TLRs are important signaling recruiters, and they can be classified into cell membrane surface receptors (e.g., TLR1, TLR2, TLR4, TLR5, and TLR6) and intracellular receptors (e.g., TLR3, TLR7, TLR8, and TLR9) depending on their location in the cell (29, 53). In aquatic animals, the identified TLRs include TLR1, TLR5, and TLR14 in fish (30–33, 54), TLR1, TLR2, and TLR4 in the Pacific oyster (Crassostrea gigas) (55), TLR3 in sea cucumbers (Apostichopus japonicus) (56), TLR2 in the green mud crab (Scylla paramamosain), and TLR6, TLR7, TLR8, and TLR9 in Pacific white shrimp. In recent years, studies on miRNAs targeting TLRs have been conducted in fish.

miR-200a-3p, miR-8159-5p, and miR-217-5p are three known miRNAs with lengths of 22 nt, 23 nt, and 23 nt, respectively, and seed regions of “-AACACUG-” (miR-200a-3p), “-CAGTAAC-” (miR-8159-5p), and “-CTGCATC-” (miR-217-5p), respectively. Several studies have reported that miR-200a-3p, miR-8159-5p, and miR-217-5p can bind to the 3’ untranslated region (UTR) of the TLR1 gene and exert negative regulation of post-transcriptional attenuation in the miiuy croaker (Miichthys miiuy) ( Figure 2A ). Pathogenic bacterial infection experiments indicated that the relative expression trends of miR-200a-3p, miR-8159-5p, and miR-217-5p were opposite to that of TLR1 in the spleen and leukocytes of miiuy croaker after infection with Vibrio anguillarum or stimulation with lipopolysaccharide (LPS), suggesting that miR-200a-3p, miR-8159-5p, and miR-217-5p may be involved in the immune response against pathogenic bacteria in fish by regulating the canonical NF-κB pathway via targeting TLR1 (30, 31).

miR-142a-3p (32), miRn-115 (32), and miR-182-3p (33) have been identified as miRNAs targeting TLR5 homologous genes in aquatic animals. miR-142a-3p, miRn-115, and miR-182-3p are 23 nt, 18 nt and 20 nt in length, respectively, with seed regions of “-GUAGUGU-” (miR-142a-3p), “-GAAGGCC-” (miRn-115), and “-GUUCUAG-” (miR-182-3p), respectively ( Figure 2B ). Dual luciferase reporter gene assays showed that both miR-142a-3p and miRn-115 could bind to the 3’ UTR of grass carp (Ctenopharyngodon Idella) TLR5 and that they have a negative regulatory relationship ( Figure 2B ). Overexpression of miR-142a-3p or miRn-115 resulted in a significant decrease in the relative expression of the TLR5 gene in the kidneys of grass carp infected with Aeromonas hydrophila, and this in turn suppressed the expression of downstream genes (e.g., interleukin-1β (IL-1β), interleukin-8 (IL-8), and tumor necrosis factor-α (TNF-α)) to avoid an excessive inflammatory response, suggesting that miR-142a-3p or miRn-115 could indirectly suppress the inflammatory response mediated by the canonical NF-κB pathway via negatively regulating the expression of TLR5 homologous genes. miR-182-3p is another widely studied immunomodulatory-related miRNA in fish that has a length of 20 nt and a seed region of “-GUUCUAG-” ( Figure 2C ). As has been reported, miR-182-3p effectively inhibited the relative expression of TLR5M (the membrane form of TLR5) in the spleen cells of E. coioides during the immune response induced by flagellin of Staphylococcus parapsilosis (57) and consequently exerted organismal antibacterial immunity via inhibiting the activation of the TLR5M/MAPK/NF-κB pathway (32, 33).

miR-122 is a 22 nt miRNA that has a seed region of “-GGAGTG-”. miR-122 is involved in cell cycle regulation, cell differentiation, cell proliferation, and apoptosis processes in mammals (58–60). In aquatic animals, miR-122 is considered as a biomarker that contributes to the non-invasive diagnosis of liver injury in fish (61). TLR14 is a novel TLRs member that has been specifically identified in fish, including Japanese pufferfish (Takifugu rubripes) (GenBank accession No. AC156431.1), mandarin fish (Siniperca chuatsi) (GenBank accession No. MT594450.1), and miiuy croaker (M. miiuy) (GenBank accession No. KR709254.1) (54). A dual luciferase reporter gene assay indicated the presence of the seed region of miR-122 in the 3’ UTR of the TLR14 gene (which belongs to the TLR2 subfamily) in miiuy croaker ( Figure 2D ). It was reported that the relative expression levels of miR-122 and TLR14 exhibited opposite trends in the spleen and macrophages of miiuy croaker after V. anguillarum infection and LPS stimulation, suggesting that miR-122 may affect the TLR14/NF-κB signaling cascade response by negatively regulating the relative expression of TLR14 (54).

TRIF, also known as TICAM-1 (62), is involved in immune response and disease regulation. Currently, TRIF homologs have been identified in a variety of aquatic organisms, including the Northeast Chinese lamprey (Lethenteron morii) (63), channel catfish (Ictalurus punctatus) (64), zebrafish (65), grass carp (66), orange-spotted grouper (67), and large yellow croaker (Larimichthys crocea) (34).

miRNAs targeting TRIF homologous genes in aquatic animals include miR-181b-2 (35), miR-21-1 (35), and miR-15a-5p (68) ( Figure 2E ). miR-181b-2, miR-21-1, and miR-15a-5p are 22 nt, 22 nt, and 21 nt in length, respectively, with highly similar seed regions of “-ACAUUCA-” (miR-181b-2), “-AACAGCA-” (miR-21-1), and “-AGCAGCA-” (miR-15a-5p), respectively. Dual luciferase reporter assays demonstrated that the seed regions of miR-181b-2, miR-21-1, and miR-15a-5p were all present in the 3’ UTR of TRIF in miiuy croaker ( Figure 2E ). The results of pathogenic infection experiments showed that the relative expression levels of miR-181b-2, miR-21-1, and miR-15a-5p in the macrophages and brain cells of miiuy croaker challenged by LPS stimulation and V. anguillarum or Siniperca chuatsi rhabdovirus (SCRV) infection showed opposite trends to those of TRIF (35). These observations suggested that miR-181b-2, miR-21-1, and miR-15a-5p could negatively regulate the expression of TRIF homologous genes and in turn regulate TRIF/NF-κB, ultimately inhibiting antibacterial and antiviral immune responses in fish (35).

MyD88, a key node in the canonical NF-κB signaling pathway, is also an important target for several miRNAs (3). To date, MyD88 homologs have been identified in a variety of aquatic animals, including a marine gastropod (Littorina littorea) (69), a tropical sea cucumber (Holothuria leucospilota) (70), and a sea cucumber (A. japonicus) (36).

miR-148 (37), miR-214 (38), miR-19a (71), and miR-3570 (17) are known miRNAs that are 21 nt, 21 nt, 23 nt, and 22 nt in length, respectively, and contain seed regions “-CAGTGCA-” (miR-148), “-CAGCAGG-” (miR-214), “-GTGCAAA-” (miR-19a), and “-ACAATCA-” (miR-3570), respectively. These miRNAs have been validated to target and regulate the expression of MyD88 homologs in different aquatic animals ( Figure 2F ). For example, dual luciferase assays have confirmed that the seed regions of miR-148, miR-214, miR-19a, and miR-3570 were simultaneously present in the 3’ UTR of MyD88 in miiuy croaker (17, 37, 38, 71). Pathogenic bacterial infection experiments indicated that the relative expression trends of miR-148, miR-214, miR-19a, and miR-3570 were contrary to the relative expression levels of MyD88 in the spleen, macrophages, and kidney of miiuy croaker after Vibrio harveyi infection and LPS stimulation, suggesting that miR-148, miR-214, miR-19a, and miR-3570 can negatively regulate the expression of MyD88. These observations also indicated that miR-148, miR-214, miR-19a, and miR-3570 may be involved in pathogenic bacteria-induced immune responses in fish through inhibiting the canonical NF-κB pathway via targeting MyD88 homologs. Interestingly, it was reported that miR-19b (a member of the miR-19 family) with a seed region “-GUGCAAA-” significantly enhanced NF-κB activity in human HEK293 cells and mouse embryonic fibroblasts (72). Since there is no report concerning the role of miR-19b in aquatic animals, further study is necessary to clarify the regulation between miR-19b and the NF-κB pathways in aquatic animals.

IRAK1 and IRAK4 are the only two members of the IRAK family with kinase activity (73–75), and IRAK1 can mediate NF-κB signaling by being recruited by MyD88, in turn leading to the production of inflammatory factors such as IL-8 and TNF-α (76). IRAKs have been identified in many aquatic organisms; for example, IRAK1 homologs have been identified in red tailed shrimp (Fenneropenaeus penicillatus) (77) and Pacific white shrimp (78), and IRAK4 homologs have been identified in an abalone (Haliotis discus) (GenBank accession No. KU351646.1), a thick shell mussel (Mytilus coruscus) (79), and a sea cucumber (A. japonicus) (39). Dual luciferase reporter assay data showed that 3’ UTRs of both IRAK1 and IRAK4 in aquatic animals have multiple miRNA target binding sites ( Figures 2G, H ). miR-148-1-5p (40), miR-133 (41), miR-203 (42), miR-21 (43), and miR-27c-3p (80) are known miRNAs with lengths of 23 nt, 23 nt, 22 nt, 22 nt and 21 nt, respectively, and the seed regions “-AGUUCUG-” (miR-148-1-5p), “-UUGGUCC-” (miR-133), “-TGAAATG-” (miR-203), “-AACAGCA-” (miR-21), and “-UCAGACU-” (miR-27c-3p), respectively. miR-148-1-5p and miR-133 can regulate the expression of IRAK1, and miR-203, miR-21, and miR-27c-3p regulate the expression of IRAK4 ( Figure 2H ). Studies have shown that the expression levels of miR-148-1-5p in the brain cells of miiuy croaker and that of miR-133 in the coelomocytes of A. japonicus showed clear contradictory trends to those of IRAK1 homologous genes after pathogenic bacterial infection as well as to the expression trends of miRNAs (miR-203, miR-21, and miR-27c-3p) and IRAK4 in the liver, spleen, macrophages, kidney, and intestine cells of miiuy croaker. These observations indicated that miR-148-1-5p and miR-133 may act as negative regulators to inhibit the activation of the canonical NF-κB pathway by downregulating IRAK1 homologous gene expression and that miR-203, miR-21, and miR-27c-3p could ultimately inhibit the antibacterial and antiviral immune effects of aquatic organisms via downregulation of IRAK4 homologous gene expression.

TRAF6 is an important intracellular multifunctional signaling molecule, and it is one of the most widely studied members of the TRAF family (81, 82). It has been reported that TRAF6 activated the IKK complex, in turn leading to the activation of NF-κB and the expression of inflammatory cytokines (83). Based on current research, TRAF6 has been identified in a variety of aquatic organisms, including Pacific white shrimp (69), Zhikong scallop (Chlamys farreri) (84), and Hong Kong oyster (Crassostrea hongkongensis) (GenBank accession No. MK799968.1).

miR-2187 (16), miR-30c-3-3p (44), and miR-146a (85) are 21 nt, 22 nt, and 23 nt in length, respectively, with seed regions “-UACAGGC-” (miR-2187), “-TGGGAGA-” (miR-30c-3-3p), and “-GUAGAUA-” (miR-146a), respectively. Dual luciferase reporter assays showed that TRAF6 homologs in aquatic animals have binding sites ( Figure 2I ) on the 3’ UTR that exactly match the seed sequences of miR-2187, miR-30c-3-3p, and miR-146a. It has been reported that overexpression of miR-2187, miR-30c-3-3p, or miR-146a significantly suppressed the expression of TRAF6 in the liver and spleen cells of miiuy croaker infected with V. anguillarum and red-spotted grouper nervous necrosis virus (RGNNV), indicating a negative regulatory relationship between these miRNAs and TRAF6. In addition, overexpression of miR-2187, miR-30c-3-3p, or miR-146a also promoted the replication and occurrence of SCRV or RGNNV, suggesting that miR-2187, miR-30c-3-3p, or miR-146a could be induced and utilized by SCRV or RGNNV to be conducive to their infection and reproduction. Furthermore, it was also reported that miR-2187, miR-30c-3-3p, or miR-146a may possibly depress the expression of inflammatory factors (e.g., TNF-α, IL-8, or IL-1β) in an indirect way in the liver, kidney, and spleen cells of miiuy croaker or in the spleen of E. coioides after LPS stimulation or infection by RGNNV or SCRV (16, 44, 85). Combining all of the above observations, we conclude that miR-2187, miR-30c-3-3p, and miR-146a may enhance infection by SCRV and RGNNV through suppressing the NF-κB pathways and subsequently initiating inflammatory responses via targeting TRAF6 homologs in fish.

TAK1 is a member of the MAPK kinase family, and it can be activated by TNF, LPS, and Epstein-Barr virus latent membrane protein 1 (LMP1) (86–89). Additionally, one of the most important roles of TAK1 is acting as an upstream activator of the NF-κB pathways (90). In aquatic animals, TAK1 homologs have been identified in crustacean species and echinoderms such as the Pacific white shrimp (GenBank accession No. KU522004.1), a mud crab (Scylla paramamamosain) (GenBank accession No. MK319934.1), and a sea urchin (Paracentrotus lividus) (45).

miR-217 is the only known miRNA to date that may interact with TAK1 homologs in aquatic animals. This mRNA is 23 nt in length and has a “-CUGCAU-” seed region. Zhang et al. identified a binding site in the 3’ UTR of miiuy croaker TAK1 that exactly matched the seed sequence of miR-217 by using dual luciferase validation (91) ( Figure 2J ). In the Chinese mitten crab (Eriocheir sinensis), overexpression of miR-217 affects the replication of white spot syndrome virus (WSSV) and plays an active role in WSSV infection (92). Further in vivo and in vitro pathogenic infection experiments showed that the expression trends of miR-217 and TAK1 were contrary in the spleen and macrophages of miiuy croaker, indicating that miR-217 may be involved in the antibacterial and antiviral immune responses of fish through suppressing the NF-κB pathways via targeting TAK1 (91).

The p65 protein is located at the end of the canonical NF-κB pathway. The transcription of inflammatory cytokines is modulated by binding to p50 and forming a “p65/p50” dimer. The “p65/p50” dimer enters the nucleus and binds to the κB site in the promoter or enhancer of the target gene (93–97). In aquatic animals, p65 homologous genes have been identified in teleost fishes such as zebrafish (9), olive flounder (Paralichthys olivaceus) (46), and common carp (Cyprinus carpio) (GenBank accession No. MN167531.1).

Currently, miR-216a is the only identified miRNA that can bind to the 3’ UTR of p65 homologs in fish (98) ( Figure 2K ). miR-216a is 22 nt in length and has the seed region “-AATCTC-”. A study by Xu et al. (98) showed that the expression of miR-216a was significantly upregulated in the spleen and macrophages of miiuy croaker. Overexpression of miR-216a could also affect the expression of inflammatory cytokines such as TNF-α, IL-1β, interleukin-6 (IL-6), and IL-8. In vivo and in vitro experiments demonstrated that miR-216a could downregulate the activation of the NF-κB pathways by negatively regulating the expression of miiuy croaker p65 at the post-transcription level. All of the above-mentioned studies indicate that miR-216a could suppress excessive and prolonged inflammatory responses in the organism through negatively regulating the NF-κB pathways via directly targeting p65 homologous genes in fish (98).

NOD1 is one of the most representative members of the NLR family, and it serves as an important intracellular receptor that effectively detects pathogenic components produced by various gram-negative bacteria in mammals (102, 103). NOD1 is involved in the antibacterial or antiviral-induced immune response by activating the NF-κB pathways and subsequent inflammatory responses (104, 105). In addition, NOD1 can also act as a receptor to enhance the immune response during viral infection (106). Currently, NOD1 has been identified in several aquatic organisms, including teleost fishes such as rainbow trout (Oncorhynchus mykiss) (GenBank accession No. KF484402.1), zebrafish (GenBank accession No. KC207831.1), mandarin fish (S. chuatsi) (GenBank accession No. KY974318.1), olive flounder (GenBank accession No. JF830013.1), and miiuy croaker (104).

miR-144 and miR-217 are 22 nt and 23 nt in length, respectively, with seed regions “-ACAGUAU-” (miR-144) and “-ACUGCAU-” (miR-217), respectively. Dual luciferase reporter assays have shown that NOD1 homologs in aquatic animals have binding sites in the 3’ UTR that exactly match the seed sequences of miR-144 and miR-217 ( Figure 2L ). Overexpression of miR-144 and miR-217 significantly inhibited the expression of miiuy croaker NOD1 at the post-transcription level in the spleen and macrophages of miiuy croaker infected with V. harveyi and stimulated by LPS, respectively, indicating a negative regulatory relationship between the above-mentioned miRNAs and NOD1 (100). Taken together, the above-mentioned studies suggest critical roles for miR-144 and miR-217 that may involve inhibiting organismal and antibacterial immunity via negatively regulating the NF-κB pathways and subsequent inflammatory responses by suppressing NOD1 expression (100).

RIPK2 is a key factor involved in the pathogen-induced immune response (107–109). As the downstream bridging protein in the NLR signaling pathway, RIPK2 can influence cellular signaling and cytokine production induced by NOD1 (110). Currently, RIPK2 homologs have been identified in a number of aquatic animals, including cyclostome and teleost fishes such as the Reissner lamprey (Lethenteron reissneri) (111), goldfish (Carassius auratus) (GenBank accession No. KJ636470.1), and cyprinid fish (Schizothorax prenanti) (GenBank accession No. MW113673.1).

miR-210 and miR-3570 are two known miRNAs having regulatory relationships with RIPK2 homologs (101). miR-210 and miR-3570 are both 22 nt in length, with seed regions “-CCACUG-” (miR-210) and “-CUGUU-” (miR-3570), respectively. Dual luciferase reporter assays revealed that both miR-210 and miR-3570 could precisely bind to the 3’ UTR of the RIPK2 gene in miiuy croaker (101) ( Figure 2M ). Pathogenic bacterial infection experiments showed that in the spleen and macrophages of miiuy croaker infected by V. harveyi and subjected to LPS stimulation, the relative expression trends of miR-210 and miR-3570 were significantly in contrast to those of RIPK2. Considering all of the above results, we can conclude that miR-210 and miR-3570 may participate in the immune response against pathogenic bacterial or LPS stimulation in fish by negatively regulating the NF-κB pathways via targeting RIPK2 (101).

MAVS acts as a mitochondrial antiviral signaling adapter that bridges the gap between RIG-I and MDA5 sensing of viral infection and downstream signaling. Activation of MAVS leads to the rapid production of antiviral cytokines. MAVS homologs have been identified in teleost fishes such as zebrafish (116) and Atlantic salmon (Salmo salar) (117).

miR-3570 (25), miR-122 (26), and miR-21-3p (27) have lengths of 22 nt, 22 nt, and 22 nt, respectively, with seed regions “-ACAATCA-” (miR-3570), “-GGAGUG- “ (miR-122), and “-GACAACA-” (miR-21-3p) ( Figure 2N ). Dual luciferase reporter gene assays showed that miR-3570, miR-122, and miR-21-3p can bind to the 3’ UTR of the miiuy croaker MAVS gene and exert a negative regulatory effect on post-transcriptional attenuation. Pathogenic infection and poly(I:C) challenge experiments indicated that in macrophages, intestine cells, and kidney cells of miiuy croaker after SCRV infection, the relative expression of levels miR-3570, miR-122, or miR-21-3p were completely opposite to that of MAVS. Hence, the expression level promoted viral replication by inhibiting the production of IFNs and antiviral genes (e.g., TNF-α, IL-1β, and Mx1), ultimately inhibiting the organism’s antiviral response and promoting the replication of SCRV (25–27). Overall, these studies indicated that miR-3570, miR-122, and miR-21-3p acted as negative regulators in suppressing organismal immune and antiviral responses through depressing the NF-κB pathways via directly targeting MAVS (25–27).

MITA is also known as a stimulator of interferon genes (STING) (118). As a crucial member of the RLR signaling pathway, MITA is involved in the regulation of signal transduction and the innate antiviral response in mammals (118). Compared to studies in mammals, there are relatively few studies related to MITA in aquatic animals.

miR-210, miR-21-3p, and miR-29a-3p are all 22 nt in length, with seed regions “-GCCACUG-” (miR-210), “-GACAACA-” (miR-21-3p), and “-AGCACCA-” (miR-29a-3p), respectively. Dual luciferase reporter assays demonstrated that MITA of miiuy croaker had binding sites ( Figure 2O ) in the 3’ UTR that precisely match the seed sequences of miR-210, miR-21-3p, and miR-29a-3p. Overexpression of miR-210, miR-21-3p, or miR-29a-3p suppressed the expression of MITA at both the mRNA and protein levels in the macrophages, intestinal cells and spleen cells of miiuy croaker after SCRV infection (50–52). Meanwhile, the expression of inflammatory cytokines (e.g., TNF-a, IL-6, and Mx1) was significantly inhibited. Taken together, these results suggest that miR-210, miR-21-3p, and miR-29a-3p may be involved in reducing cell proliferation, promoting viral replication, and inhibiting the organismal antiviral response by negatively regulating the NF-κB pathways via targeting MITA (50–52).

TBK1 is a vital immune-related kinase involved in the production of IFNs to prevent invasion by pathogenic microorganisms (119, 120). To date, TBK1 homologous genes have been identified in several groups of aquatic animals, including mollusks and teleost fishes, in species such as marine gastropods (69), Pacific oyster (121), and zebrafish (122).

miR-2187-3p is the only miRNA that has a relationship with TBK1 homologs in aquatic animals (115). This miRNA is 21 nt in length, with a seed region “-UACAGGCU-”. Dual luciferase reporter gene assays demonstrated that TBK1 of miiuy croaker has a binding site in the 3’ UTR that completely matches the seed sequence of miR-2187-3p ( Figure 2P ) (115). In intestinal cells of miiuy croaker after SCRV infection, overexpression of miR-2187-3p downregulated the expression of TBK1 at both transcriptional and post-transcriptional levels. These observations suggested that miR-2187-3p may act as a negative regulator suppressing the organismal antiviral response through the NF-κB pathways via downregulating the relative expression of TBK1 (115).

As mentioned above, miRNAs involved in NF-κB pathways have now been identified in a variety of aquatic organisms, especially in teleost fishes (123–154) ( Table 3 ). Notably, most of these identified miRNAs have corresponding homologous sequences in mammals. Phylogenetic analysis data indicated a high conservation of examined miRNA homologous sequences in both aquatic animals and mammals ( Figure 3 ), suggesting that these miRNAs might have similar regulatory mechanisms in different species. Furthermore, some miRNAs (miR-133, miR-21 and miR-210) have been identified both in vertebrate and non-vertebrate aquatic animals, whereas some have only been identified in teleost fishes ( Tables 3 , 4 ). As the current relevant studies and evaluated aquatic species are still relatively few, whether there are differences in miRNAs regulating NF-κB pathways between vertebrate and non-vertebrate aquatic animals needs further exploration and justification.