A calmodulin targeted by miRNA scaffold659_26519 regulates IL-17 expression in the early immune response of oyster Crassostrea gigas.

Abstract

Calmodulin (CaM) is a highly conserved second messenger protein transducing calcium signals by binding and modulating intracellular calcium ions (Ca2+), and involves in the Ca2+-dependent physical processes including host defense in vertebrates. In the present study, a CaM homologue (designated as CgCaM) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgCaM cDNA was of 471 bp encoding a polypeptide of 156 amino acid residues. There were four EFh domains predicted in CgCaM, which shared high homologies with those in CaMs from oyster C. virginica and other invertebrates. The mRNA transcripts of CgCaM were constitutively expressed in all the tested tissues including labellum, mantle, gonad, gills, adductor muscle, haemocytes and hepatopancreas, with the highest expression level in haemocytes. The mRNA expression level of CgCaM in haemocytes decreased significantly (0.31-fold of that in blank, p\u202f<\u202f0.05) at 3\u202fh after LPS stimulation, while the intracellular Ca2+ (1.57-fold of that in blank, p\u202f<\u202f0.05) and the mRNA expression of cytokine CgIL17-1 (4.87-fold of that in blank, p\u202f<\u202f0.05) both increased in haemocytes. Meanwhile, an oyster miRNA scaffold659_26519 was identified, and it was proved to target the 3 -untranslated regions (3 -UTR) of CgCaM mRNA by luciferase reporter assay. The expression of scaffold659_26519 increased significantly at 3\u202fh (43.523-fold of that of blank, p\u202f<\u202f0.05) and 6\u202fh (55.91-fold of that of blank, p\u202f<\u202f0.05) after LPS stimulation. When the expression of scaffold659_26519 was inhibited by transfection with its inhibitor in vitro, the expression of CgIL17-1 declined significantly to 0.58-fold of that in LPS stimulation group. These findings indicated that the miRNA scaffold659_26519 targeted CaM was involved in the early inflammatory response of oyster immunity, and provided a new evidence for CaM-mediated immune mechanism in molluscs.