Identification and Differential Expression of MicroRNAs during Metamorphosis of the Japanese Flounder (Paralichthys olivaceus)

BACKGROUND: MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20-25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA. METHODOLOGY/PRINCIPAL FINDINGS: Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis. CONCLUSIONS/SIGNIFICANCE: We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder

Wnt signaling in triple-negative breast cancer

Wnt signaling regulates a variety of cellular processes, including cell fate, differentiation, proliferation and stem cell pluripotency. Aberrant Wnt signaling is a hallmark of many cancers. An aggressive subtype of breast cancer, known as triple-negative breast cancer (TNBC), demonstrates dysregulation in canonical and non-canonical Wnt signaling. In this review, we summarize regulators of canonical and non-canonical Wnt signaling, as well as Wnt signaling dysfunction that mediates the progression of TNBC. We review the complex molecular nature of TNBC and the emerging therapies that are currently under investigation for the treatment of this disease

Super-induction of Dicer-2 expression by alien double-stranded RNAs: an evolutionary ancient response to viral infection?

Dicer-2 is a ribonuclease involved in the insect RNAi pathway. On attempting to knockdown Dicer-2 expression in the insect Blattella germanica by RNAi, we found that treatment with Dicer-2 dsRNA upregulated the targeted mRNA. This unexpected result was also observed after treating with a nucleopolyhedrovirus dsRNA. Experiments with this alien dsRNA showed an all-or-none response with a threshold for inducing Dicer-2 upregulation between 0.4 and 0.04 μg in terms of dsRNA concentration and between 50 and 20 bp in terms of dsRNA length. The response seems specific of dsRNA given that equivalent experiments carried out with dsDNA did not affect Dicer-2 expression. In insects, Dicer-2 is postulated to be a sensor of viral infections and a key antiviral defense element. The upregulation of Dicer-2 expression after dsRNA administration fits well with this sensor role, and the occurrence of this mechanism in B. germanica, a phylogenetically basal insect, suggests that sensing alien RNAs might be an ancestral function of Dicer-2 proteins.</p

MicroRNA-dependent metamorphosis in hemimetabolan insects

How does a juvenile insect transform into an adult? This question, which sums up the wonder of insect metamorphosis, has fascinated mankind since ancient times. Modern physiology has established the endocrine basis regulating these transformations, which mainly depend on two hormone types: ecdysteroids, which promote molts, and juvenile hormones, which repress the transformation into the adult stage. The interplay of these two hormones regulates the genes involved in juvenile and adult programs and the shift from one to the other. microRNAs (miRNAs) are small noncoding RNAs, which participate in many biological processes, and we wondered whether they might be also involved in insect metamorphosis. In insects, Dicer-1 ribonuclease transforms miRNA precursors into mature miRNAs. Thus, using systemic RNA interference (RNAi) to silence the expression of Dicer-1 in the hemimetabolan insect Blattella germanica, we depleted miRNA contents in the last instar nymph. This practically inhibited metamorphosis after the next molt, as the resulting specimens showed nymphoid features and were able to molt again. The experiments show that miRNAs play a key role in hemimetabolan metamorphosis, perhaps regulating genes that are juvenile hormone targets

Role of Methoprene-Tolerant (Met) in Adult Morphogenesis and in Adult Ecdysis of <i>Blattella germanica</i>

<div><p>Juvenile Hormone (JH) represses metamorphosis of young instars in insects. One of the main players in hormonal signalling is Methoprene-tolerant (Met), which plays the role of JH receptor. Using the Polyneopteran insect <i>Blattella germanica</i> as the model and RNAi for transcript depletion, we have confirmed that Met transduces the antimetamorphic signal of JH in young nymphs and plays a role in the last nymphal instar moult in this species. Previously, the function of Met as the JH receptor had been demonstrated in the Eumetabola clade, with experiments in Holometabola (in the beetle <i>Tribolium castaneum</i>) and in their sister group Paraneoptera (in the bug <i>Pyrrhocoris apterus</i>). Our result shows that the function of Met as JH receptor is also conserved in the more basal Polyneoptera. The function of Met as JH transducer might thus predate the evolutionary innovation of metamorphosis. Moreover, expression of Met was also found in last nymphal instar of <i>B. germanica</i>, when JH is absent. Depletion of Met in this stage provoked deficiencies in wing growth and ecdysis problems in the imaginal moult. Down-regulation of the ecdysone-inducible gene E75A and Insulin-Like-Peptide 1 in these Met-depleted specimens suggest that Met is involved in the ecdysone and insulin signalling pathways in last nymphal instar, when JH is virtually absent.</p></div

The microRNA toolkit of insects

Is there a correlation between miRNA diversity and levels of organismic complexity? Exhibiting extraordinary levels of morphological and developmental complexity, insects are the most diverse animal class on earth. Their evolutionary success was in particular shaped by the innovation of holometabolan metamorphosis in endopterygotes. Previously, miRNA evolution had been linked to morphological complexity, but astonishing variation in the currently available miRNA complements of insects made this link unclear. To address this issue, we sequenced the miRNA complement of the hemimetabolan Blattella germanica and reannotated that of two other hemimetabolan species, Locusta migratoria and Acyrthosiphon pisum, and of four holometabolan species, Apis mellifera, Tribolium castaneum, Bombyx mori and Drosophila melanogaster. Our analyses show that the variation of insect miRNAs is an artefact mainly resulting from poor sampling and inaccurate miRNA annotation, and that insects share a conserved microRNA toolkit of 65 families exhibiting very low variation. For example, the evolutionary shift toward a complete metamorphosis was accompanied only by the acquisition of three and the loss of one miRNA families.This work was supported by the Spanish Ministry of Economy and Competitiveness (grants CGL2012-36251 and CGL2015-64727-P to X.B.), Spanish Ministry of Science and Innovation (grant BFU2011-22404 to M.D.P.) and Catalan Government (2014 SGR 619). The research has also benefited from European Fund for Economic and Regional Development (FEDER) funds. B.F. was supported by the South-Eastern Norway Regional Health Authority grant #201404