The female-biased expression, transcriptional regulation and knock-down effect of insulin-like growth factor binding protein 7 in Chinese tongue sole, Cynoglossus semilaevis

Abstract

Insulin-like growth factor binding protein 7 (igfbp7) is a member of the insulin-like growth factor binding protein (IGFBP) superfamily, which is involved in the regulation of insulin-like growth factor (IGF) and insulin signal transduction. Recently, igfbp7 was found to be female-biased expressed in the gonad of Chinese tongue sole (Cynoglossus semilaevis), a marine flatfish exhibited female-biased sexual dimorphism. This study aimed to explore the roles of igfbp7 in the growth and sex differentiation of C. semilaevis. Firstly, C. semilaevis igfbp7 was cloned, and the coding sequence (CDS) was 666 bp encoding 221 amino acids. The phylogenetic and structural analyses revealed that C. semilaevis igfbp7 was clustered with Atlantic halibut (Hippoglossu shippoglossus), sailfin molly (Poecilia latipinna), and so on. The subsequent temporal and spatial expression analyses showed that igfbp7 was mainly expressed in female gonad. During the different gonad developmental stages, the expression of igfbp7 displayed the highest level at 6-month. Methylome data showed that female igfbp7 exhibited lower methylation levels than male and pseudomale across the whole gene region in the gonad. Dual-luciferase experiment revealed that C. semilaevis igfbp7 promoter exhibited strong transcription activity. In addition, pituitary-specific positive transcription factor 1 (POU1F1) and steroid hormone-testosterone significantly enhanced the transcriptional activity of the igfbp7 promoter. In vitro transfection experiment revealed the activation of igfbp7 on the nuclear factor kappa B (NF-κB) signaling pathway. Finally, the activation of insulin-like growth factor 1 receptor (igf1r), serine/threonine kinase 1 (akt) and heat shock 60 protein 1 (hspd1) was observed in C. semilaevis ovary cells transfected by igfbp7 small interfering RNA (siRNA). These results provided causal clues for igfbp7 in growth regulation by influencing igf1r, akt and NF-κB signal.