Implementation of circFISH to quantify the expression and cellular localization of circNFIX in cerebral palsy
Date
2023
Authors
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Publisher
University of Delaware
Abstract
Cerebral Palsy (CP) is the most common disability of motor function in children, characterized by impairments in movement, normal activity, and posture. Children with spastic CP are affected by muscle tissue defects, including reduced cross-sectional area, decreased muscle mass and volume, increased sarcomere length, disrupted neuromuscular junctions (NMJ) and reduced satellite-cell (SC) number. However, the underlying mechanisms contributing to such muscle alterations remain elusive. Regulatory non`-coding RNAs called circular RNAs (circRNAs) have been studied to play important roles in different physiological processes and dysregulation of circRNAs have been associated with different cancers and many other pathologies. Recently circNFIX has been observed to be a regulatory factor in myogenic differentiation, showing differential expression in various stages of myogenesis, and can be potentially characterized as a key player in myogenic alterations in CP. This led to the hypothesis that circNFIX might be a key regulator in CP associated musculoskeletal development. In this study, circFISH was implemented to simultaneously image circNFIX and its linear isoform, linNFIX in SC-MBs and differentiated myotubes of spastic CP patients and healthy controls. Significant differences in circNFIX counts and cellular localization pattern in SC-MBs and myotubes between CP and healthy controls were observed. Additionally, RNAse R treatment resulted in significant reduction in linNFIX levels with minimal change in circNFIX levels. CircNFIX knockdown SCs showed significant reduction in circNFIX counts. Significant differences in nuclear and cytoplasmic distribution of circNFIX in both proliferating and differentiated cells point towards regulatory functions of circNFIX that might be at play. The results further reinforce the potential involvement of circNFIX in the regulation of CP muscles. Further investigation of circNFIX with respect to its involvement in muscle development and cellular functions can aid in shedding light on the underlying mechanisms of CP associated muscle defects.
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Keywords
Cerebral Palsy, Neuromuscular junctions, Satellite-cells, CircRNAs, Myogenesis, Skeletal muscle