Hsa_circ_0000313/miR-1224-3p/MKNK2 Axis Modulates CD4⁺ T Cells by Activating p38 MAPK Signaling in Myasthenia Gravis

Myasthenia gravis (MG) is an autoimmune disorder in which circular RNAs (circRNAs) are increasingly implicated, with growing evidence supporting their critical role in autoimmune pathogenesis. The role of hsa_circ_0000313 in MG, including its biological functions and mechanisms, remains unknown.

Agarose gel electrophoresis, RNase R digestion, and Sanger sequencing were employed to verify the circular structure of hsa_circ_0000313, while nucleoplasmic separation experiment was used to determine its subcellular localization. Analysis of hsa_circ_0000313, miR-1224-3p, and MKNK2 expression was conducted via quantitative real-time PCR (qRT-PCR). CCK-8 assay and flow cytometry were employed to evaluate the proliferative capacity and apoptotic rate of Jurkat cells. ELISA detected inflammatory cytokine secretion. Potential interactions involving miR-1224-3p with either hsa_circ_0000313 or MKNK2 were predicted using bioinformatics tools and subsequently validated through dual-luciferase reporter assays. FISH assay was used to detect subcellular colocalization of hsa_circ_0000313 and miR-1224-3p.

We observed that compared to healthy controls, MG patients exhibited increased expression of hsa_circ_0000313 and MKNK2, along with decreased expression of miR-1224-3p. Knockdown of hsa_circ_0000313 and MKNK2, along with overexpression of miR-1224-3p, inhibited the proliferation and secretion of inflammatory factors in Jurkat cells and promoted their apoptosis. Additionally, miR-144-3p was identified as the target miRNA of hsa_circ_0000313, and MKNK2 was identified as the target gene of miR-1244-3p. Hsa_circ_AA0000313 was confirmed to be circular and resistant to RNase R digestion. There was significant colocalization of hsa_circ_0000313 with miR-1224-3p within the cytoplasm. Inhibition of miR-1244-3p reversed the effects of hsa_circ_0000313 knockdown and MKNK2 knockdown on Jurkat cells proliferation, apoptosis, and inflammatory cytokine secretion. In LPS-stimulated Jurkat cells, hsa_circ_0000313 knockdown suppressed the p38 MAPK pathway via the miR-1224-3p/MKNK2 axis, which reduced inflammatory cytokine secretion and cell proliferation as well as promoted apoptosis.

Hsa_circ_0000313 is involved in MG progression by regulating the miR-1224-3p/MKNK2 axis and can act on the p38MAPK pathway to participate in the progression of MG concomitant inflammatory infections, which may provide a promising therapeutic target in MG.