The results of profiling miRNA using genomic methods have proven to be very platform dependent. This can be explained by differences the probe design methods which at times address all possible isoforms or sometimes just target one very specific isoform.
Some of the differences observed between Agilent and Exiqon may be related to this fact. Agilent arrays use hairpin probes that require a perfect match of the 3′ end of the probe, therefore being very specific for the major reference mature isoform and less likely to detect isomiRs different from the reference isoform. On the other hand, Exiqon probe design in principle could cross-hybridize to pre-miRNA and different isomiRs from a specific class. This could explain why more miRNAs show differential expression in Exiqon. For example, miR-21 was not found to be differentially expressed on the Agilent platform, and this could be explained by the fact that the miRBase reference sequence interrogated by the hairpin loop probes is the minor isoform in HeLa cells.
Small RNA-seq is a powerful methodology that provides a qualitative improvement on the microarray technology. We have shown that it is able to detect changes similar to those detected by microarrays, as well as revealing the underlying isoform complexity that cannot be captured using microarray based assays.
In our hands, both platform approaches, high-throughput sequencing and microarrays, are complementary techniques and not mutually exclusive. We observed high agreement between both strategies for the highest ranked targets using Rankprod, which were further cross-validated using RT-qPCR.
We compared our results with those reported in a recent study describing the temporal response to EGF treatment of HeLa cells and found little overlap (Avraham et al., 2010). We believe this is due to the fact that in our study we compare the treated and untreated transcriptome at the time of interest whereas the experimental design of this other work compares to a baseline at the onset of treatment. Whereas our design would focus on the specific EGF dependent response, the comparison to the baseline may be biased toward temporal rather than treatment specific responses. In agreement with this, subtraction of control 15 minute time point in our dataset showed similar temporal profiles for most genes with or without exposure to EGF, indicating that exposure to reduced serum used in EGF exposure experiments may account for most of the responses reported previously, and therefore the response attributed to EGF in the work of Avraham may be confounded. This would explain why in our hands the number of EGF responsive miRNAs may be reduced relative to theirs.
In agreement with our observations, recent results derived from lung cancer cell lines where EGFR transcripts were targeted by shRNA expression resulted in miRNA targets highly overlapping with our own targets (including miR-221, miR-222, miR-29a,b,c) also found to be targets of MET tyrosine kinase receptor . At the functional level this would identify such targets as core to EGFR (and MET) tyrosine kinase signaling in other cell types.
EGF-dependent miRNAs in cancer
EGF treatment leads to the activation of a network of signaling pathways which leads to the up-regulation of anti-apoptotic proteins or to the inactivation of pro-apoptotic proteins. The post-transcriptional and expression events implicated in these pathways have been a matter of intense study during the last decade [5–7, 16]. A critical question about signal transduction is how a weak or transient activation achieves a robust and long-term switch in gene expression. The discovery of miRNAs opened a new door in the study of the regulation of the downstream events related to these pathways that may help disclose some of these open questions, such us the inhibitory mechanisms leading to attenuation of gene expression . This hypothesis is supported by the fact that Gene Ontology analysis indicates that several of the genes targeted by the up-regulated miRNAs after 6 hours of EGF treatment in our study are involved in the mechanisms that down-regulate the MAP kinase pathway after a mitogenic stimulus.
Recent studies have emphasized causative links between microRNA deregulation and cancer development. In fact, many oncogenes and tumor suppressor genes are regulated by miRNAs ([8, 30–33] for review) and significant down-regulation of most of the miRNAs has been reported in various tumors as compared to normal tissues .
Few studies so far have focused on the potential link between EGF signaling and miRNA [7, 17] although a direct link between EGF signaling and miRNA biosynthesis has been described. Argonaute-2 expression is regulated by epidermal growth factor receptor and mitogen-activated protein kinase signaling and correlates with a transformed phenotype in breast cancer cells  and miR-7 has been found to mediate EGF receptor signaling and promote photoreceptor differentiation in the Drosophila eye . In addition, miR-7 down-regulates EGFR mRNA and protein expression in cancer cell lines (lung, breast, and glioblastoma) and the AKT pathway [37, 38] inhibiting schwannoma cell growth  and inducing cell cycle arrest and cell death, which suggests a negative regulatory mechanism acting on the MAPK pathway . Furthermore, increasing evidence shows that the links between microRNA deregulation and cancer development may be often mediated through the MAPK pathway [41–44]. Interestingly, the 8 miRNAs described to be up-regulated in the present study had already been described to be involved in several processes such as cell growth and proliferation, cell cycle and cellular development among others, besides their implication in several types of cancers.
In vitro studies indicate that miR-21 promotes proliferation and invasion in cultured cells . miR-21 has also been found to be overexpressed under many pathophysiological conditions, especially in cancer ( for review). Indeed, miR-21 has been implicated in various aspects of carcinogenesis, including cellular proliferation, apoptosis, and migration . miR-21 is up-regulated in breast cancer  and this overexpression is correlated with specific breast cancer biopathological features such as advanced tumor stage, lymph node metastasis and poor survival of the patients. miR-21 is also up-regulated in pancreatic cancer  and in hepatocellular carcinoma (HCC) tumors . Furthermore, an aberrant expression of miR-21 can contribute to HCC growth and spreading by modulating Phosphatase and TENsin homolog (PTEN) expression which mediates phenotypic characteristics of cancer cells such as cell growth, migration and invasion .
miR-21 has been also found to target tumor suppressor Programmed Cell Death 4 (PDCD4) and stimulates invasion, intravasation and metastasis in colorectal cancer  and its inhibition in breast cancer cells causes reduced cell growth . Interestingly PDCD4, suppresses tumor progression in human colon carcinoma cells by down-regulating the MEK kinase kinase 1 (MAP4K1) gene transcription , with the consequent inhibition of c-Jun activation and Activator Protein-1 (AP-1)-dependent transcription. Given the role of PDCD4 as a negative regulator of AP-1, the miR-21-mediated down-regulation of PDCD4 seems to be essential for the maximal induction of AP-1 activity in response to RAS transformation . Interestingly we have also found that c-jun expression was down-regulated after 6 h of EGF treatment . Recent studies have implicated the down-regulation of the tumor suppressor gene acidic (leucine-rich) nuclear phosphoprotein 32 family, member A (ANP32A)  and Spry2  of miR-21 to its oncogenic function.
miR-29 regulates the anti-apoptotic Bcl-2 family member, Mcl-1  and DNA methyltransferases (DNMT) 3A and 3B  and is down-regulated in lung cancer where DNMT is frequently up-regulated. In addition, several studies have implicated miR-29 in several types cancers such as acute myeloid leukemia , chronic lymphocytic leukemia , hepatocellular carcinoma  and cutaneous melanoma .
miR-132 is elevated in pancreatic cancer  and in the monocytic leukemia cell line THP-1 by Lipopolysaccharide (LPS) treatment. LPS triggers activation of NF-κB and AP-1 transcription factors and results in up-regulation of immune-responsive genes. REST (Re1 silencing transcription factor) and its cofactor complex are targets of miR-132 . Given the role of both REST and miRNA as repressors, this suggests a double-negative feedback loop between REST and the miR-132 for stabilizing and maintaining neuronal gene expression. miR-132 is also induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light .
miR-31 is a pleiotropic miRNA up-regulated in colorectal tumors and its expression levels are correlated with the stage of the tumor . On the contrary, it has been found to be down-regulated greater than two-fold in breast cancer . miR-31 also inhibits breast cancer metastasis  and over-expression of miR-31 significantly enhanced proliferation and tumorigenicity of lung cancer cells . An interesting recently published study indicates that restoring miR-31 function in already-established metastases induces metastatic regression and opens the possibility of intervention strategies centered in restoring miR-31 function in order to combat metastasis .
Distinct up-regulations of miR-222, miR-221, and miR-31 have been observed in HCC . miR-221 and miR-222 are potent regulators of proliferation, mediated by regulation of p27Kip, a cell cycle inhibitor and tumor suppressor . High levels of miR-221 and miR-222 appear in human thyroid papillary carcinomas , prostate cancer  and in glioblastomas [36, 73] and correlate with low levels of p27kip. Indeed, sustained activation of ERK1/2 by NGF induces miR-221 and miR-222 in PC12 cells .
Finally, miR-215 has been described to be a p53-responsive miRNA  able to induce cell cycle arrest. miR-215 suppresses Denticleless protein homolog expression, inducing a decreased cell proliferation by causing G2-arrest  and contributing to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support .
Blocking of miR-29a, miR-29b, miR-221 and miR-222 expression by the selective MEK inhibitor U0126 in an analogous manner to AG1478 and the overexpression of these miRNAs in RasV12 transfected cells suggest that the MAPK pathway is a critical effector downstream of EGFR for the transmission of the information leading to miRNAs regulation. The same effect but to a lesser extent is observed after PI3K/AKT pathway inhibition with wortmannin. Indeed, both MAPK and PI3K/AKT pathways are activated by EGF-treatment controlling cell fates such as cell proliferation, survival and apoptosis [78, 79]. Expression of most of the miRNAs regulated by EGF found in the present work induces a decrease (miR-7, miR-215) or increase in cell proliferation (miR-21, miR-221, miR-222, miR-31).
Further studies are required to identify their in vivo targets and to elucidate the specific regulatory networks where they are involved.
The results obtained from isomiR analysis show the presence of sequence heterogeneity for all the miRNA analyzed, supporting the idea of a novel degree of complexity for the miRNA transcriptome [67, 80] as a result of inexact Dicer processing. Furthermore, some of the miRNA species studied have a majority of mature miRNA sequences that differ from that listed on databases. Lee et al. catalogued miRNA end heterogeneity from different biological samples and found that from 34% to 51% of the detectable miRNA species have a predominant sequence differing from that listed in databases . However, the equal proportion of the different types of variants in serum-starved versus EGF-treated indicates that the molecular mechanisms involved in the generation of these variants are not regulated by EGF. Several studies already confirmed the presence of these variants in multiple and diverse kinds of samples, tissues and species, however, up to this date, there is no data regarding the functional role of isomiR diversity as well as their generation mechanisms besides a recent study suggesting that 5′ end variations may produce specificity changes in the type of AGOs with which these miRNA isoforms preferentially associate . In addition a biological role for isomiRs is supported by the finding of regulation of variability in isomiR expression .