Deregulated microRNAs in myotonic dystrophy type 2 - PubMed (original) (raw)

Deregulated microRNAs in myotonic dystrophy type 2

Simona Greco et al. PLoS One. 2012.

Abstract

Myotonic Dystrophy Type-2 (DM2) is an autosomal dominant disease caused by the expansion of a CCTG tetraplet repeat. It is a multisystemic disorder, affecting skeletal muscles, the heart, the eye, the central nervous system and the endocrine system. Since microRNA (miRNA) expression is disrupted in Myotonic Dystrophy Type-1 and many other myopathies, miRNAs deregulation was studied in skeletal muscle biopsies of 13 DM2 patients and 13 controls. Eleven miRNAs were deregulated: 9 displayed higher levels compared to controls (miR-34a-5p, miR-34b-3p, miR-34c-5p, miR-146b-5p, miR-208a, miR-221-3p and miR-381), while 4 were decreased (miR-125b-5p, miR-193a-3p, miR-193b-3p and miR-378a-3p). To explore the relevance of DM2 miRNA deregulation, the predicted interactions between miRNA and mRNA were investigated. Global gene expression was analyzed in DM2 and controls and bioinformatic analysis identified more than 1,000 miRNA/mRNA interactions. Pathway and function analysis highlighted the involvement of the miRNA-deregulated mRNAs in multiple aspects of DM2 pathophysiology. In conclusion, the observed miRNA dysregulations may contribute to DM2 pathogenetic mechanisms.

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Conflict of interest statement

Competing Interests: F. Martelli and M.C. Capogrossi are members of the PLoS One Editorial Board. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. All the other authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Experimental Plan.

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.

Figure 2

Figure 2. Profiling of miRNAs in DM2 patients and CTR.

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).

Figure 3

Figure 3. Validation of miRNA modulations in DM1 and DM2 patients.

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).

Figure 4

Figure 4. Discrimination between DM2 and control groups using the DM2 miRNA score.

(A) Dot plot of the DM2 miRNA score in DM2 patients and CTR. The segments among the dots indicate median values for each group and the difference is statistically significant (DM2 = 13, CTR n = 13; p<0.0001). (B) ROC curve displaying DM2 miRNA score discrimination between the DM2 and CTR groups (AUC = 0.96).

Figure 5

Figure 5. Correlation of atrophy and hypertrophy with DM2 miRNAs score.

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 (blue arrows) and hypertrophic (asterisks) myofibers, as well as nuclear clumps (red arrows) and internal nuclei (black arrows) 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).

Figure 6

Figure 6. Validation of miRNA/mRNA interactions in DM2 patients.

Validation by qPCR of the expression of randomly selected mRNAs either up-(A) and down-(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).

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