Regulation of atrogene expression by miRNA-411-5p in a dexamethasone-induced atrophy model in C2C12 cells.

Abstract

Introduction
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the post-transcriptional level and are involved in various cellular processes, including the development, maintenance, and regeneration of skeletal muscle. Several miRNAs located within the DLK1-DIO3 locus have been associated with the proliferation and differentiation of myoblasts. C2C12 cells, a murine myoblast cell line, are widely used as an in vitro model to study these processes, as they can differentiate into myotubes and replicate physiological and pathological events of muscle tissue.

One of the most commonly used in vitro models to induce muscle atrophy is exposure to dexamethasone, a synthetic glucocorticoid that mimics the muscle-wasting effects observed with prolonged steroid treatments. This form of atrophy is characterized by increased expression of catabolic genes known as atrogenes. Recent evidence suggests that certain miRNAs, including miRNA-411-5p from the DLK1-DIO3 locus, may play a role in the regulation of these genes. Therefore, studying this miRNA in muscle atrophy models may help identify new therapeutic targets for combating muscle loss.

Objective
To determine the involvement of miRNA-411-5p from the DLK1-DIO3 locus in the regulation of atrogenes in a dexamethasone-induced muscle atrophy model using C2C12 cells.

Methodology
C2C12 myoblasts were cultured and induced to differentiate into myotubes using a specific differentiation medium. After differentiation, the cells were treated with dexamethasone (100 µM) for 48 hours to induce muscle atrophy. Total RNA was then extracted to assess the expression of atrophy-related genes (KLF5, FOXO1, Atrogin-1, and MuRF1) and miRNA-411-5p, using quantitative PCR (qPCR). Additionally, morphological analysis was performed via optical microscopy to observe cellular changes associated with atrophy. Finally, bioinformatic analyses were conducted to identify potential therapeutic targets of miRNA-411-5p.

Results
Dexamethasone treatment clearly induced atrophy in differentiated myotubes, evidenced by a significant increase in the expression of atrogenes KLF5, FOXO1, Atrogin-1, and MuRF1, along with a reduction in MHC (myosin heavy chain), a structural marker of skeletal muscle. In parallel, a significant decrease in miRNA-411-5p expression was observed. Bioinformatic analyses indicated that FOXO1 could be a direct target of this miRNA, suggesting a relevant functional relationship in the regulation of muscle atrophy.

Conclusion
miRNA-411-5p is significantly downregulated in a dexamethasone-induced atrophy model, coinciding with the upregulation of key atrogenes such as FOXO1. These findings suggest that miRNA-411-5p may negatively regulate pathways associated with muscle degradation and could represent a potential therapeutic target for counteracting glucocorticoid-induced muscle atrophy.