| Skeletal muscle function is a vital requirement for an enriched healthy life. For years the molecular processes governing myogenesis and the development of skeletal muscle were primarily associated with proteins. In recent years, however, non-coding RNAs including microRNAs (miRNAs) and long non coding RNAs (lncRNAs) have emerged as integral components of gene regulation that take part in cellular differentiation, development and disease. HOTAIR (HOX antisense intergenic RNA) is a long non-coding RNA (lncRNA) that is transcribed from the antisense strand of the homeobox C (HOXC) gene locus present on chromosome 12. HOTAIR has been shown to have a regulatory role in gene silencing as it coordinates with chromatin-modifying enzymes. Although there is a substantial amount of data suggesting important functions of HOTAIR in several human and mouse cells and tissues, very little knowledge exists about HOTAIR functions in skeletal muscle. To explore the impact of HOTAIR on skeletal muscle cell growth and development, we knockdown HOTAIR in mouse skeletal muscle cells and analyzed the impact on cell proliferation and differentiation. HOTAIR expression was reduced with a synthetically designed HOTAIR antisense DNA and siRNA in separate experiments. To investigate the underlying effect of HOTAIR in C2C12 cells, a series of gene expression, gene array, immunostaining and calcium imaging analyses were employed during cell differentiation. Partial knockdown of HOTAIR in C2C12 cells resulted in fewer and smaller myotubes as confirmed by immunostaining at days 3 and 4 of differentiation with Myosin Heavy Chain (MHC) which only stains myocytes and myotubes but not myoblasts. RT-qPCR revealed a dysregulation of myogenic genes expression that is reflective of abnormal myogenesis. Intracellular calcium (Ca 2+) measurements of the siRNA-treated C2C12 cells showed a decrease in maximum amplitude peak response to caffeine, reduced peak level and shorten relaxation phase, suggesting that less Ca2+ is available for release and re-uptake due to the partial silencing of HOTAIR, correlating with impaired myogenesis. To investigate the molecular signaling machinery induced by partial HOTAIR knockdown we used a real-time PCR gene array to monitor the activity of 10 signaling pathways. We discovered that HOTAIR knockdown modulated the NFkappaB and Oxidative Stress pathways (Ccl5 and Nqo1). These results suggest that HOTAIR may exert its function on skeletal muscle via the regulation of the NFkappaB and Oxidative Stress pathways which are critical for skeletal muscle homeostasis. These studies provide the basis for in-depth studies of HOTAIR function in skeletal muscle by extending its exploration to primary murine and human models as well as animal models. |
