From Limited Samples to Mechanistic Insights: Exploratory Identification and Functional Validation of a hsa_circ_0062400/hsa_circ_0002397-miR-338-3p-NRP1 Axis in Myasthenia Gravis

introductionCircRNAs are implicated in various autoimmune diseases, such as Myasthenia Gravis (MG), yet their regulatory mechanisms remain poorly understood. This research investigated the regulatory network of circRNAs, miRNAs, and mRNAs in modulating MG progression. MATERIALS AND

methodsThree MG patients (male: female = 1:2) and three healthy controls were enrolled for microarray analysis. The “Limma” package was employed to identify the differentially expressed circRNAs (DECs). Target miRNAs of DECs were predicted via the CircInteractome database, while target genes of miRNAs were predicted utilizing miRWalk, miRTarbase, and TargetScan databases. Cytoscape software was applied to establish the circRNAs-miRNAs-mRNAs network. Thereafter, a dual-luciferase reporter assay was conducted to validate the targeted regulatory relationship between hsa_circ_0062400, hsa_circ_0002397, and miR-338-3p. The expression of NRP1 was measured by qRT-PCR and Western blotting, and the cell viability of Jurkat cells was evaluated via CCK-8 assay. The levels of cytokines (IL-2, IL-6, IFN-γ, TNF-α) after NRP1 knockout were detected by ELISA.

results4 circRNAs, 2 miRNAs, and 11 target genes associated with MG pathogenesis were identified to construct a ceRNA regulatory network. In-vitro assays validated the targeted interactions between hsa_circ_0062400, hsa_circ_0002397, and miR-338-3p. miR-338-3p negatively regulated both protein and mRNA levels of NRP1. Further, silencing hsa_circ_0062400 or hsa_circ_ 0002397 markedly suppressed NRP1 expression and Jurkat cell proliferation, which was reversed by miR-338-3p inhibitor. Moreover, NRP1 affected the levels of cytokines in Jurkat cells. DISCUSSION: The circRNAs were demonstrated to be closely associated with MG etiology, which was also supported by our current discovery that hsa_circ_0062400 and hsa_circ_0002397 regulated the level of NRP1 by sponging miR-338-3p in MG. However, the roles of hsa_circ_0062400 and hsa_circ_0002397 in MG have been rarely reported, which requires further validation. Limitations of this study included a small, gender-imbalanced sample size for microarray analysis and incomplete verification of the ceRNA network. In addition, functional experiments were limited to Jurkat cells, and more in-vivo validation assays were lacking.

conclusionCollectively, the present study revealed MG pathogenesis and also provided potential treatment targets for the disease.