The discovery of microRNAs (miRNAs, miRs) led to a profound change on our vision about the regulation of gene expression in eukaryotes. MicroRNAs are an emerging class of small non-coding endogenous RNAs that participate on the fine tuning of gene expression at the post-transcriptional level. First discovered at the early 90s in the nematode
Neuroblastoma is the most common extracranial solid tumor in childhood and the most common tumor in infants, which originates from aberrant development of primordial neural crest cells. Several lines of evidence support the idea that microRNA deregulation could contribute to neuroblastoma pathogenesis and progression [2, 3], and the usefulness of miRNA profiles for neuroblastoma diagnostics, classification and prognosis has been recently reported . Neuroblastoma cell lines can be induced to differentiate
In this article we want to review the evidences supporting the contribution of miRNA regulation to RA-induced differentiation of neuroblastoma cells. We will show that miRNA contribute to the gene-expression changes associated with neuroblastoma cell differentiation and that specific RA-induced miRNAs target the expression of relevant genes in the context of neural differentiation. In addition RA-regulated miRNAs contribute to the reduction in the biological aggressiveness elicited by RA
2. The molecular bases of miRNA action
2.1. Biogenesis of miRNAs
miRNAs use complementary base pairing and the RNA induced silencing complex (RISC) to bind and either block translation and/or promote degradation of their target mRNAs. miRNAs are 19-22 nt-long RNA molecules transcribed mainly from non-coding regions of the genome, although some are embedded within genes, primarily as part of intronic sequences . In addition, clusters of miRNAs were also found in the genome . miRNAs are transcribed as large hairpin-containing molecules, called pri-miRNA, that are cleaved in the nucleus by the microprocessor complex, involving Drosha and Pasha/DGCR8 proteins [16, 17]. The result of this cleavage is a shorter precursor hairpin (approx. 70 nt), called pre-miRNA. Pre-miRNAs are exported through RAN GTPase and exportin-5 to the cytoplasm  where undergo further cleavage by Dicer to yield a transient intermediate imperfect duplex of approx 19-22 bp miRNA . Subsequently, the duplex unwinds and miRNA strand is loaded into RISC complex together with proteins of the Argonaute (Ago) family . The miRNA strand in RISC acts as a guide strand to find the complementary site in mRNA, and thereby suppressing the translational activity of the target mRNA. The complementary strand (known as miRNA* or as passenger strand) is degraded when the duplex is unwound, although recent evidences show that in some cases miRNA* accumulated at physiological levels and support the idea of a role for miRNA* on gene regulation . (see Figure 1)
2.2. miRNA target binding
miRNAs interact primarily with the 3’-untranslated (3’UTR) region of their target mRNAs, although recent evidences show that miRNAs can also associate with sites located within the coding region of target genes . In fact, complex arrays of multiple binding sites for either the same or different miRNAs located both in the 3’UTR as well as in the coding region of the target genes have been reported . The base pairing of miRNA and mRNA in vertebrates requires only partial homology, with a preference for contiguous pairing occurring only at the
2.3. Suppression of mRNA translation and/or mRNA degradation mediated by miRNAs
The binding of miRNA-RISC complex to its cognate mRNA target leads to mRNA silencing through suppression of mRNA translation and/or mRNA decay. [25, for review] Several mechanisms involving different protein complexes have been proposed. mRNA translation could be blocked at initiation step as well as post-initiation stages. The miRNA-RISC complex inhibits translation initiation by interfering with eIF4F-cap recognition and 40S small ribosomal subunit recruitment or by antagonizing 60S subunit joining and preventing 80S ribosomal complex formation. The interaction of the GW182 protein with the poly(A)-binding protein (PABP) might interfere with the closed-loop formation mediated by the eIF4G-PABP interaction and thus contribute to the repression of translation initiation. The miRNA-RISC might inhibit also translation at post-initiation steps by inhibiting ribosome elongation, inducing ribosome drop-off, or promoting proteolysis of nascent polypeptides. To promote mRNA degradation, the miRNA-RISC complex interacts with the CCR4-NOT1 deadenylase complex to facilitate deadenylation of the mRNA poly(A) tail. Deadenylation requires the direct interaction of the GW182 protein with PABP. Following deadenylation, the 5′-terminal cap (m7G) is removed by the DCP1-DCP2 decapping complex. Although miRNA-mediated deadenylation followed by mRNA degradation appear to be widespread events, not all miRNA-targeted mRNAs are destabilized. miRNA-targeted translationally repressed mRNAs can accumulate in discrete cytoplasmic foci, such as P or GW bodies, or stress granules. A fraction of GW bodies co-localizes with multivesicular bodies (MVBs), membrane structures that play a role in miRNA-mediated repression. Compelling evidences support a role for miRNAs at the nucleus, acting on transcriptional regulation via chromatin remodeling and epigenetic mechanisms .
3. Profiling miRNA expression during retinoic-acid-induced neuroblastoma cell differentiation
3.1. Profiling miRNA expression during retinoic-acid induced neuroblastoma cell differentiation
Several studies have addressed the changes in the expression of miRNAs upon RA-dependent induction of differentiation of neuroblastoma cells, with somewhat different results depending on the cell line, treatment duration, analysis platform used, etc. [2, 27-30]. To analyze the contribution of microRNA regulation to RA-induced differentiation of neuroblastoma cells, we have studied the changes in the pattern of expression of 667 different human miRNAs upon RA treatment of SH-SY5Y neuroblastoma cells. We used miRNA profiling with TaqMan RT-PCR Low Density Arrays, and we found that 452 miRNAs were expressed above detection level. From them, 42 specific miRNAs change significatively their expression levels (26 upregulated and 16 downregulated) during RA-induced differentiation (Figure 2). This suggests miRNAs as an additional post-transcriptional regulatory layer under RA control .
3.2. A role for miRNAs-10a and -10b in RA-dependent regulation of neuroblastoma differentiation
We have focused our study on the closely related miR-10a and -10b, that showed the most prominent expression changes in SH-SY5Y cell line. Similar results have been reported for other neuroblastoma cell lines, like LA-N-1, LAN5 and SK-N-BE [29, 30].
Loss of function experiments with anti-sense anti-miRs antagonists could show that miR-10a and -10b contribute to the regulation of RA-induced differentiation. RA-induced neurite outgrowth was impaired in cells with experimentally reduced levels of miR-10a or -10b, and the expression of several neural differentiation markers like Tyrosine Kinase receptors
Conversely, the downregulation of the members of the ID gene family,
3.3. miRNAs-10a and -10b contribute to the reduction on the biological aggressiveness of neuroblastoma cells induced by RA
It has been reported that RA treatment of neuroblastoma cells results in a reduction in their biological aggressiveness, by decreasing their migratory and invasive abilities [8-10]. We wanted to analyze whether RA-induced expression of miR-10a and -10b could be related to the reduction in migratory and invasive potential of neuroblastoma cells. To test the migratory potential of SH-SY-5Y cells we used a modified, light-opaque Boyden chamber assay (Falcon HTS FluoroBlok, 8 μm pore size). Cells were transfected with anti-miR-10a or -10b or the corresponding Negative Control anti-miR, treated with 1 μM RA or vehicle in culture medium during 96 h, and labeled in the plate with Calcein AM. Labeled cells were counted and added to the upper chamber of the Boyden chamber, and allow to migrate towards de lower chamber, filled with medium containing 10% FBS as chemoattractant. The results show that indeed RA-treatment reduced the migration of neuroblastoma cells. However suppressing miR-10a or -10b expression not only abolished that reduction but increased migration over basal levels, supporting a contribution of RA-induced miR-10a and 10b to the reduction of migratory activity produced by RA . (Figure 4A)
For invasion assays we used a similar assay, with the difference that the porous membrane separating the upper and lower chambers of the Boyden chamber was covered with BD
To analyze the effects of RA treatment on the metastatic potential of neuroblastoma cells we used the chicken embryo chorioallantoic membrane assays (also known as CAM assay; ). This assay is useful to study intravasation and metastasis
In good agreement with our results, it has been reported that miR-10a and -10b reduces the ability of neuroblastoma cells to form colonies in soft agar , a phenotype that is characteristic of malignant cells. All these results support the idea that miR-10a and -10b expression contribute to reduction of migratory, invasive and metastatic activities induced by RA. In a recent report it has been shown that a protein involved in cell migration, Tiam1, is targeted by miR-10b in mammary tumor cells. Overexpression of miR-10b suppresses the ability of breast carcinoma cells to migrate and invade . Consistent to that, it has been reported an association between lower miR-10a expression and lower overall survival for a subclass of neuroblastoma tumors (11q- tumor cohort) . However other reports seem to involve the members of the miR-10 family as promoters of migration and metastasis in different tumors [33-40]. This apparent controversy may suggest that the role of the microRNAs from the miR-10 family in tumorigenesis and metastasis would depend on their molecular targets and therefore would depend on the cellular context.
4. Molecular targets of miR-10a and -10b in the differentiation of neuroblastoma cells
4.1. The search for the molecular targets for miRNAs
The identification of molecular targets for miRNAs is a crucial step towards the understanding of miRNA function. Because an ever growing number of experimentally validated targets for miRNAs are being reported, a simple way to identify miRNA targets is to search for validated targets in the literature or in databases as TarBase . A validated target for miR-10b in breast cancer cells is the homeobox gene
A lot of effort has been made to generate computational miRNA target prediction tools [reviewed in 43], mainly based on the search for complementary sequences in the genome. However, that is not an easy task, because short sequences are problematic for the algorithms usually developed for complementarity analysis. As indicated in 2.2, the base pairing of miRNA and mRNA in vertebrates requires only partial homology, with a preference for contiguous pairing occurring only at the “seed” region, located at nucleotides 2-7 of the guide strand, and this makes even more difficult to find the right target sequence in the genome. Several authors have approached this problem from different startpoints, using mainly complementarity analysis of the complete miRNA sequence, complementarity analysis of the seed sequence, or adding thermodynamic stability analysis of duplex sequences or 3’UTR sequence conservation to the complementarity analysis. Nowadays a set of miRNA target prediction resources are available, mainly as web-based tools. However it becomes striking to the new users of these tools how different results can be obtained when using the same sequence with different prediction tools. In addition, prediction tools generate lists of hundreds of genes for each of the miRNAs, and the fact of having sequence diversity at the 3’UTR by alternative polyadenylation sites could also complicate the analysis [for discussion, see 44].
To find relevant targets for miR-10a and-10b in neuroblastoma cells we choose to combine bioinformatic prediction tools together with experimental analysis. We created a list of potential miR-10a and -10b targets by including the common predicted genes using three different prediction resources: miRbase targets , TargetScan  and PicTar . Only mRNAs that contained evolutionarily conserved miRNA binding sequences on their 3’UTR were considered. This list was crossed with the dataset of an Affymetrix microarray experiment containing the genes downregulated after 48 h RA treatment. In the resulting list, two members of the Arginine/serine-rich splicing factors,
4.2. Regulation of
SFRS1 (SF2/ASF) by miR-10a and -10b
The regulation of
4.3. Regulation of
NCOR2 (SMRT) by miR-10a and -10b
The regulation of
MicroRNAs are essential players in the process on neural differentiation of neuroblastoma cells, and contribute to the transduction of Retinoic Acid signaling. In addition, miRNAs have been reported to participate in the pathogenesis and progression of human neuroblastoma tumors [2, 3], and miRNA profiles have been recently proven to be useful for classification and prognosis . Finally miRNAs open new avenues for the treatment of neuroblastoma cells, and proof of concept experiments showing a therapeutic action of miRNA-based treatments in animal models of neuroblastoma [52-54] and other tumors  have been reported. Therefore, we have to expect in the next years an increased interest in the study of microRNAs among the neuroblastoma researchers community.
AcknowledgmentsThe research work described in this article was financed through grants of the Spanish National Plan for Research, Development and Innovation (SAF2007-60780, SAF2010-15032 and SAF2011-23869), Generalitat Valenciana (ACOMP 09/212) and Genoma España to D. Barettino. S. Meseguer was the recipient of an EACR training and travel fellowship award and a CSIC I3P predoctoral fellowship/contract.
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