MicroRNA-301a directly targets IL-4/13A to negatively regulate Th2 immune responses in large yellow croaker (Larimichthys crocea)

Abstract

Interleukin-4 (IL-4) and IL-13, predominantly secreted by T helper type 2 (Th2) cells, are pivotal cytokines involved in the Th2 response via the MAPK and mTOR signaling pathways. MicroRNAs (miRNAs) modulate various biological processes by inhibiting mRNA translation or facilitating mRNA degradation. However, research on miRNAs targeting IL-4 and IL-13 and their associated downstream signaling molecules remains limited. In this study, the miR-301a was identified for the first time as a potential regulator of IL-4/13A in Larimichthys crocea (Lc) through comprehensive analyses of miRNA-mRNA transcriptomes. The Lcpre-miR-301a comprises 86 nucleotides, including a 21-bp mature miRNA sequence. Secondary structure prediction showed that Lcpre-miR-301a can form a classic hairpin structure. Multiple sequence alignment revealed that the mature LcmiR-301a is highly conserved across fish, mammals, amphibians, and birds. The miR-301a was demonstrated to directly bind to the 3′-UTR of LcIL-4/13A. Upon induction with phytohemagglutinin (PHA), the expression levels of LcIL-4/13A were significantly upregulated, leading to the activation of T cell proliferation and differentiation, as well as B cell proliferation in primary head kidney leukocytes (PKLs). Conversely, the overexpression of miR-301a in PKLs significantly inhibited the PHA-induced upregulation of LcIL-4/13A expression level. Additionally, the stimulatory effects of PHA on T cells and B cells were diminished by the overexpression of LcmiR-301a. Moreover, after transfection with miR-301a mimics and subsequent induction with PHA, the phosphorylation levels of MEK1/2 and 4E-BP1, two critical signaling molecules within the MAPK and mTOR pathways, were significantly reduced in PKLs compared to induction with PHA alone. These findings suggest that miR-301a plays a crucial role in negatively regulating the Th2 immune response by targeting LcIL-4/13A. This study enhances our understanding of the mechanisms underlying miRNA regulation in the adaptive immune response of fish.