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Transcriptomic data and analyses of shMeg3 muscle in vitro and in vivo

Citation

Naya, Francisco et al. (2020), Transcriptomic data and analyses of shMeg3 muscle in vitro and in vivo, Dryad, Dataset, https://doi.org/10.5061/dryad.k0p2ngf77

Abstract

Formation of skeletal muscle is among the most striking examples of cellular plasticity in animal tissue development, and while muscle progenitor cells are reprogrammed by epithelial-mesenchymal transition (EMT) to migrate during embryonic development, regulation of EMT in postnatal myogenesis remains poorly understood. Here, we demonstrate that the long noncoding RNA (lncRNA) Meg3 regulates EMT in myoblast differentiation and skeletal muscle regeneration. Chronic inhibition of Meg3 in C2C12 myoblasts induced EMT, and supressed cell state transitions required for differentiation. Furtheremore, adenoviral Meg3 knockdown compromised muscle regeneration, which was accompanied by abnormal mesenchymal gene expression and interstitial cell proliferation. Transcriptomic and pathway analyses of Meg3-depleted C2C12 myoblasts and injured skeletal muscle revealed a significant dysregulation of EMT-related genes, and identified TGFβ as a key upstream regulator. Importantly, inhibition of TGFβR1 and its downstream effectors, and the EMT-related transcriptional repressor Snai2, restored many aspects of myogenic differentiation in Meg3-depleted myoblasts in vitro. We further demonstrate that reduction of Meg3-dependent Ezh2 activity results in epigenetic alterations associated with TGFβ activation. Thus, Meg3 regulates myoblast identity to facilitate progression into muscle differentiation.

Methods

C2C12 myotubes were collected on day 3 differentiation using Trizol and active lysis. TA muscle tissue was harvested 7 days post-injury, and injured muscle tissue was snap-frozen, and treated with Trizol with Polytron dissociation. RNA was purified by phenol-chlorophorm extraction and isopropanol precipitation. A minimum of 100 ng RNA per sample was provided to the Boston University Microarray and Sequencing Resource Core. Briefly, samples were evaluated for quality by Bioanalyzer to confirm RIN scores were above 7. Subsequently, second-strand synthesis was prepared using TruSeq mRNA stranded library preparation kit, and samples were subsequently sequenced by high-output 75 bp paired-end read Illumina NextSeq500 sequencing. Raw counts were then preprocessed using the Illumina BaseSpace RNA Express application, to assess for quality via FastQC and RSeQC, and aligned to mouse genome build mm10 via STAR. Quantification of pairs uniquely aligned to each Ensembl gene (via HTSeq) produced counts, which were converted to regularized algorithm (rlog) transformed expression values (via DESeq2) for statistical and expression analyses. IPA summary files in this dataset were conducted by Tiffany Dill by restricting the gene set list to significantly dysregulated transcripts (p < 0.05). All other files in this dataset were produced by Adam Gower at the Boston University Microarray and Sequencing Resource Core.