The override of ROD1 down-modulation by hypoxia increased apoptosis.

<p>HEK-293 were transfected with plasmids encoding ROD1 (pCMV-ROD1) or with vector alone (pCMV) and the next day were exposed to 1% hypoxia for the indicated time. A) HEK-293 growth curve. Data are expressed as % of T0 normoxic control. Significant differences between pCMV-ROD1 and pCMV transfected cells in the same experimental condition are indicated (n = 5–11; *p<0.001; #p<0.008). B) Cell death assessed by Trypan blue exclusion assay. Data are expressed as % of dead cells for each experimental point. Differences between hypoxic cells transfected with either pCMV-ROD1 or pCMV are statistically significant (n = 5–11; *p<0.001; #p<0.008). C) Transfected cells were exposed to hypoxia for 48 hrs and apoptosis was measured assessing the apoptotic fragmentation of cytoplasmic DNA (n = 3; *p<0.007; #p<0.03).</p

miRNAs modulated by miR-210.

<p>P.Value (moderated t-statistic) adj.P.Val (p-value adjusted for False Discovery Rate).</p

Increased or decreased miR-210 levels are associated to higher or lower ROD1 transcript levels in the RISC, respectively.

<p>HEK-293 were co-transfected with expression vectors for either miR-210 or a scramble sequence, and c-myc-Ago2 (A, miR-210 column and B). Alternatively, HEK-293 (4×10³/cm²) were co-transfected with anti-miR-210 or anti-scramble LNA-oligonucleotides and c-myc-Ago2 (A, anti-210 column and C). Then, c-myc antibody was used to immuno-precipitate c-myc-Ago2-containing complexes. A) miRNA levels in RISCs derived from transfected cells were assayed by qPCR. As expected, miR-210 was enriched or deprived in RISCs derived from cells in which miR-210 levels were up- or down-modulated, respectively, whereas other miRNAs did not show any significant modulation. Average values are expressed using a log2 scale. Green and red colors indicate down- or up-regulation, respectively (n = 3; p<0.01). B) ROD1 mRNA levels in RISCs derived from miR-210-enriched cells were assayed by qPCR. Background controls were represented by c-myc-immuno-precipitates derived from cells transfected with miR-210, but not mAgo2 (n = 7; *p<0.001). C) ROD1 mRNA levels in RISCs derived from miR-210-deprived cells were assayed by qPCR. Background controls were represented by c-myc-immuno-precipitates derived from cells transfected with anti-miR-210 LNA-oligonucleotides, but not mAgo2 (n = 3; *p<0.001; #p<0.01).</p

Pathways and Functions Analysis by IPA.

<p> <b>Table legend:</b></p><p><b>ratio</b> = number of modulated genes/total number of genes present in the relevant IPA category.</p><p><b>enrichment index</b> =  ratio between observed genes and number of genes expected by chance in each category.</p

Characteristics of Profiling Cohorts (Mean±Se).

<p><b>Abbreviations: MRC scale</b> = Medical Research Council scale (0–5 grade); <b>CPK</b> = Creatine PhosphoKinase; <b>FT3</b> = free-triiodothyronine; <b>FT4</b> = free-thyroxine; <b>TSH</b> = thyroid stimulating hormone; <b>ECG</b> =  electrocardiogram; <b>QRS duration</b> = QRS complex of ECG corresponds to the depolarization of the right and left ventricles of the human heart; <b>EF</b> = ejection fraction; <b>ICM</b> = ischemic cardiomyopathy.</p

Validation of miRNA/mRNA interactions in DM2 patients.

<p>Validation by qPCR of the expression of randomly selected mRNAs either up-(<b>A</b>) and down-(<b>B</b>) regulated in DM2 patients vs CTR. Expression level compared to CTR of the targeting miRNA is indicated for each mRNA (DM2 = 12, CTR = 10;*p≤0.05; **p≤0.01; ***p≤0.001; ****p≤0.0001).</p

Correlation of atrophy and hypertrophy with DM2 miRNAs score.

<p>A) Representative stainings of transverse sections of frozen DM2 muscle biopsies. Top panel shows a hematoxylin and eosin staining; bottom panel shows an immunostaining for fast myosin heavy chain. Atrophic (<i>blue arrows</i>) and hypertrophic (<i>asterisks</i>) myofibers, as well as nuclear clumps (<i>red arrows</i>) and internal nuclei (<i>black arrows</i>) are highlighted. B) Spearman’s correlation between DM2 miRNAs score and Atrophy (AR) or Hypertrophy (HR) indexes in fast and slow fibers (DM2 n = 13; CTR n = 9).</p

Experimental Plan.

<p>miRNA (blue) and mRNA (red) expression patterns were determined in DM2 and CTR, and significantly modulated miRNAs and mRNAs were identified. Validation was performed by qPCR for both miRNAs and mRNAs in 13 DM2 patients and 13 CTR. miRNA targets among the mRNAs identified by Class Comparison analysis were predicted by dedicated softwares. Only miRNA/mRNA couples displaying inverse significant correlation were selected and then analyzed by Ingenuity Pathway Analysis software that allowed to identify enriched molecular pathways and functions.</p

Profiling of miRNAs in DM2 patients and CTR.

<p>In the heat map on the left, mean miRNAs expression values are shown in a log₂ scale (-ΔΔCt), where red and green indicate positive and negative modulation respectively. The table on the right shows the same values in a linear scale (DM2 = 13, CTR  = 13; *p≤0.05 **p≤0.01).</p

Validation of miRNA modulations in DM1 and DM2 patients.

<p>In the heat map on the left, the mean values of miRNA expression in both DM1 and DM2 compared to controls are shown in a log₂ scale (-ΔΔCt), where red and green indicate positive and negative modulation respectively. On the right, the table shows the same values in a linear scale; statistically significant differences are highlighted in bold (DM2 n = 13, DM1 = 16, CTR n = 13; *p≤0.05 **p≤0.01 ***p≤0.001).</p