Functional analysis of circSTX8 in chicken macrophages under lipopolysaccharide stimulation

Abstract

Circular RNAs (circRNAs) have a regulatory role in gene expression, development, differentiation, and immune response. In a previous study, circular RNA STX8 (circSTX8) exhibited low expression in chicken lungs during lipopolysaccharide (LPS) stimulation. PCR amplification and Sanger sequencing showed that circSTX8 was created by back-splicing of exons 5 to 6 of STX8. RNase R exonuclease treatment indicated that circSTX8 was a stable circular RNA. RT-qPCR showed that circSTX8 was highly expressed in cecum, spleen, harderian gland, stomach, thymus, liver, small intestine, and lung instead of that in muscle, cerebrum, and cerebellum (n = 8). Chicken macrophages were then divided into four groups: control, overexpression of circSTX8 group, LPS group, and overexpression of circSTX8 + LPS group. CCK8 and RT-qPCR showed that circSTX8 can exacerbate the cellular injury induced by LPS, resulting in a reduction of cell viability and an increase of the pro-inflammatory cytokines expression. In addition, four miRNAs were identified to interact with circSTX8, potentially targeting 914 genes, which were significantly enriched in the pathways of Tight junction, mTOR signaling pathway, MAPK signaling pathway, TGF-beta signaling pathway, Notch signaling pathway, ErbB signaling pathway, and Cell adhesion molecules. These findings showed that circSTX8 was able to regulate the LPS induced cellular immune and inflammatory response.