Abstract
Nuclear factor (NF)-κB pathway is an evolutionally conserved pathway in activating immune response, in which IκBs can repress the activation. In the present study, cgi-miR-2d, an invertebrate-specific microRNA, was proved to regulate CgIκB2 expression and haemocyte phagocytosis during bacterial infection in oyster Crassostrea gigas. The expression of cgi-miR-2d was significantly up-regulated after Vibrio splendidus challenge, while CgIκB2 transcripts decreased. Significant decreases in both luminescence and CgIκB2 3′UTR level was observed after transfection of cgi-miR-2d in CgIκB2 3′UTR luciferase reporter assay. CgIκB2 mRNA level decreased significantly (0.51-fold of control group, p < 0.05) in gain-of-function assay of cgi-miR-2d in vivo while it increased markedly (1.27-fold, p < 0.05) when cgi-miR-2d was repressed (0.10-fold, p < 0.01). A significant increase of haemocyte phagocytosis rate was observed in cgi-miR-2d overexpression group (p < 0.01), consistent with results in CgIκB2 knock-down group (p < 0.01). Moreover, the apoptosis rate of haemocytes was found significantly declined (28.57%, p < 0.01) in gain-of-function assay of cgi-miR-2d. Together, those results not only depicted the functional conservation of miR-2d family in anti-apoptosis of oysters but also highlighted its interaction with phagocytosis by modulating NF-κB pathway, which might dedicate critically to the well-balance of host immune response.
Highlights
Phagocytosis of immunocytes is an essential process in host immune response against invaded pathogens[1]
The phagocytosis rate of oyster haemocytes was determined at 8 h, 12 h and 24 h post V. splendidus challenge
Within mass of immune-related pathways, the Nuclear factor (NF)-κBpathway has been well investigated as a global regulator of immune response including phagocytosis, where inhibitor of κBs (IκBs) genes are regarded as hallmarks[4]
Summary
Phagocytosis of immunocytes is an essential process in host immune response against invaded pathogens[1]. The phagocytosis against infiltrated pathogens, as found, can be robustly activated after challenge and strictly modulated afterward by diverse genes or pathways, keeping the well-balance of host immune system[3] Among those regulators, nuclear factor-κB(NF-κB) family[4] is the most important ones as a global activator of phagocytosis-related genes[5]. The NF-κB family is mainly composed by a family of five structurally related transcriptional factors, including NF-κB1 (p105/p50), NF- κB2 (p100/p52), RelA (p65), RelB, and c-Rel[5,6] Those NF- κB proteins dimerize with each other to form homo- and hetero-dimers and can modulate diverse biological responses, such as phagocytosis and apoptosis, by regulating target gene transcription[4,7]. The purposes of the present study were to (1) survey the phagocytic changes of oyster haemocytes after Vibrio splendidus challenge, (2) revise the phylogeny of cgi-miR-2d, (3) investigate the interaction between of CgIκBs and cgi-miR-2d during challenge, and (4) reveal the modulation on haemocyte phagocytosis by cgi-miR-2d and hopefully provide new hints for the miRNA-mediated immunomodulation mechanism in oysters
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