396 - Epigenetic Control of Muscle in Spastic Cerebral Palsy: Identification of a Novel Circular-RNA, Micro-RNA, mRNA Regulatory Axis

Publication Number: 396.4906

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; Mona Batish, University of Delaware, Newark, DE, United States

Background: Cerebral palsy (CP) is a set of movement disorders, with spastic CP accounting for >80% of cases. CP is characterized by a static brain lesion with musculoskeletal function that deteriorates over time, making it challenging for individuals to move, maintain balance, and exert postural control. The cell & molecular mechanisms responsible for motor dysfunction in spastic CP are not well understood, but altered muscle growth and function have been implicated. Pilot studies from our group showed that the expression of a circular RNA (circRNA) called circNFIX is significantly reduced in muscle cells of CP patients. CircNFIX is a non-coding RNA with crucial roles in muscle by modulating gene expression primarily through its ability to “sponge” regulatory microRNAs (miRNAs) controlling myogenic factor function.

Objective: We explored the role of circNFIX in regulating the expression of the critical myogenic factor MEF2C in primary myoblasts (MBs), and myotubes (MTs).

Design/Methods: With IRB-approval, MBs were isolated from muscle biopsies of individuals with spastic CP and control subjects (CN). Total RNA was isolated and qRT-PCR analysis was performed to estimate the level of circNFIX and MEF2C in MBs, MTs, and muscle tissue. Bioinformatic analyses were performed to identify miRNAs targeting circNFIX and MEF2C. To validate the regulatory role of circNFIX, the circular RNA was knocked down in CN MBs. Additionally, MEF2C protein expression was assessed in MBs and MTs by immunofluorescence. The expression of downstream targets of MEF2C was assessed by qRT-PCR. The relationship of the circRNA/miRNA/mRNA axis to MEF2C was evaluated by dual-luciferase assay using a mimic of the target microRNA miR-373-3p and a negative control miRNA.

Results: We observed reduced circNFIX and MEF2C levels in CP MBs, MTs, and muscle tissue compared to controls. The protein level of MEF2C was significantly decreased in both CP and KD MB cells, reducing the level of downstream MEF2C targets. Additionally, we found that miR373-3p, which regulates MEF2C levels, was expressed in cells from both CP and CN samples. These findings suggest that circNFIX may differentially regulate the translation of MEF2C protein by sponging of miR-373-3p.

Conclusion(s): The regulatory circNFIX/miRNA373-3p/MEF2C could be associated with motor dysfunction in CP. The decrease in circNFIX levels may lead to an increase in available miR-373-3p, resulting in the development of shortened and overstretched sarcomeres due to ME2C downregulation. CircNFIX could be explored as a potential non-invasive diagnostic biomarker or therapeutic target for CP.