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

Zirong Han; Jialuo Li; Weilin Wang; Jiaxin Li; Qi Zhao; Meijia Li; Lingling Wang; Linsheng Song

doi:10.1016/j.dci.2021.104180

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

Dev Comp Immunol. 2021 Nov:124:104180. doi: 10.1016/j.dci.2021.104180. Epub 2021 Jun 24.

Authors

Zirong Han¹, Jialuo Li¹, Weilin Wang¹, Jiaxin Li¹, Qi Zhao¹, Meijia Li¹, Lingling Wang², Linsheng Song³

Affiliations

¹ Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China.
² Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Functional Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China; Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China.
³ Liaoning Key Laboratory of Marine Animal Immunology, Dalian Ocean University, Dalian, 116023, China; Functional Laboratory of Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266235, China; Liaoning Key Laboratory of Marine Animal Immunology and Disease Control, Dalian Ocean University, Dalian, 116023, China; Dalian Key Laboratory of Aquatic Animal Disease Prevention and Control, Dalian Ocean University, Dalian, 116023, China. Electronic address: lshsong@dlou.edu.cn.

PMID: 34171368
DOI: 10.1016/j.dci.2021.104180

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 < 0.05) at 3 h after LPS stimulation, while the intracellular Ca²⁺ (1.57-fold of that in blank, p < 0.05) and the mRNA expression of cytokine CgIL17-1 (4.87-fold of that in blank, p < 0.05) 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 h (43.523-fold of that of blank, p < 0.05) and 6 h (55.91-fold of that of blank, p < 0.05) 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.

Keywords: Calmodulin (CaM); Crassostrea gigas; IL-17 expression; Immune response; miRNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

3' Untranslated Regions
Amino Acid Sequence
Animals
Calcium / immunology
Calmodulin / genetics*
Calmodulin / immunology
Crassostrea / genetics
Crassostrea / immunology*
Gene Expression / immunology
Hemocytes / drug effects
Hemocytes / immunology
Interleukin-17 / genetics*
Interleukin-17 / immunology
Lipopolysaccharides / immunology
MicroRNAs / genetics*
MicroRNAs / immunology
Phylogeny
RNA, Messenger / genetics
RNA, Messenger / immunology
Tissue Distribution / immunology

Substances

3' Untranslated Regions
Calmodulin
Interleukin-17
Lipopolysaccharides
MicroRNAs
RNA, Messenger
Calcium