401 - Multifaceted roles of a key circular RNA in Epigenetic regulation of Spastic Cerebral Palsy

Publication Number: 401.5848

Mona Batish, University of Delaware, Newark, DE, United States; Brigette M. Romero Carpio, University of Delaware, Newark, DE, United States; Parsa Hoque, University of Delaware, Rochester, MN, United States; Karyn Robinson, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Stephanie Lee, Nemours Children's Hospital, Wilmington, DE, United States; Vijay Parashar, University of Delaware, Newark, DE, United States; Wade Shrader, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States; Robert Akins, NemoursAlfred I. duPont Hospital for Children, Wilmington, DE, United States

Background: Spastic cerebral Palsy (CP) is most common pediatric motor disability involving muscle hypertonia and musculoskeletal deformities that worsen over time and require surgical correction. Unfortunately, the molecular and cellular mechanisms leading to motor dysfunction in spastic CP remain largely unknown. A new class of regulatory RNA called circular RNAs (circRNAs) play important roles in musculoskeletal disorders, however, their role in CP is not fully explored.

Objective: Functional characterization of circRNAs in satellite cells-derived myoblasts (SC-MBs) and myotubes (SC-MTs).

Design/Methods: SC-MBs were isolated from skeletal muscle biopsies from CP and control subjects. The expression of reference markers PAX7 and MYF5 was used to validate the isolated SC-MBs. SC-MBs were cultured and differentiated into myotubes. Total RNA was isolated and qRT-PCR analysis was done using divergent primers to identify and validate circular RNAs that were differentially expressed. The selected circRNAs was imaged to determine copy number and cellular localization. RNA binding proteins and mRNAs interacting with selected circRNAs were identified using the CRISPR-assisted detection of RNA-protein detection.

Results: A total of 42 and 106 differentially expressed circRNAs were identified in SC-MBs, and SC-MTs, respectively. Among these, 11 circRNAs showed amplification and differential expression between control and CP groups. Particularly, circular nuclear factor IX, (circNFIX), a high copy number circRNA, was significantly downregulated in muscle cells from CP as compared to control subjects. It was found mainly in cytoplasm but showed nuclear enrichment in CP. A microRNA miR373-3p and protein YBX-1 protein were found as interacting partners.

Conclusion(s): Future work is aimed at identifying other interacting partners of circNFIX so that it can be used as a potential therapeutic target. Additionally, we aim to perform targeted RNA seq specific for circRNAs to identify and characterize other dysregulated circRNAs for their function and potential for therapeutic targets. circRNAs are highly enriched into extracellular vesicles and thus this study opens the potential to identify dysregulated circRNAs that can be used as biomarkers for early, specific and non-invasive diagnosis for CP to improve outcomes of therapy and quality of life of CP patients.