Sequence characterization and regulatory mechanisms of the EGFL7/miR-126b promoter in Larimichthys crocea

MicroRNAs (miRNAs) play a crucial role in modulating various biological pathways by repressing mRNA translation or promoting mRNA degradation. Specifically, miR-126b is implicated in the negative regulation of Th2 immune response by targeting the IL-4Rα1 in Larimichthys crocea (Lc). However, the regulatory mechanisms of LcmiR-126b are unclear. Gene mapping identified LcmiR-126b as being located within the sixth intron of the host gene EGFL7 in L. crocea, and they share a common promoter. The 5' flanking region of EGFL7 gene was cloned, revealing the presence of typical promoter elements, including a transcription start site (TSS), TATA box, CAAT box, and multiple transcription factor binding sites. The EGFL7/miR-126b promoter exhibited basal transcriptional activity, which was significantly enhanced following stimulation with inactivated Pseudomonas plecoglossicida. Promoter truncation and site-directed mutagenesis identified AP-1 and GATA3 binding sites as essential elements for maintaining EGFL7/miR-126b promoter activity. Overexpression of transcription factors c-Jun (a subunit of AP-1) and GATA3 significantly increased the promoter activity, while this effect was not observed in the constructs with mutations at the corresponding binding sites. Chromatin immunoprecipitation (ChIP) assays substantiated the specific binding of c-Jun and GATA3 to the EGFL7/miR-126b promoter region. Additionally, the expression levels of c-Jun, GATA3, EGFL7 and miR-126b were up-regulated in large yellow croaker kidney cell line (LYCK) by inactivated P. plecoglossicida stimulation. Both promoter activity and endogenous expression levels of LcmiR-126b and EGFL7 were enhanced by c-Jun and GATA3 overexpression under inactivated P. plecoglossicida treatment. Conversely, the knockdown of c-Jun and GATA3 resulted in a decreased expression in EGFL7 and LcmiR-126b expression in LYCK cells. Collectively, these data indicate that AP-1 and GATA3 function as positive regulators of EGFL7/miR-126b transcription during P. plecoglossicida infection, offering novel insights into the sequence characterization and regulatory mechanisms of miR-126b in fish.