Jingjing Han

The genome of the miiuy croaker reveals well-developed innate immune and sensory systems

The miiuy croaker, Miichthys miiuy, is a representative Sciaenidae known for its exceptionally large otoliths. This species mainly inhabits turbid aquatic environments with mud to sandy mud bottoms. However, the characteristics of the immune system of this organism and its specific aquatic environment adaptations are poorly understood. Thus, we present a high-quality draft genome of miiuy croaker. The expansions of several gene families which are critical for the fish innate immune system were identified. Compared with the genomes of other fishes, some changes have occurred in the miiuy croaker sensory system including modification of vision and expansion of taste and olfaction receptors. These changes allow miiuy croaker to adapt to the environment during the long-term natural selection. The genome of miiuy croaker may elucidate its relatively well-developed immune defense and provide an adaptation model of the species thriving in turbid deep aquatic environments.

Identification and characteristic analysis of TLR28: A novel member of the TLR1 family in teleost

Toll-like receptors (TLRs) play a critical role in the innate immune response of fish to recognize microorganisms. Fish TLRs have significant variety and distinct features. This study focuses on a novel TLR member that belongs to the TLR1 family and was first discovered in miiuy croaker (designated as TLR28, mmiTLR28). In phylogenetic analysis, the mmiTLR28 clustered in the TLR1 family. Further characteristic analysis showed a high homology with TLR2 despite some differences between them. The predicted tertiary structure of mmiTLR28 possesses a hydrophobic pocket in the ectodomain region. Expression analysis showed the high expression level in the liver of miiuy croaker. Further functional experiments on the liver after Vibrio anguillarum, Staphylococcus aureus, lipopolysaccharides (LPS), and poly (I:C) stimulation showed significant upregulation; these results indicate the potential role of mmiTLR28 in immune response. For LPS stimulation in miiuy croaker leukocytes, mmiTLR28 also displayed significant upregulation. The discovery of mmiTLR28 will enrich the information on TLR family; the functional experiments have shown the role of mmiTLR28 in immunity. The results of this study lay the foundation for future research on fish immune systems.

Comparative analysis of the small RNA transcriptomes of miiuy croaker revealed microRNA-mediated regulation of TLR signaling pathway response to Vibrio anguillarum infection

MicroRNAs (miRNAs) are an abundant class of endogenous noncoding small RNAs (sRNAs) that are partially complementary to their target messenger RNA (mRNA), which post-transcriptionally regulate various biological processes by repressing mRNA translation or inducing mRNA degradation. MiRNAs have been demonstrated to play crucial roles in the host response to infection by diverse pathogens. As an important bacterial pathogen, Vibrio anguillarum has been caused great economic losses in miiuy croaker aquaculture. To identify immune-related miRNAs of miiuy croaker in response to V. anguillarum infection, we constructed two sRNA libraries with or without bacterial infection. High-throughput deep sequencing and subsequent bioinformatic analysis identified 241 conserved and 137 novel miRNA precursors in miiuy croaker based on its whole genome sequences, encoding 293 and 124 mature miRNAs, respectively. Then we compared the expression patterns of miRNAs in the two libraries. There were significant differences in the expression of 12 miRNAs between the infection group (IG) and control group (CG). Further, the expressions of six miRNAs were validated by real-time quantitative PCR. The target gene prediction and function analysis were conducted for the 12 differential miRNAs. This analysis revealed that these miRNAs participated in the regulation multiple immune-related signaling pathways. Transcription factors in TLR signaling, such as AP-1, IRF5, NF-κB and IRF3, were activated by these miRNAs via post-transcriptionally regulating the expression of TLRs and TLR-associated signaling proteins, inducing effective host immune response to eradicate infectious pathogens. This is the first study of the identification and characterization of miiuy croaker miRNAs in response to V. anguillarum infection. The comprehensive analysis of the expression of miRNAs and the target gene and function prediction of differently expressed miRNAs may help to understand the regulatory mechanisms of miRNA in fish during the interaction between host and bacterial pathogens.

The miiuy croaker microRNA transcriptome and microRNA regulation of RIG-I like receptor signaling pathway after poly(I:C) stimulation

MicroRNAs (miRNAs) as endogenous small non-coding RNAs play key regulatory roles in diverse biological processes via degrading the target mRNAs or inhibiting protein translation. Previously many researchers have reported the identification, characteristic of miRNAs and the interaction with its target gene. But, the study on the regulation of miRNAs to biological processes via regulatory the key signaling pathway was still limited. In order to comprehend the regulatory mechanism of miRNAs, two small RNA libraries from the spleen of miiuy croaker individuals with or without poly(I:C) infection were constructed. The 197 conserved miRNAs and 75 novel miRNAs were identified, and 14 conserved and 8 novel miRNAs appeared significant variations. Those differently expressed miRNAs relate to immune regulation of miiuy croaker. Furthermore, expressions of four differently expressed miRNAs were validated by qRT-PCR, and the result was consistent with sequencing data. The target genes of the differently expressed miRNAs in the two libraries were predicted, and some candidate target genes were involved in the RIG-I-like receptor (RLR) signaling pathway. The negative regulation of miRNAs to target genes were confirmed by comparing the expression pattern of miRNAs and their target genes. The results of regulating target genes were that firstly directly or indirectly activating the downstream signaling cascades and subsequent inducting the type I interferon, inflammatory cytokines and apoptosis. These studies could help us to deeper understand the roles of miRNAs played in the fish immune system, and provide a new way to investigate the defense mechanism of fish.

Comparative genomic evidence for duplication of TLR1 subfamily and miiuy croaker TLR1 perceives LPS stimulation via MyD88 and TIRAP

Being indispensable pattern recognition receptors in innate immune responses in host protection, Toll-like receptors (TLRs) play an important role in pathogen recognition. Fish TLRs exhibit high variety and distinct features, although little is known about their function on ligand recognition and signaling pathway in fish. This paper reports the evolutionary spectrum of the TLR1 subfamily (referred to as TLR1, TLR6, and TLR10) as determined using the comparative genomic approach. We hypothesized that the TLR1 subfamily underwent two rounds of gene duplication events; the first duplication occurred prior to the divergence of amphibians, and the second one occurred prior to the divergence of eutherians. To further study the function of fish TLR1, we identified miiuy croaker (Miichthys miiuy) TLR1 (mmiTLR1) and determined its potential ability to perceive Vibrio anguillarum and lipopolysaccharide stimulation. Data further suggested that mmiTLR1 is dependent on TIRAP and MyD88 for signal transmission. In addition, immunocytochemistry showed the speculative interaction between MyD88 and mmiTLR1 TIR domain. Overall, we systematically and comprehensively analyzed evolution of TLR1 subfamily and the function of mmiTLR1, which will provide the basis for future scientific research on fish TLRs.

The evolution and functional characterization of miiuy croaker cytosolic gene LGP2 involved in immune response

The laboratory of genetics and physiology 2 (LGP2) is a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors), which may participate in the immune regulation process. The role of LGP2 on modulating signaling was ambiguous, some researchers suggested that the regulation mechanism of LGP2 to melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene-I (RIG-I) were different. In this study, the bioinformatics and functions of LGP2 from miiuy croaker (mmLGP2) were characterized. Comparative genomic analysis showed that the evolution of LGP2 in mammals was more conserved than it in fish. LGP2 contains three structural domains: ResIII, HelicaseC and RD, and ResIII structural domain of LGP2 was extremely conservative. The mmLGP2 was ubiquitously expressed in the tested miiuy croaker tissues and the expressions were significantly upregulated after stimulation with poly(I:C), indicating that LGP2 might participate in the immune response, especially antiviral immunity. Furthermore, immunofluorescence of miiuy croaker LGP2 presents in the cytoplasm in Hela cells. The overexpression of mmLGP2 can activate ISRE, but cannot activate NF-κB luciferase reporter, implying that mmLGP2 might act as a positive regulator in immune responses through activating ISRE to induce the expression of IFN. The research of mmLGP2 will enrich the information of fish LGP2, and the functional experiments will be helpful for the future research about fish immune systems.

microRNA-145 regulates the RLR signaling pathway in miiuy croaker after poly(I:C) stimulation via targeting MDA5.

ABSTRACT MicroRNAs (miRNAs) are endogenous small non‐coding RNAs that participate in diverse biological processes via degrading the target mRNAs or repressing translation. In this study, the regulation of miRNA to the RLR (RIG‐I‐like receptor) signaling pathway by degrading the target mRNAs was researched in miiuy croaker. MDA5, a microRNA‐145–5p (miR‐145–5p) putative target gene, was predicted by bioinformatics, and the target sites from the 3′untranslated region of MDA5 transcripts were confirmed using luciferase reporter assays. Pre‐miR‐145 was more effective in inhibiting MDA5 than miR‐145–5p mimic, and the effect was dose‐ and time‐dependent. The expression patterns of miR‐145–5p and MDA5 were analyzed in liver and kidney from miiuy croaker. Results implied that miR‐145–5p may function via degrading the MDA5 mRNAs, thereby regulating the RLR signaling pathway. Studies on miR‐145–5p will enrich knowledge of its functions in immune response regulation in fish, as well as offer a basis for regulatory networks that are composed of numerous miRNAs. HIGHLIGHTSTarget gene of miR‐145–5p is MDA5 in miiuy croaker.Pre‐miR‐145 showed remarkable inhibitory effects on MDA5 than mimic.The regulation of miR‐145–5p to MDA5 presented dose and time dependent.

Genomic organization, evolution and functional characterization of soluble toll-like receptor 5 (TLR5S) in miiuy croaker (Miichthys miiuy)

&NA; Toll‐like receptors (TLRs) play the key role in host defense of invasion of pathogens, not only in the innate immunity, but also in adaptive immunity. There are significant varieties and distinct features in fish TLRs, the TLR5 subfamily have two members (TLR5M and TLR5S). However, the exact role of TLR5 was lack of research in fish. In this study, a soluble form of TLR5 (TLR5S) was identified in miiuy croaker. The bioinformatics analysis showed that miiuy croaker TLR5S lacked the transmembrane domain and TIR domain. In other words, mmiTLR5S only has leucine‐rich repeats (LRRs) domain, it is one of differences between TLR5M and TLR5S. Comparative genomic analysis showed that TLR5S might have happened an evolution between species. Expression analysis showed that mmiTLR5S was expressed in all tested miiuy croaker tissues and the mmiTLR5S expressions were significantly upregulated at 12 h in liver and kidney after Vibrio harveyi infection. Further functional experiments showed that NF‐&kgr;B can be actived by mmiTLR5S, TLR5S might be an indispensable role in organism immune response. In short, the study of mmiTLR5S enriches the information of TLR5S and lays the foundation for future research on teleost TLRs system. HighlightsTLR5S was identified in miiuy croaker.Miiuy croaker TLR5S highly expressed in liver.Miiuy croaker TLR5S can activate NF‐&kgr;B signaling pathway.

microRNA-210 participates in regulating RIG-I signaling pathway via targeting DUBA in miiuy croaker after poly(I:C) stimulation

ABSTRACT MicroRNAs (miRNAs) are endogenous small non‐coding RNAs that participate in the regulation of various biological processes. A series of microRNAs have been shown to be important regulators of both innate and adaptive immune responses, including RIG‐I signaling pathway. In this study, we evaluated the regulation role of miR‐210 in the RLRs signaling pathway of miiuy croaker. Upon poly(I:C) stimulation, the expression of miR‐210 in both miiuy croaker spleen tissues and macrophages were significantly upregulated. By means of the dual luciferase reporter assay, a direct interaction between miR‐210 and the 3‐untranslated region (UTR) of Deubiquitinating enzyme A (DUBA) was confirmed, and we found that miR‐210 could reduce the luciferase levels of wild‐type DUBA 3′UTR, whereas mutant‐type led to a complete abrogation of the negative effect. Furthermore, the negative regulatory effects of pre‐miR‐210 on DUBA have been indicated in a dose‐ and time‐dependent manners. As DUBA is an important regulator involved in the RLRs signaling pathway and could bind with and regulate TRAF3, we also examined the expression patterns of DUBA and TRAF3 in vivo and in vitro. We found that the expression of both DUBA and TRAF3 were significantly changed upon poly(I:C) stimulation in miiuy croaker. The expression patterns between miR‐210 and DUBA showed a negative correlation, which indicated that miR‐210 can target and downregulate the expression of DUBA. Overall, these results will enrich the knowledge of immune response related miRNAs in miiuy croaker, which will be useful for better understanding the complicated regulatory networks in fish species. HighlightsmiR‐210 can regulate TRAF3 via targeting DUBA in miiuy croaker.Expression profiles of miR‐210, DUBA and TRAF3 were conducted in vitro and vivo.miR‐210 participates in antiviral immune response in miiuy croaker.