMS [11, 20–24], as recently summarized by Novak et al. .
Epigenetic DNA changes, such as DNA promoter hypermethylation and histone modifications, have critical roles in chromatin remodelling and general regulation of gene expression in mammalian development and human diseases . In particular, DNA methylation of CpG islands in promoter regions has been correlated with silencing of tumour suppressor genes and other tumour-related genes, and it has been recognised as a crucial component of the mechanism underlying cancer development . DNA methylation-associated silencing of miRNAs in different human tumours, including RMS, has also been reported [27, 28].
In this study, deep sequencing technology was utilised to quantify the absolute abundance of miRNAs in ARMS and ERMS tumours as well as in normal skeletal muscle (NSM), and to identify an RMS-specific miRNA expression pattern. The majority of miRNAs were found to be down-regulated, as predicted. Interestingly, miR-378a/b/c/d/e/f/h/i molecules, belonging to a large family of evolutionary conserved miRNAs, were strongly under-represented in ARMS and ERMS tumours in comparison to NSM. Transient transfection of miR-378a-3p in an ARMS-derived cell line (RH30) induced apoptosis and decreased viability/proliferation by repressing the IGF1R/AKT pathway. Importantly, elevated levels of miR-378a-3p impaired RH30 cell migration and promoted myogenic differentiation. Demonstration that epigenetic modifications may be involved in RMS tumourigenesis was achieved by restoring miR-378a-3p levels. In addition, 5-aza-2′-deoxycytidine (5-aza-dC) treatment induced apoptosis, cell cycle arrest in G2 phase and decreased cell viability compared to untreated RH30 cells. Interestingly, RH30 5-aza-dC-treated cells changed their morphology and expressed muscle differentiation markers, partially overlapping the effect of miR-378a-3p transfection.
Taken together, these data provide the first evidence for an anti-tumour activity of miR-378a-3p in RMS, suggesting that this miRNA could be a potential therapeutic target in RMS. Furthermore, the importance of epigenetic regulation in RMS was confirmed, which may have important clinical implications in this malignancy.
Patient clinical and tumour histopathological characteristics
Clinico-pathological features of the analysed tumour cases
Samples were immediately frozen in liquid nitrogen after surgery and stored at −80°C. Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer’s instructions. Samples were enriched for small RNAs up to 200 bp by size selection using Pure Link miRNA Isolation Kit (LifeTech). RNA purity, integrity and size distribution were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies).
Small RNA library generation and sequencing
Enriched RNA samples were processed using the Small RNA Expression Kit according to the manufacturer’s protocol (Small RNA expression kit, rev. C, Applied Biosystems). Briefly, RNA was first hybridized and ligated with the adapter mix “A”, subsequently reverse transcribed and treated with RNAse H. The obtained cDNA libraries were PCR amplified, purified and size-selected by PAGE, resulting in libraries containing inserted small RNA sequences of 20–40 bp length. Size, integrity and purity of the libraries were verified by the Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA libraries were barcoded using the Solid RNA barcoding kit and amplified onto beads using emulsion PCR. Templated beads were deposited on slides and analysed using the Applied Biosystems SOLiD 4 Sequencer.
Statistical and bioinformatics analyses
The quality filtered reads were mapped against all annotated human mature miRNA sequences (miRBase v19.0)  using the Lifetech Lifescope 2.5.1 Small RNA pipeline, filtering for rRNA, primers, and small non-miRNA non-coding transcribed sequences such as tRNAs and snoRNAs. Sequence counts were extracted and reformatted with perl scripts from the pipeline output. Differential expression analysis was performed with the edgeR Bioconductor statistical library  version 3.0.8 on R version 2.15.3. TMM-normalized sequence counts in the libraries were transformed in Counts Per Millions (CPM) according to the formula: CPM = (normalized counts/total miRNA matches) *1,000,000. After having estimated the tagwise dispersion, genewise exact test  as implemented in edgeR was used to measure the significance of differential expression, using the gene “Pseudo-counts”. Sequences were deemed significantly differentially expressed if (1) the p-value given by this method was < 0.05, (2) the total count was greater than 50 CPM in at least one group of samples, and (3) there was at least a two-fold change in normalised sequence counts between the two groups.
The validated target prediction of a panel of regulated microRNAs was performed by interrogating the ‘Validated Target’ option of the miRWalk web software . Target genes of the miR-378 family were predicted using TargetScan Human 6.2 (http://www.targetscan.org/), miRanda (http://www.microrna.org/microrna/home.do) and DIANA-microT version 3.0 (http://diana.cslab.ece.ntua.gr/microT/) algorithms. Annotation and enrichment of functional pathways associated with the miR-378 target genes were evaluated using the Reactome database and associated analytical tools (http://www.reactome.org/). The same miR-378 target gene list was used as the starting dataset for the generation of a Functional Interaction network analysis and related Gene Ontology enrichment analysis as described [34, 35]. Preliminary isoMIR analysis was performed by selecting from the YM500 miR-Seq database (http://ngs.ym.edu.tw/ym500v2/index.php) the hsa-miR378a-3p isoforms which had been found with at least 50.000 sequence reads in 40 different experiments, allowing 3 nt 5′ extensions, 3′ extensions and one mismatch with respect to the reference miRBase mature sequence. The resulting 14 isomirs were identified and counted in all the NSM, ARMS and ERMS sequences using the bowtie 0.12.8 database search software and ad-hoc created perl scripts.
Nucleotide sequence pattern analysis of the miR-378a-3p promoter region was performed using the program CpG plot of the EMBOSS sequence analysis suite (http://emboss.sourceforge.net/), with standard parameters, and other softwares such as CpG island searcher (http://cpgislands.usc.edu/) on the genome region chr5: 149107388–149112388 corresponding to the putative has-miR378a-3p putative promoter.
Each experiment was repeated three times independently. All results were expressed as means ± standard deviation (SD), and a p-value < 0.05 was used for significance. One-way ANOVA analysis for independent samples was used to determine statistical significance in different assays.
Human ARMS RH30 and ERMS RD cell lines were maintained in high-glucose Dulbecco’s modified Eagle’s medium(DMEM-HG) supplemented with 10% foetal bovine serum (FBS), 1% v/v L-glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin, and grown at 37°C in a humidified atmosphere of 5% CO2.
RH30 and RD cells were seeded at 4 × 105 cells/well in 6-well plates. After 24 h, 5-aza-dC (Sigma-Aldrich, St. Louis, MO) was added to a final concentration of 20 μM. Following different times of treatment, cells were collected for cell cycle analysis, apoptosis, MTT, migration, Q-PCR, western blot or immunofluorescence. Mock treatments were carried out treating cells in the same medium with DMSO (Ctr).
RH30 and RD cells were seeded at 8 × 105 cells/well in 6-well plates; miRNA mimics (miR-378a-3p, Dharmacon Research) or negative control (miR-Ctr, Dharmacon Research) were transfected using Lipofectamine 2000 reagent (Life Technologies) at 50 nM final concentration, following the manufacturer’s protocol. Following different times of treatment, cells were collected for cell cycle analysis, apoptosis, MTT, migration, Q-PCR, western blot or immunofluorescence.
Quantitative Real Time PCR (Q-PCR)
Reverse transcription (RT) for human miR-378a-3p, miR-378a-5p, miR-483-3p and miR-503-5p was carried out with TaqMan MicroRNA Assay kit (Life Technologies) using 20 ng of total RNA sample and the specific stem-loop primer according to manufacturer’s protocols. Quantitative Real Time PCR (Q-PCR) analysis was performed on a StepOne Real Time System (Life Technologies) machine using miRNA-specific TaqMan MGB primers/probe (Life Technologies). PCR reactions were run at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. Data were normalized to U6 small nuclear RNA (RNU6) levels. Each sample was run in triplicate. The amount of each miRNA was calculated by the comparative Ct method and expressed as fold change (2-ΔΔCT) compared to NSM using the DataAssist v3.01 software (Life Technologies).
RH30 cells were treated with 5-aza-dC or transfected with miRNA mimics, and cell viability was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay. Cells (5×103) were plated onto 96-well plates in sextuplicates and, after 24 h, treated with 5-aza-dC or transfected with synthetic miR-378-3p; untreated and blank cell-free controls were included. At designated times after treatment or transfection (0-24-48-72 h), 10 μl of MTT (5 mg/mL, Sigma-Aldrich) were added to each well and plates were incubated at 37°C for 4 h. Media were removed and 150 μl dimethyl sulfoxide (DMSO) were added into each well to dissolve the dark blue formazan crystals. Absorbance was measured at wavelength of 550 nm, with reference at 630 nm, using a microtitre plate reader (Select Science). The results were plotted as means ± SD of two separate experiments having six determinations per experiment for each experimental condition.
Cell cycle analysis
RH30 and RD cells treated with 5-aza-dC were collected and washed twice with phosphate buffered saline (PBS). After fixation in 70% ice-cold ethanol overnight at +4°C, cell pellets were washed twice with ice-cold PBS and treated with RNase A for 15 min at 37°C. Propidium iodide (PI) was added to each sample and DNA content was determined by collecting 10,000 events using a BD FACS Calibur Flow Cytometer (BD Biosciences). Data were analysed using CellQuest Pro software (BD Biosciences). Experiments were performed three times.
Cell apoptosis was analysed by flow cytometry with PE Annexin V Apoptosis Detection Kit I (BD Pharmingen). Briefly, RH30 and RD cells were seeded overnight in 6-well plate and treated with 5-aza-dC or transfected with miRNA mimics for 72 h. Cells were washed twice in cold PBS and resuspended in 1x Annexin V Binding Buffer at a concentration of 1×106 cells/ml. Cells were stained with Annexin V and 7-Amino-Actinomycin D (7-AAD) for 15 min at room temperature (RT) in darkness according to the manufacturer’s instructions. Annexin V and 7-AAD fluorescence intensities of control or treated samples were analysed using a BD FACSCalibur Flow Cytometer (BD Biosciences). Data were analysed using Cell Quest Pro software (BD Biosciences). Experiments were performed three times.
RH30 cells were cultured in complete medium with 5-aza-dC or miRNA mimics for 72 h before plating 5×104 cells per well into BD FalconTM Cell Culture Inserts with 8 μm pore polycarbonate filters (Falcon). Chambers with cells contained medium without serum, whilst the lower well had DMEM supplemented with 10% FBS, used as chemoattractant. After 24 h, migrated cells at the base of the inserts were fixed in 100% methanol and stained with 2% crystal violet dye. Cells were photographed under a light microscope at 20× or 40× magnifications; 8 randomly selected fields were examined and counted manually. The average number of migrated cells was calculated. Experiments were performed in triplicate and repeated twice.
Western blot analysis
RMS cells were seeded overnight in 6-well plates. Cells were treated with 5-aza-dC or transfected with miRNA mimics for 72–96 h and lysed with Hepes buffer (20 mM Hepes, 250 mM NaCl, 0.1% Triton X-100, 2 mM EDTA, 10 μg/mL leupeptin, 10 μg/mL aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 4 mM sodium orthovanadate, 1 mM DTT). Total protein extracts (30 μg) were separated on 8-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, Bedford, MA). Filters were blocked with 5% non-fat dry milk in PBS-Tween for 30 min at RT and incubated with the primary antibody. The following antibodies were incubated over-night at +4°C: anti-IGFR1 (Cell Signaling), anti-phospho-Akt (Cell Signaling), anti-MyoR(Santa Cruz Biotechnology), anti-MyOD1 (Millipore), anti-Myf5 (Millipore), anti-desmin (Millipore) and anti-MyHC (Millipore). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) were used for 1 h at RT. Protein-antibody complexes were detected with ECL Super Signal (Pierce). Tubulin (Sigma-Aldrich) was used as a normalization control for equal loading. Experiments were performed at least three times.
RMS cells were seeded overnight in 24-well plates. Cells were treated with 5-aza-dC or transfected with miRNA mimics for 72 h and then seeded into 8-chamber culture slides (Falcon). After two additional days, cells were rinsed with PBS and fixed with 4% paraformaldehyde at RT for 30 min. After treatment with 0.1 M glycine and permeabilisation with 0.1% Triton X-100, cells were subjected to immunofluorescence staining with the anti-MyHC (Millipore) antibody for 1 h 30 min at RT. Cells were washed with cold PBS three times and incubated with Texas Red-anti-mouse secondary antibody (1:100, Jackson Laboratories) at RT for 30 min. The actin cytoskeleton was visualized with TRITC-phalloidin (1:50, Sigma) at RT for 1 h 30 min. Nuclei were counter-stained with 1 μg/ml Hoechst (Sigma). Labelled sections were examined and analysed by using a Zeiss ApoTom epifluorescent microscope (Carl Zeiss) and Axio-Vision software. Experiments were replicated twice.
DNA Methylation analysis
Genomic DNA from RH30 cells treated or not with 5-aza-dC for 72 h was obtained by phenol:chloroform:isoamyl alcohol method. About 1 μg of DNA was modified using the Epitect DNA Bisulfite Kit (Qiagen), according to the manufacturer’s protocol. For sequencing, the bisulfite-treated DNA was amplified by PCR with two different primer sets for the human miR-378a promoter: BS1 forward 5′-GGGGAAAAGttAGGtTGGA-3′ and BS1 reverse 5′-aCTaACATTTTTaaTaaCTaCTTaTCCCAaC-3′; BS2 forward 5′-GGGTAAtTGGGGGTTttAG-3′ and BS2 reverse 5′-CAaCAACAaCACTCTaaaaACT-3′. PCR products, purified using a commercially available kit (Qiagen), were sequenced on an ABI sequencer with dye terminators (Applied Biosystems). Analysis of primary bisulfite sequencing was carried out with BISMA software (http://services.ibc.uni-stuttgart.de/BDPC/BISMA) by uploading the unconverted reference sequence and the sequencing results. For methylation-specific PCR (MSP), the bisulfite-modified genomic DNA was amplified using primers based on methylated (M) or unmethylated (UM) cytosines in CpG islands in the miR-378a promoter region: M forward 5′-AGtTAGCGGtttTGCGGtAGtC-3′ and M reverse 5′-aCCCGaaaaaAaaaAaCCAaCGAaCG-3′; UM forward 5′-tttGtttttGtAGtTAGtGGtttTGtGGtAGttG-3′ and UM reverse 5′-CaAaCCaaCCCaaaaaaAaaaAaCCAaCaAaCa-3′. PCR products were run on 2% agarose gel. All primers were designed using the MSPprimer algorithm. Experiments were performed three times.
Small RNA library generation, sequencing, identification and quantification of annotated miRNAs
Distribution in abundance classes of different miRNA transcripts in tumours and normal muscle
1 - 102
102 - 104
The distribution of miR-387a-3p isomirs between NSM and RMSs was assessed. One YM500 database isoform with an extension at 3′ and a mismatch in the extension (ACTGGACTTGGAGTCAGAAGGCG[C]T) was represented in both NSM and ARMS/ERMS samples, but with a noticeable difference in frequency which will warrant further investigation.
Differentially expressed miRNAs
miRNA expression in RMS tumours
log 2 FC
log 2 CPM
log 2 FC
log 2 CPM
log 2 FC
log 2 CPM
log 2 FC
log 2 CPM
miR-378a-3p negatively regulates IGF1R levels
Human miR-378 family members
miR-378-3p induces apoptosis and impairs cell migration
miR-378a-3p correlates with myogenic differentiation
5-aza-2′-deoxycytidine (5-aza-dC) treatment up-regulates miR-378a-3p levels and induces apoptosis, G2-cell arrest and decreased migration in RMS cells
These results indicate that demethylation inhibits viability and proliferation of RMS cells due to G2 cell cycle arrest and programmed cell death, and that it might also affect RMS progression by reducing cell migration.
5-aza-dC treatment induces myogenic differentiation
Finally, to assess whether 5-aza-dC treatment can also promote myogenic differentiation in RMS, RH30 cells were exposed to the demethylating drug followed by assessment of myogenic markers by immunofluorescence experiments. RH30 cells treated with 5-aza-dC exhibited marked changes in the cytoskeleton structure, as evident by phalloidin staining, and a differentiated phenotype, supported by the MyHC positivity (Figure 6E).
Taken together, these data are partially consistent with those following miR-378a-3p transfection, although the effects in RMS cells are more pronounced, this being due to the fact that 5-aza-dC action is not limited to the regulation of an individual miRNA but that it facilitates the re-expression of several epigenetically silenced genes.
RMS is a malignant tumour occurring most commonly in the paediatric population, with a survival of around 20% when metastatic disease is present at diagnosis . MiRNAs seem to play a crucial role in RMS development and progression, and might represent novel tools for improving the outcome of this malignancy [12, 43, 44]. In this study, the expression levels of miRNAs were measured in a panel of alveolar and embryonal tumours by using deep sequencing technology in order to identify key drivers of RMS specific processes. The global miRNA abundance was reduced in RMS, this contributing to the neoplastic transformation by allowing an increased expression of proteins with oncogenic potential, as previously observed . Notably, many relevant myo-miRNAs, such as miR-1, miR-133a and miR-29, were markedly down-regulated in tumour samples in comparison to NSM, as already shown in previous studies carried out by microarray or Q-PCR technologies [21, 37]. Interestingly, mir-206 was present with a detectable negative fold change in all comparisons but without being associated with a significant statistical index in the final analysis, as also reported in recent published microarray experiments carried out in the same biological model . MiRNAs expression was relatively uniform in ARMS and ERMS tumours, and only a restricted number of small regulatory molecules seemed to discriminate the two RMS subtypes. Further analyses need to be undertaken to understand the possible differential role of specific miRNAs and related pathways in ARMS and ERMS oncogenic transformation, and to determine the potential role of miRNAs as diagnostic biomarkers in the two RMS subtypes.
To our knowledge, this is the first study that shows a marked down-regulation of miR-378 family members in RMS. This finding is probably due to the improved specificity and sensitivity of deep sequencing compared to microarray and Q-PCR analyses, deep sequencing also allowing identification of novel MiRNA isoforms. MiR-378 molecules were strongly under-expressed both in ARMS and ERMS tumours, this showing that the regulation of these miRNAs does not differentiate among RMS subtypes. Interesting preliminary results showed a significant difference in the abundance of one specific miR-378a-3p 3′ isoform between RMS and NSM. miR-378 molecules are encoded by different genes but they share identical seed sequences for mRNA target recognition. The most important regulatory small RNA of this family is miR-378a-3p, whose expression is highly variable in different cancers, reflecting the complexity and specificity of the miRNA response [46–48]. In addition, some studies suggest that miR-378a-5p, the opposite strand of miR-378a-3p, also has a functional relevance [49, 50]. Our data confirmed that miR-378a-3p was significantly reduced not only in RMS tumours but also in human RMS cell lines, this finding suggesting a role of miR-378a-3p as a tumour suppressor. Among the main pathways identified as potential targets for miR-378a-3p in our bioinformatics analysis, we chose to focus on IGF1R/AKT signalling, which has a well-established role in cellular growth and survival as well as in myogenic differentiation [40, 51, 52]. Ectopic expression of miR-378a-3p in RH30, an ARMS-derived cell line, markedly reduced IGF1R protein levels and promoted apoptosis via phospho-AKT inhibition and concomitant caspase-3 activation. Furthermore, miR-378a-3p-transfected cells moderately reduced cell proliferation and markedly cell migration. Accordingly, miR-378a-3p has been shown to be involved in carcinogenesis and an anti-oncogenic function has been suggested both in nasopharyngeal  and gastric cancer  through the negative regulation of VEGF expression, a protein promoting cell migration and inhibiting apoptosis. In a study by Martins et al. , RMS development seemed to involve increased IGF1R expression, which in turn enhanced AKT and Bcl-XL-mediated cell survival. The moderate effects on apoptosis and cell growth we observed in RH30 cells could be explained by the presence of the t(2;13)(q35;q14) translocation, since PAX3/FOXO1 chimeric protein is directly involved in the regulation of the IGF1R expression.
In accordance with the observation that in RMS proliferation and migration are inversely related to myogenic differentiation [3, 55], we found that miR-378a-3p mimic transfection was also able to promote myogenesis, as shown by the modulated levels of specific differentiation markers and by the changes in the cell morphology, from star-round to a more elongated appearance. In particular, normalization of miR-378a-3p expression reduced the levels of MyoR, a MyoD1 inhibitor, thus contributing to reactivate myogenesis; this was reflected by the MyHC levels, which is specifically related to committed muscle cells. Furthermore, miR-378a-3p transfected RH30 cells showed a more concentrated assembly of actin bundles, a characteristic of the intermediate muscle differentiation stage . In accordance with these results, recent works have reported miR-378a-3p as a new candidate in skeletal muscle development [37, 57]. In C2C12 murine proliferating myoblasts, up-modulation of miR-378 has been proposed as allowing the efficient myotube formation by repressing antagonists of differentiation, like MyoR . Likewise, the expression pattern of miR-378 in porcine longissimus muscles is closely related to myogenesis regulation, mainly with fibre formation . Re-activation of skeletal muscle development in RMS seems to be linked to the IGF1R inhibition, as demonstrated by miR-1 and miR-133a [51, 58]. Indeed, the marked down-regulation of miR-378a-3p, miR-1 and miR-133a, and the concomitant up-regulation of IGF1R observed in RMS tumours could explain why cancer cells are prohibited from undergoing terminal differentiation despite their commitment to a myogenic pathway.
This study also underlines the relationship between aberrant epigenetic regulation of tumour suppressors, including miRNAs, and cancer development. The presence of CpG sites in the promoter regions of the miR-378 family members has recently been suggested and partially experimentally demonstrated in other neoplasms [47, 59–61]. In our in vitro RMS model, treatment with 5-aza-2′-deoxycytidine, a demethylating agent, was able to restore the expression of mature miR-378a-3p in a dose-dependent manner but without direct effect on DNA methylation status. Therefore, the hyper-methylation of CpG islands at the miR-378a-3p promoter region does not represent a causative mechanism for the marked down-regulation of this miRNA in RMS. Indeed, many CpG islands located at genes that have a tissue-restricted expression pattern, can remain methylation-free even when their associated genes are silent. Inactive unmethylated CpG island promoters show elevated levels of dimethylation at histone H3 lysine 4 (H3K4), suggesting that this chromatin mark may protect DNA from methylation . In line with this model, recruitment of H3K4 methylases by unmethylated CpGs has recently been suggested . Notwithstanding, our data imply that epigenetic modifications may play an indirect role on miR-378a expression by acting on specific transcription factors able to bind the miR-378a promoter region, which also warrant further study. Consistent with the up-regulation of miR-378a-3p levels, 5-aza-dC exposure affected apoptosis, proliferation and migration of RMS cells, with more dramatic effects. In particular, the resulting DNA hypomethylation arrested the cell cycle at G2-phase with a concomitant induction of myogenic differentiation, as demonstrated by the more elongated cell morphology and enhanced MyHC expression. Most likely, the fact that azacytidine has a much broader effect on the RH30 cellular phenotype reflects the fact that the transcriptional activation is not limited to miR-378a-3p but involves several epigenetically silenced genes. Aberrant DNA methylation and histone deacetylation was reported to have a key role in silencing plakoglobin gene expression in ARMS cells, whose over-expression was shown to suppress tumorigenicity in different cancer cell lines . Moreover, recent findings suggest that suppression of BMP2 by epigenetic silencing may play a critical role in the genesis of RMS . Based on these findings, 5-aza-dC might be a promising therapeutic agent in patients with RMS. To date, azacytidine is FDA-approved for the treatment of patients with acute myeloid leukemia and myelodysplastic syndrome [66, 67]. Data in sarcomas are still limited , but ongoing clinical trials in the field of epigenetic treatment suggest that this approach may be a potential option for patients with sarcomas, including RMS .
This study suggests that miR-378a-3p is involved in RMS pathogenesis, exerting tumour suppressor-like activity. Since miR-378a-3p is down-regulated in other malignancies, the identification of the regulated tumorigenic pathways might have important implications in oncology. A better understanding of the biological mechanisms underlying RMS development represents a fundamental step in order to identify novel diagnostic and prognostic markers, as well as to develop targeted therapies in children and adolescents with RMS. Indeed, a combination of molecules inhibiting IGF1R signalling and promoting demethylation might be a possible therapeutic strategy worth to be further investigated in this tumour.
This work was partially supported by “Associazione Onlus Fabrizio Procaccini”, “Io…domani, Associazione Onlus per la Lotta contro i Tumori Infantili” (ALTI), “Isle of Man Anti-Cancer Association”, Italian “Ministero dell'Istruzione, dell'Università e della Ricerca” (MIUR), and Genomnia srl R&D funds.
The Authors are grateful to: Dr. David N. Shapiro for providing RH4 and RH30 ARMS cell lines; Prof. Pier Luigi Lollini for providing RD and RD18 ERMS cell lines; Ms. Olga Mannarino for technical assistance; Dr. Simona Ceccarelli and Dr. Saula Checquolo for Apotom image acquisition; and Dr. Bettina Nadorp for useful discussions on miR-378 family promoter characterisation.
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