Overexpression of microRNA-206 in the skeletal muscle from myotonic dystrophy type 1 patients

Stefano Gambardella, Fabrizio Rinaldi, Saverio M Lepore, Antonella Viola, Emanuele Loro, Corrado Angelini, Lodovica Vergani, Giuseppe Novelli, Annalisa Botta, Stefano Gambardella, Fabrizio Rinaldi, Saverio M Lepore, Antonella Viola, Emanuele Loro, Corrado Angelini, Lodovica Vergani, Giuseppe Novelli, Annalisa Botta

Abstract

Background: MicroRNAs are highly conserved, noncoding RNAs involved in post-transcriptional gene silencing. They have been shown to participate in a wide range of biological processes, including myogenesis and muscle regeneration. The goal of this study is to test the hypothesis that myo-miRs (myo = muscle + miR = miRNA) expression is altered in muscle from patients affected by myotonic dystrophy type 1 (DM1), the most frequently inherited neuromuscular disease in adults. In order to gain better insights about the role of miRNAs in the DM1 pathogenesis, we have also analyzed the muscular expression of miR-103 and miR-107, which have been identified in silico as attractive candidates for binding to the DMPK mRNA.

Methods: To this aim, we have profiled the expression of miR-133 (miR-133a, miR-133b), miR-1, miR-181 (miR-181a, miR-181b, miR-181c) and miR-206, that are specifically induced during myogenesis in cardiac and skeletal muscle tissues. miR-103 and miR-107, highly expressed in brain, heart and muscle have also been included in this study. QRT-PCR experiments have been performed on RNA from vastus lateralis biopsies of DM1 patients (n = 7) and control subjects (n = 4). Results of miRNAs expression have been confirmed by Northern blot, whereas in situ hybridization technique have been performed to localize misexpressed miRNAs on muscle sections from DM1 and control individuals.

Results: Only miR-206 showed an over-expression in 5 of 7 DM1 patients (threshold = 2, fold change between 1.20 and 13.22, average = 5.37) compared to the control group. This result has been further confirmed by Northern blot analysis (3.37-fold overexpression, R2 = 0.89). In situ hybridization localized miR-206 to nuclear site both in normal and DM1 tissues. Cellular distribution in DM1 tissues includes also the nuclear regions of centralized nuclei, with a strong signal corresponding to nuclear clumps.

Conclusions: This work provides, for the first time, evidences about miRNAs misexpression in DM1 muscle tissues, adding a new element in the pathogenesis of this complex genetic disease.

Figures

Figure 1
Figure 1
QRT-PCR quantification of myo-miRs and miR-133 and miR-107 in biopsies from vastus lateralis of 7 DM1 patients compared with 4 controls. Fold change values of miR-206: DM1-1 = 3,73; DM1-2 = 7,02; DM1-3 = 7,77; DM1-4 = 2,70; DM1-5 = 1,20; DM1-6 = 13,22; DM1-7 = 1,95
Figure 2
Figure 2
Northern blot analysis of myo-miRs expression. DM1 patients were pooled into two groups: DMa (DM1-1, DM1-2, DM1-3, DM1-4) and DMb (DM1-5, DM1-6, DM1-7), 4 healthly subjects were pooled as well. The U6-snRNA was used as control for normalization of samples. Figure 2a: Northern Blot results of the 3 pooled samples (Ctr, DMa and DMb) for the 4 miRNA analyzed (miR 181, miR 1, miR 206 and miR 133) compared to U6-snRNA. Figure 2b: Densitometry of autoradiograms performed using OptiQuant image analysis software (Packard) showing miR/U6 ratios.
Figure 3
Figure 3
Western blot analysis showing the Utrophin and α-Tubulin protein expression levels in 5 DM1 patients and 4 controls. Utophin/Tubulin ratios in the analyzed samples are: DM1-1 = 0,63, DM1-2 = 0,74, DM1-3 = 0,31, DM1-4 = 1,66, DM1-5 = 0,66, CTR 1 = 0,15, CTR 2 = 0,85, CTR 3 = 0,50, CTR 4 = 0,34. Figure 3a: Western blot of Utrophin and α-Tubulin protein expression in the 5 DM1 patients showing the miR-206 upregulation and in 4 controls. 50 μg of sample was loaded on each lane. Figure 3b: Densitometric analysis of Western blot autoradiograms performed using OptiQuant image analysis software (Packard) showing Utophin/Tubulin ratios.
Figure 4
Figure 4
In situ hybridisation showing miR-206 localization in transversal section of vastus lateralis muscle from one DM1 patient and one control subject. 4a: Tissue distribution of miR-206 in an healthy subject. miR-206 was expressed mostly in nuclear regions. 4b: Tissue distribuition of miR-206 in a DM1 patient. The miR-206 strongest signal corresponds to nuclear clumps (red arrow). Expression of miR-206 was also observed in nuclear regions of centralized nuclei. 4c: hybridization of U6-siRNA LNA used as positive control. 4d: LNA probe with a scrambled sequence, which is not present in the human genome, has been used to test the specificity of the probes. Green signal corresponds to lipofuscin-derived autofluorescence of the muscle tissue and does not localize with the nuclei.

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