<i>sox6</i> and <i>rod1</i> gene expression (miR-499 targets) in red and white skeletal muscle fibers of Nile tilapia.

<p>RT-qPCR shows gene expression levels of <i>sox6</i>, <i>rod1</i>, and <i>myh7b</i> (miR-499 host gene) between muscle cell types (A). Relative quantification values are shown on Log 2 basis. *Statistical significance in comparison to white muscle (P<0.05). RM = red muscle; WM = white muscle; RQ = relative amount of transcripts. Fluorescent <i>in situ</i> hybridization demonstrates the spatial location of <i>sox6</i> mRNA at <i>200x</i> magnification (B and C) and <i>400x</i> magnification (D and E) and <i>rod1</i> mRNA at <i>200x</i> magnification (F and G) and <i>400x</i> magnification (H and I) in red and white muscle fibers.</p

miR-499 binding sites at 3´UTR of <i>sox6</i> and <i>rod1</i> genes from 22 species of vertebrates.

<p>Red letters correspond to the seed regions of miR-499. Blue letters stand for complementary base pairing regions (microRNA recognition elements or MRE) at 3´UTR of <i>sox6</i> and <i>rod1</i> mRNAs. Dashes are manually inserted gaps, and dots represent continuity of the target mRNAs at both the 5' and 3' ends.</p

Comparative analysis of protein domains and <i>in silico</i> prediction of protein-to-protein interactions (PPI) in the miR-499 gene regulatory network.

<p>(A) Alignment of SOX6 and ROD1 sequences retrieved from representatives of the main vertebrate groups. (B) mir-499 gene regulatory network based on PPI filtered using gene ontology. miR-499 are indicated as blue nodes and other myomiRs as white nodes, whereas green nodes correspond to miR-499 target genes evaluated. Transcription factors are shown as dashed-line border nodes, and validated interactions are shown as thick-line bridges.</p

MicroRNA-499 Expression Distinctively Correlates to Target Genes <i>sox6</i> and <i>rod1</i> Profiles to Resolve the Skeletal Muscle Phenotype in Nile Tilapia

<div><p>A class of small non-coding RNAs, the microRNAs (miRNAs), has been shown to be essential for the regulation of specific cell pathways, including skeletal muscle development, maintenance and homeostasis in vertebrates. However, the relative contribution of miRNAs for determining the red and white muscle cell phenotypes is far from being fully comprehended. To better characterize the role of miRNA in skeletal muscle cell biology, we investigated muscle-specific miRNA (myomiR) signatures in Nile tilapia fish. Quantitative (RT-qPCR) and spatial (FISH) expression analyses revealed a highly differential expression (forty-four-fold) of miR-499 in red skeletal muscle compared to white skeletal muscle, whereas the remaining known myomiRs were equally expressed in both muscle cell types. Detailed examination of the miR-499 targets through bioinformatics led us to the <i>sox6</i> and <i>rod1</i> genes, which had low expression in red muscle cells according to RT-qPCR, FISH, and protein immunofluorescence profiling experiments. Interestingly, we verified that the high expression of miR-499 perfectly correlates with a low expression of <i>sox6</i> and <i>rod1</i> target genes, as verified by a distinctive predominance of mRNA destabilization and protein translational decay to these genes, respectively. Through a genome-wide comparative analysis of SOX6 and ROD1 protein domains and through an <i>in silico</i> gene regulatory network, we also demonstrate that both proteins are essentially similar in vertebrate genomes, suggesting their gene regulatory network may also be widely conserved. Overall, our data shed light on the potential regulation of targets by miR-499 associated with the slow-twitch muscle fiber type phenotype. Additionally the results provide novel insights into the evolutionary dynamics of miRNA and target genes enrolled in a putative constrained molecular pathway in the skeletal muscle cells of vertebrates.</p></div

Immunoexpression of SOX6 and ROD1 in red and white skeletal muscle fibers of Nile tilapia.

<p>Fluorescent immunostainning demonstrates the SOX6 protein expression at <i>200x</i> magnification (A and B) and <i>400x</i> magnification (C and D) and ROD1 protein expression at <i>200x</i> magnification (E and F) and <i>400x</i> magnification (G and H) on red and white muscle fibers. Both of them are expressed only in muscle fiber. RM = red muscle; WM = white muscle.</p

MyomiR expression profile in red and white skeletal muscle fibers of joined males and females adults of Nile tilapia fish.

<p>RT-qPCR shows gene expression levels of myomiRs miR-1, -133a, -133b, -206, and -499 between red and white muscle (A). Relative quantification (RQ) values are relative amount of transcripts shown on linear basis. *Statistically significant in comparison to white muscle (P<0.05). MicroRNA fluorescence <i>in situ</i> hybridization with LNA probes demonstrates the tissue location of miR-1 at <i>200x</i> magnification (B and C) and <i>400x</i> magnification (D and E); miR-206 at <i>200x</i> magnification (F and G) and <i>400x</i> magnification (H and I); and miR-499 at <i>200x</i> magnification (J and K) and <i>400x</i> magnification (L and M) on red and white skeletal muscle fibers. The miR-1 and miR-206 are clearly expressed in muscle fibers and also connective tissue cells (arrow-head), whereas miR-499 is muscle-fiber-specific. RM = red muscle; WM = white muscle.</p

mRNA and protein levels of the skeletal muscle proteolysis inhibitor PGC1α.

<p>A and D: mRNA levels in the soleus (A) and plantaris (D) muscles; protein content in the soleus (B) and plantaris (E) muscles; representative blots of soleus (C) and plantaris (F) muscles in Sham-UN, Sham-ET, AS-UN, and AS-ET groups. Data are presented as the mean ± SD; *Significant difference from the Sham-UN group, p<0.05; †p<0.05 from the AS-UN group.</p

mRNA and protein levels of skeletal muscle hypertrophy components.

<p>A and D: mRNA levels in the soleus (A) and plantaris (D) muscles; protein content in the soleus (B) and plantaris (E) muscles; representative blots of soleus (C) and plantaris (F) muscles from Sham-UN, Sham-ET, AS-UN, and AS-ET groups. Data are expressed as the mean ± SD; *Significant difference from the Sham-UN group, p<0.05; #p<0.05 vs. Sham-ET; †p<0.05 from the AS-UN group.</p

Cross sectional area (µm²) of the two major soleus muscle fiber types after ET.

<p>Data are expressed as mean ± SD.</p><p>*p<0.05 vs. Sham-UN;</p>†<p>p<0.05 vs. AS-UN.</p><p>One-way ANOVA + Tukey test.</p><p>Cross sectional area (µm²) of the two major soleus muscle fiber types after ET.</p

Deregulated miRNAs in adenocarcinoma of Vater ampulla (AMP).

Deregulated miRNAs in adenocarcinoma of Vater ampulla (AMP).</p