MiRNA-mediated Modulation In The Stress Response Of Oyster Crassostrea Gigas

The oyster Crassostrea gigas is a dominant sessile mollusc in intertidal zone throughout the world and weights greatly in aquaculture.The life span of adult oysters,unlike other intertidal organisms,is geologically limited to rock surfaces after their settlement at spat stage.Therefore,the oysters have evolved a complex mechanism to respond against the vigorously changing environments and were regarded as an ideal model in stress adaptation,especially the adaptation against pathogen infection and desiccation.As an important class of gene regulators at post-transcriptional level,miRNAs have been identified in numbers of organisms and proved indispensable in almost all biological processes.However,less was known in invertebrate miRNAs especially their role in the adaptation mechanism of oysters living in intertidal zone.“Neural-endocrine-immune?NEI?system” is a major modulation network among the nervous system,endocrine system and immune system and plays vital roles in organisms during stress and infection.The oyster C.gigas is an intertidal mollusc and believed to evolve a primary NEI system.Six small RNA libraries from haemocytes of oyster stimulated with acetylcholine?ACh?and norepinephrine?NE?were sequenced to survey the immunomodulation role of neurotransmitter-responsive miRNAs.A total number of 132 new miRNA were identified in the present study.Along with 199 miRNAs identified previously,331 mi RNAs were identified in oyster haemocytes so far.Among those miRNAs,a sum of 21 and 16 miRNAs expressed differentially?FDR < 0.05?after ACh and NE stimulation,respectively.Besides,21 miRNAs expressed differently between ACh stimulation group and NE stimulation group.A total of 570 pairs of neurotransmitter-responsive mi RNAs and target genes were predicted,representing 35 miRNAs and 355 genes.Immune-related GO terms,including death,immune system process and response to stimulus were annotated with those targets genes.Moreover,immune-related genes,including PRRs,intracellular receptors,signaling transducers and immune effectors,were also predicted to be modulated by those NeurimmiRs.Tumor necrosis factor alpha?TNF-??mediated inflammatory response plays indispensable roles in organisms defending against the invaded bacteria,during which microRNAs have been found crucial by controlling multiple TNF-?-related genes.In the present study,cgi-mi R-92 d was annotated as a member of miR-17⁹2 family and could target the CDS region of lipopolysaccharide?LPS?-induced TNF-? factor?CgLITAF3?in oyster C.gigas.It was observed that cgi-miR-92 d could be vigorously modulated by Vibrio splendidus or LPS stimulation while CgLITAF3 altered oppositely.Two putative binding sites of cgi-mi R-92 d were then found at CDS region of CgLITAF3.The interaction between cgi-miR-92 d and CgLITAF3 was subsequently verified both in vitro and in vivo.As a result,a significant decrease of cellular luminescence was observed in CgLITAF3 luciferase reporter assay when cgi-miR-92 d was overexpressed.The luminescent decrease was then recuperated when cgi-miR-92 d inhibitor was co-transfected with miRNA mimics.Besides,CgLITAF3 transcripts were significantly down-regulated when cgi-miR-92 d was overexpressed in vivo during V.splendidus challenge.Gain-of-function assay of CgLITAF3 was then conducted in HEK293 T cells to verify its function.Consequently,a significant increase of TNF-? was observed during the assay.At the meantime,CgTNF was also down-regulated in gain-of-function assay of cgi-mi R-92 in vivo,which was a member of TNF superfamily in oysters which could be robustly induced after pathogen stimulation.Together,these results verify the interaction between CgLITAF3 and cgi-miR-92 d,which might dedicate crucially in the repaid activation of CgTNF expression during inflammatory response of oysters.Dozens of species-specific mi RNAs have been identified in oyster C.gigas while their functions remain largely unknown.In the present study,an oyster species-specific miRNA scaffold42648₅080 was found responsive to LPS stimulation and might target a total of 31 genes possibly involved in cell communication,cellular localization and cellular response to stimulus of oysters.Besides,in gain-of-function assay of scaffold42648₅080 in vivo,the phagocytosis?30.90% in mi RNA group verse 23.20% in miRNA control group?,apoptosis?3.10% in miRNA group verse 5.30% in miRNA control group?and migration rate?13.88% in miRNA group verse 21.03% in miRNA control group?of oyster haemocytes were found significantly altered after the injection of scaffold42648₅080 mimics.Among the target genes,integrin-linked kinase?CgILK?was considered crucial in cell migration and its interaction with scaffold42648₅080 was then verified both in vitro and in vivo.Consequently,a significant decrease of relative luciferase ratio was observed in CgILK 3'-UTR luciferase reporter assay after transfection of scaffold42648₅080 mimics?0.70-fold of that in blank group,p < 0.01?.Meanwhile,when scaffold42648₅080 was overexpressed in vivo?5.41-fold of miRNA control group,p < 0.01?,the expression of CgILK declined significantly to 0.25-fold of miRNA control group?p < 0.01?.Comparatively,a significant decrease of the haemocyte migration rate?19.76% verse 34.82% in siEGFP control group,p < 0.01?was observed after knock-down of CgILK in vivo.The present study,as far as we know,for the first time revealed the immunomodulation role of an oyster species-specific miRNA,which might provide new insights into miRNA-mediated adaptation mechanism of oysters.Nuclear factor?NF?-?B pathway is an evolutionally conserved pathway in activating immune response,in which I?Bs can repress the activation.In the present study,cgi-miR-2d,an invertebrate-specific mi RNA,was proved to regulate CgI?B2 expression and haemocyte phagocytosis during bacterial infection in oyster C.gigas.The expression of cgi-miR-2d was significantly up-regulated after V.splendidus challenge,while CgI?B2 transcripts decreased.Significant decreases in both luminescence and CgI?B2 3'UTR level was observed after transfection of cgi-miR-2d in CgI?B2 3'UTR luciferase reporter assay.CgI?B2 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?,which was consistent with results in CgI?B2 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-?B pathway,which might dedicate critically to the well-balance of host immune response.Acetylcholine?ACh?is the main neurotransmitter in cholinergic neuroendocrine system and plays indispensable role in modulating diverse immune response.As an important transporter in choline uptake,choline transporter-like proteins?CTLs?can control ACh synthesis and release indirectly in multiple organisms.In the present study,cgi-miR-2d,an invertebrate-specific miRNA in oyster C.gigas,is proved to repress the synthesis/release of ACh by targeting CgCTL1 and choline uptake in haemocytes during the early-stage of pathogen infection.In short,an opposite expression pattern between CgCTL1 and cgi-miR-2d is observed during V.splendidus infection,accompanied with changes in haemolymph ACh.And expression level of CgCTL1 is found significantly repressed after cgi-miR-2d overexpression in vivo.Meanwhile,gain-of-function of cgi-miR-2d also leads to a strong decrease in both haemocyte choline and haemolymph ACh in vivo,which is similar with that in CgCTL1 knock-down assay.As a result,the expression of two tumor necrosis factor like proteins and the bacteriostatic activity of oyster haemocytes,are found altered significantly by either gain-of-function cgi-miR-2d or knock-down of CgCTL1.To our knowledge,this is the first miRNA identified in invertebrates that can target ancient cholinergic system and augment immune response during infection.The adaptation mechanism of environmental stress in oyster involves multiple levels,and miRNA is one of the most important regulators in post-transcriptional level.In the present study,an oyster norepinephrine-responsive mi RNA cgi-miR-365 was proved to contribute to the host adaptation against desiccation by directly promoting the expression of CgHSP90AA1.Briefly,a significant increase of cgi-miR-365 was observed from the first day after aerial exposure and the up-regulation was vigorously repressed when oysters were injected with adrenoceptors antagonists.A total of 15 genes involved in biological regulation,metabolic process and response to stimulus were predicted to be modulated by cgi-miR-365.Among these genes,CgHSP90AA1 was up-regulated significantly during desiccation and could be down-regulated after simultaneous injection of adrenoceptors antagonists.The interaction between cgi-miR-365 and CgHSP90AA1 was subsequently verified in vitro,and a significant promotion of CgHSP90AA1 transcripts was observed after overexpressing cgi-miR-365 in either in vitro luciferase reporter assay or primarily cultured haemocytes.Meanwhile CgHSP90AA1 transcripts decreased in vivo when cgi-miR-365 was repressed by its inhibitor during desiccation.Collectively,it was suggested that cgi-miR-365 could be induced by norepinephrine during desiccation and promote CgHSP90AA1 expression directly after binding to its 3'-UTR.This study provided new evidences in miRNA-mediated adaptation mechanism in oysters against intertidal stresses.Oysters were found tolerant against desiccation and could survive for days before succumbed in overwhelming inflammation.In the present study,the expression levels of CgTNFs were surveyed during desiccation and also found vigorously up-regulated at the late-stage of stress.As reported,NO could modulate the expression of cytokines in multiple organisms.Here the relative activity of NOS and concentration of NO were surveyed simultaneously during desiccation and found altered synchronously with CgTNFs or CgIL17.CgNOS was then blocked by L-NMMA injection,and expression levels of CgTNF6440 and CgIL17-4 were found inhibited at the same time.CaM was an important class of Ca2+ sensor and could activate NOS.Here CgCaMs were found modulated during aerial exposure in a post-transcriptional way.Meanwhile,NOS activity and NO concentration could be significantly repressed by inhibition of CgCaMs.Expression levels of CgTNFs were also remarkably down-regulated during the inhibition of CgCaMs.To figure out the underlying modulation mechanism of CgCaMs,miRNAs targeting CgCaMs were searched globally.It was found that eight mi RNAs could target CgCaM24243 while six of them shared opposite expression pattern during stress.Gain-and loss-of function assay of above six miRNAs were then conducted in HEK293 T cells or oysters.As a result,CgCaM24243 could be significantly repressed by the coordination of miRNAs.Meanwhile,NOS activity and NO concentration could also be modulated.Tissue injuries of oyster hepatopancreas were then found greatly relieved by continuous inhibition of either CgCaMs or NOS during desiccation.miRNAs are likely to play a vital role in the intertidal life of oysters especially during their responses against pathogen infection or desiccation stress.