5 - Identification and characterization of the role of non-coding RNAs in spastic cerebral palsy

Poster Number: 5
Publication Number: 5.109

Brigette M. Romero Carpio, University of Delaware, Newark, DE, United States; Karyn Robinson, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Stephanie K. Lee, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Jonathan R. Hicks, NemoursAlfred I. duPont Hospital for Children, Newark, DE, United States; Robert Akins, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Mona Batish, University of Delaware, Newark, DE, United States

Background: Spastic CP accounts for more than 75% of cases of CP and involves muscle hypertonia, contracture, weakness, and musculoskeletal deformities that often worsen over time. However, the fundamental mechanisms contributing to motor dysfunction in spastic CP are not understood leading to delays in its identification and treatment options. Studies indicate that epigenetic and related non-canonical regulatory mechanisms could play major determinants role for functional differences in CP muscle cells. Regulatory noncoding RNAs, which include microRNAs (miRNAs), long ncRNAs (lncRNAs), and circular RNAs (circRNAs) have been shown to be physiologically- important and potentially heritable in multiple systems, including muscle where role of miRNAs in modulating muscle regeneration and satellite cell behavior is reported but role of circRNAs and lncRNAs is not characterized.

Objective: We sought to identify the role and mechanism of action of circRNAs in satellite cells (SCs) isolated from individuals with spastic CP.

Design/Methods: Differentially regulated circRNAs between CP and non-CP study participant were identified from existing RNAseq data from RNA isolated from satellite cells and erector spinae muscle of control and CP subjects. Satellite cell lines derived from these participants were cultured and differentiated to identify and validate circular RNAs that were differentially expressed using qRT PCR. Bioinformatic tools like Target Scan and Circinteractome were utilized to identify if selected circRNA candidates act as miRNA sponges. The expression level of target miRNAs and the mRNA targets of these miRNAs in control and CP RNA was confirmed using qRT PCR.

Results: 42 differentially expressed circRNAs were identified in proliferating cells, 106 circRNAs in differentiated cells, and 227 circRNAs in tissue sections. Of these, 11 circRNAs showed amplification and differential expression between control and CP RNA. The expression level of circRNAs/miRNA/mRNA in proliferating SCs was opposite to expression level in differentiated SCs. As expected, we found the level of circRNA, and mRNA targets showed similar trend while the level of target miRNA was inversely related. Thus, we identified the potential circRNA:miRNA:mRNA axis that regulate gene expression and clinical outcomes in CP. Conclusion(s): Non-coding RNA expression was found to be altered in satellite cells derived from subjects with CP. Regulatory circRNAs/miRNA/mRNA could represent a new potential mechanism associated with dysfunction and pave the way for new avenues of research into biomarkers and therapeutic targets.