miR-322/-503 rescues myoblast defects in myotonic dystrophy type 1 cell model by targeting CUG repeats

Abstract Myotonic dystrophy type 1 (DM1) is the most common type of adult muscular dystrophy caused by the expanded triple-nucleotides (CUG) repeats. Myoblast in DM1 displayed many defects, including defective myoblast differentiation, ribonuclear foci, and aberrant alternative splicing. Despite many were revealed to function in DM1, microRNAs that regulated DM1 via directly targeting the expanded CUG repeats were rarely reported. Here we discovered that miR-322/-503 rescued myoblast defects in DM1 cell model by targeting the expanded CUG repeats. First, we studied the function of miR-322/-503 in normal C2C12 myoblast cells. Downregulation of miR-322/-503 significantly hindered the myoblast differentiation, while miR-322/-503 overexpression promoted the process. Next, we examined the role of miR-322/-503 in the DM1 C2C12 cell model. miR-322/-503 was downregulated in the differentiation of DM1 C2C12 cells. When we introduced ectopic miR-322/-503 expression into DM1 C2C12 cells, myoblast defects were almost fully rescued, marked by significant improvements of myoblast differentiation and repressions of ribonuclear foci formation and aberrant alternative splicing. Then we investigated the downstream mechanism of miR-322/-503 in DM1. Agreeing with our previous work, Celf1 was proven to be miR-322/-503′s target. Celf1 knockdown partially reproduced miR-322/-503′s function in rescuing DM1 C2C12 differentiation but was unable to repress ribonuclear foci, suggesting other targets of miR-322/-503 existed in the DM1 C2C12 cells. As the seed regions of miR-322 and miR-503 were complementary to the CUG repeats, we hypothesized that the CUG repeats were the target of miR-322/-503. Through expression tests, reporter assays, and colocalization staining, miR-322/-503 was proved to directly and specifically target the expanded CUG repeats in the DM1 cell model rather than the shorter ones in normal cells. Those results implied a potential therapeutic function of miR-322/-503 on DM1, which needed further investigations in the future.

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Transcriptome Analysis Reveals Altered Inflammatory Pathway in an Inducible Glial Cell Model of Myotonic Dystrophy Type 1

Biomolecules ◽

10.3390/biom11020159 ◽

2021 ◽

Vol 11 (2) ◽

pp. 159

Author(s):

Cuauhtli N. Azotla-Vilchis ◽

Daniel Sanchez-Celis ◽

Luis E. Agonizantes-Juárez ◽

Rocío Suárez-Sánchez ◽

J. Manuel Hernández-Hernández ◽

...

Keyword(s):

Immune Response ◽

Alternative Splicing ◽

Myotonic Dystrophy ◽

Cell Model ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Cellular Functions ◽

Glia Cells ◽

Molecular Events

Myotonic dystrophy type 1 (DM1), the most frequent inherited muscular dystrophy in adults, is caused by the CTG repeat expansion in the 3′UTR of the DMPK gene. Mutant DMPK RNA accumulates in nuclear foci altering diverse cellular functions including alternative splicing regulation. DM1 is a multisystemic condition, with debilitating central nervous system alterations. Although a defective neuroglia communication has been described as a contributor of the brain pathology in DM1, the specific cellular and molecular events potentially affected in glia cells have not been totally recognized. Thus, to study the effects of DM1 mutation on glial physiology, in this work, we have established an inducible DM1 model derived from the MIO-M1 cell line expressing 648 CUG repeats. This new model recreated the molecular hallmarks of DM1 elicited by a toxic RNA gain-of-function mechanism: accumulation of RNA foci colocalized with MBNL proteins and dysregulation of alternative splicing. By applying a microarray whole-transcriptome approach, we identified several gene changes associated with DM1 mutation in MIO-M1 cells, including the immune mediators CXCL10, CCL5, CXCL8, TNFAIP3, and TNFRSF9, as well as the microRNAs miR-222, miR-448, among others, as potential regulators. A gene ontology enrichment analyses revealed that inflammation and immune response emerged as major cellular deregulated processes in the MIO-M1 DM1 cells. Our findings indicate the involvement of an altered immune response in glia cells, opening new windows for the study of glia as potential contributor of the CNS symptoms in DM1.

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Inhibition of Postn Rescues Myogenesis Defects in Myotonic Dystrophy Type 1 Myoblast Model

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.710112 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xiaopeng Shen ◽

Zhongxian Liu ◽

Chunguang Wang ◽

Feng Xu ◽

Jingyi Zhang ◽

...

Keyword(s):

Skeletal Muscle ◽

Myotonic Dystrophy ◽

Cell Model ◽

Neutralizing Antibody ◽

Underlying Mechanism ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Ctg Repeats ◽

Myoblast Cells

Myotonic dystrophy type 1 (DM1) is an inherited neuromuscular disease caused by expanded CTG repeats in the 3′ untranslated region (3′UTR) of the DMPK gene. The myogenesis process is defective in DM1, which is closely associated with progressive muscle weakness and wasting. Despite many proposed explanations for the myogenesis defects in DM1, the underlying mechanism and the involvement of the extracellular microenvironment remained unknown. Here, we constructed a DM1 myoblast cell model and reproduced the myogenesis defects. By RNA sequencing (RNA-seq), we discovered that periostin (Postn) was the most significantly upregulated gene in DM1 myogenesis compared with normal controls. This difference in Postn was confirmed by real-time quantitative PCR (RT-qPCR) and western blotting. Moreover, Postn was found to be significantly upregulated in skeletal muscle and myoblasts of DM1 patients. Next, we knocked down Postn using a short hairpin RNA (shRNA) in DM1 myoblast cells and found that the myogenesis defects in the DM1 group were successfully rescued, as evidenced by increases in the myotube area, the fusion index, and the expression of myogenesis regulatory genes. Similarly, Postn knockdown in normal myoblast cells enhanced myogenesis. As POSTN is a secreted protein, we treated the DM1 myoblast cells with a POSTN-neutralizing antibody and found that DM1 myogenesis defects were successfully rescued by POSTN neutralization. We also tested the myogenic ability of myoblasts in the skeletal muscle injury mouse model and found that Postn knockdown improved the myogenic ability of DM1 myoblasts. The activity of the TGF-β/Smad3 pathway was upregulated during DM1 myogenesis but repressed when inhibiting Postn with a Postn shRNA or a POSTN-neutralizing antibody, which suggested that the TGF-β/Smad3 pathway might mediate the function of Postn in DM1 myogenesis. These results suggest that Postn is a potential therapeutical target for the treatment of myogenesis defects in DM1.

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Non-invasive monitoring of alternative splicing outcomes to identify candidate therapies for myotonic dystrophy type 1

Nature Communications ◽

10.1038/s41467-018-07517-y ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 9

Author(s):

Ningyan Hu ◽

Layal Antoury ◽

Timothy M. Baran ◽

Soumya Mitra ◽

C. Frank Bennett ◽

...

Keyword(s):

Alternative Splicing ◽

Myotonic Dystrophy ◽

Myotonic Dystrophy Type 1 ◽

Invasive Monitoring ◽

Myotonic Dystrophy Type ◽

Non Invasive

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Brain-specific change in alternative splicing of Tau exon 6 in myotonic dystrophy type 1

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease ◽

10.1016/j.bbadis.2005.12.003 ◽

2006 ◽

Vol 1762 (4) ◽

pp. 460-467 ◽

Cited By ~ 34

Author(s):

Olivier Leroy ◽

Junning Wang ◽

Claude-Alain Maurage ◽

Michel Parent ◽

Thomas Cooper ◽

...

Keyword(s):

Alternative Splicing ◽

Myotonic Dystrophy ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Specific Change

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Transcriptome Analysis in a Primary Human Muscle Cell Differentiation Model for Myotonic Dystrophy Type 1

International Journal of Molecular Sciences ◽

10.3390/ijms22168607 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8607

Author(s):

Vanessa Todorow ◽

Stefan Hintze ◽

Alastair R. W. Kerr ◽

Andreas Hehr ◽

Benedikt Schoser ◽

...

Keyword(s):

Alternative Splicing ◽

Myotonic Dystrophy ◽

Therapeutic Interventions ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

Gene Set Enrichment ◽

Repeat Expansions ◽

Differentiation Stage ◽

Term Analysis

Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.

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