LncRNA pol-lnc78 as a ceRNA regulates antibacterial responses via suppression of pol-miR-n199-3p-mediated SARM down-regulation in Paralichthys olivaceus

Long non-coding RNAs (lncRNAs) function as key modulators in mammalian immunity, particularly due to their involvement in lncRNA-mediated competitive endogenous RNA (ceRNA) crosstalk. Despite their recognized significance in mammals, research on lncRNAs in lower vertebrates remains limited. In the present study, we characterized the first immune-related lncRNA (pol-lnc78) in the teleost Japanese flounder (Paralichthys olivaceus). Results indicated that pol-lnc78 acted as a ceRNA for pol-miR-n199-3p to target the sterile alpha and armadillo motif-containing protein (SARM), the fifth discovered member of the Toll/interleukin 1 (IL-1) receptor (TIR) adaptor family. This ceRNA network regulated the antibacterial responses of flounder via the Toll-like receptor (TLR) signaling pathway. Specifically, SARM acted as a negative regulator and exacerbated bacterial infection by inhibiting the expression of inflammatory cytokines IL-1β and tumor necrosis factor-α (TNF-α). Pol-miR-n199-3p reduced SARM expression by specifically interacting with the 3’ untranslated region (UTR), thereby promoting SARM-dependent inflammatory cytokine expression and protecting the host against bacterial dissemination. Furthermore, pol-lnc78 sponged pol-miR-n199-3p to ameliorate the inhibition of SARM expression. During infection, the negative regulators pol-lnc78 and SARM were significantly down-regulated, while pol-miR-n199-3p was significantly up-regulated, thus favoring host antibacterial defense. These findings provide novel insights into the mechanisms underlying fish immunity and open new horizons to better understand ceRNA crosstalk in lower vertebrates.


INTRODUCTION
Sterile alpha and armadillo motif-containing protein (SARM), the last discovered Toll/interleukin 1 (IL-1) receptor (TIR) adaptor, exhibits a highly conserved sequence across evolution, from nematodes to mammals (Jault et al., 2004;Lindsay & Wasserman, 2014;Vérièpe et al., 2015;Zhou et al., 2013).Indeed, phylogenetic analysis suggests a potential bacterial origin for animal SARM, likely arising from horizontal gene transfer (Malapati et al., 2017;Zhang et al., 2011).Like the other four TIR adaptors, myeloid differentiation primary response 88 (MyD88), MyD88 adaptor-like (Mal), TIR domaincontaining adapter-inducing interferon-β (TRIF), and TRIFrelated adaptor molecule (TRAM), SARM features a TIR domain, which facilitates its role in Toll-like receptor (TLR) signaling and innate immunity (Panneerselvam & Ding, 2015).Functional analyses demonstrated that, across all examined species, from worms and flies to mammals, SARM significantly influences immune responses to pathogenic infections, with varied outcomes despite sequence conservation.In Caenorhabditis elegans, the SARM ortholog TIR-1 regulates host defense against fungal and bacterial infections, enhancing survival without relying on the sole Toll receptor (Couillault et al., 2004).In Drosophila melanogaster, the SARM ortholog Ect4 is vital for survival post-viral infection, possibly reducing Toll signaling to restore homeostasis (Monsanto-Hearne et al., 2017).In mammals, SARM acts as a negative regulator in immune defense against live bacterial infection by suppressing TLR signaling (Carty & Bowie, 2019).Although limited in scope, investigations conducted on teleost fish, which serve as representatives of lower vertebrates, have indicated the involvement of SARM in the immune responses of grass carp to grass carp reovirus (GCRV) infection and mandarin fish to stimulation with poly (I:C) and lipopolysaccharide (LPS) (Wang et al., 2021a;Yan et al., 2015).Nonetheless, the precise role of SARM in bacterial infections in fish remains undetermined, and its immunological mechanism remains elusive.Recent research on worms and flies has revealed that SARM expression is mediated by noncoding RNAs (ncRNAs), specifically miR-956 and miR-71, respectively (Finger et al., 2019;Monsanto-Hearne et al., 2017), providing a crucial clue for elucidating the regulatory mechanisms of SARM.
Teleosts are representative of early vertebrate evolution and can serve as key models in lower vertebrate studies.However, these economically valuable aquaculture species face significant threats from bacterial diseases, amplifying their importance in immunology research.Notably, vibriosis, a prevalent aquatic disease caused primarily by Vibrio anguillarum (Egidius, 1987), has prompted great research efforts into fish defense mechanisms (Ning & Sun, 2020a, 2020b, 2021a, 2021b;Qi et al., 2021;Wang et al., 2021b;Xu et al., 2019).Nevertheless, in-depth investigations on fish defense responses remain severely impeded due to limitations in experimental techniques, including the lack of suitable cell lines.Furthermore, fish immune processes and regulatory cascades exhibit notable differences when contrasted with the well-characterized mammalian immunity.
In this study, we identified a lncRNA-miRNA-mRNA ceRNA network, consisting of pol-miR-n199-3p, pol-lnc78, and SARM, which exhibited the capacity to regulate the host immune response in the teleost Japanese flounder (Paralichthys olivaceus).SARM acted as a negative regulator, inhibiting the expression of inflammatory cytokines, and was declined upon V. anguillarum infection.Pol-miR-n199-3p suppressed SARM expression by targeting the 3' untranslated region (UTR), thereby contributing to host defense against pathogens.Moreover, pol-lnc78 functioned as a ceRNA, counteracting the inhibitory effects of pol-miR-n199-3p on SARM, and thus regulating SARM-mediated immune responses.Our results represent the first ceRNA regulatory mechanism identified in flounder.These findings should help advance our understanding of ceRNA crosstalk in lower vertebrates.

Fish and bacterial infection
Clinically healthy Japanese flounder (~50 g) were purchased from a commercial fish farm in Shandong Province, China, and maintained at 20±1 °C in aquariums equipped with aerated seawater and digital temperature sensing controllers as reported previously (Ning & Sun, 2020a).Fish were acclimated in the laboratory for at least one week before any experimental manipulation.Bacterial challenge was performed as reported previously (Ning & Sun, 2020a).Briefly, flounder individuals were injected intramuscularly with 100 μL of V. anguillarum C312, a pathogenic strain isolated from diseased flounder (Zheng et al., 2010), at a concentration of 5×10 6 colony forming units (CFU)/mL.The same volume of phosphate-buffered saline (PBS) was injected into flounder as a control.At 12 h post-infection (hpi), fish were euthanized, and spleen tissue samples were collected for RNA extraction.All experimental procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals in China and were approved by the Nanjing Normal University Animal Ethics Committee (Permit No. SYXK2015-0028).
Bio-samples from the spleen, previously utilized in a transcriptome study examining the responses of flounder to V. anguillarum over various time points (Ning & Sun, 2020a), were used to evaluate SARM and pol-lnc78 expression at 6, 12, and 24 hpi and for subsequent correlation analyses.

RNA extraction and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA from spleen samples was extracted using TRIzol reagent (Invitrogen, USA) following the manufacturer's protocols.The cDNA and miRNA cDNA templates were synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, Japan) and miRNA First-Strand cDNA Synthesis Kit (Vazyme, China), respectively, according to the manufacturer's instructions.The RT-qPCR (mRNA and lncRNA) and stem-loop RT-qPCR (miRNA) procedures were carried out with SYBR Premix Ex TaqII (TaKaRa, China) using the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, USA) according to the manufacturer's protocols.The expression level was determined using the 2 −ΔΔCt comparative Ct method (Ning & Sun, 2020b).β-actin and αtubulin were used as the internal controls for samples of uninfected and V. anguillarum-infected flounder, respectively.5S rRNA was used as the internal control for pol-miR-n199-3p.The primers used for RT-qPCR are listed in Supplementary Table S1.Correlation analysis was performed using the cor.test in R (v.3.5.2).

In vivo overexpression and effects on bacterial infection
Flounder with SARM or pol-lnc78 overexpression were constructed, with four groups of flounder used.Fish in the former two groups were injected with expression plasmid pCN3-SARM or pCN3-lnc78 with 2 μg plasmid/g fish, respectively.Fish in the latter two groups were injected with or without the same volume of empty vector pCN3 as a control.Spleen tissues were collected at 3 d post-plasmid administration, and the expression levels of SARM, pol-lnc78, and pol-miR-n199-3p were examined by RT-qPCR.
To investigate the effects of SARM or pol-lnc78 overexpression on V. anguillarum infection, flounder were injected with the plasmid pCN3-SARM or pCN3-lnc78, as described above.At 2.5 d post-plasmid administration, flounder were injected with V. anguillarum (5×10 5 CFU/fish).At 12 h post V. anguillarum injection, number of bacteria was determined using the plate count method, as reported previously (Zhang et al., 2020).

Statistical analysis
Data are presented as means±standard deviation (SD) from three independent replicates.Student's t-test was used to compare the values between different groups.Statistical analyses were performed using SPSS software (v.23.0).Statistical significance was considered at P<0.05.

Flounder SARM negatively regulates bacterial infection
Symptoms in fish challenged with V. anguillarum were first confirmed.Notably, after infection, common pathological findings of vibriosis were observed in flounder, including ulcerative skin, fin hemorrhage, and swollen spleen (Figure 1A).To evaluate the expression profiles of flounder SARM in response to V. anguillarum challenge, RT-qPCR was conducted.Results showed that SARM expression was significantly reduced during V. anguillarum infection at 6, 12, and 24 hpi (Figure 1B).In mammals, SARM is an inhibitor of TLR signaling via the suppression of IL-1β and TNF-α expression after LPS stimulation.Accordingly, we examined whether flounder SARM could inhibit these inflammatory cytokines upon bacterial infection.Fish exhibiting SARM overexpression were established using plasmid pCN3-SARM injection.Enhanced SARM expression was validated by RT-qPCR at 3 d post-administration (Supplementary Figure S1).As depicted in Figure 1C, D, in vivo overexpression of SARM significantly down-regulated IL-1β and TNF-α expression in V. anguillarum-infected flounder.To investigate the effect of SARM on pathogen infection, bacterial counts were determined in V. anguillarum-infected flounder with or without SARM overexpression.Results showed that bacterial loads in fish with SARM overexpression were significantly higher than that in fish without SARM overexpression in both the spleen and liver (Figure 1E, F).These findings suggest that antibacterial immune defense is triggered via suppression of SARM expression, which activates the TLR signaling pathway and inflammatory response, resulting in the clearance of bacterial pathogens.

SARM is a target gene of pol-miR-n199-3p
In our previous investigation on the micro-transcriptome of V. anguillarum-infected flounder, SARM was predicted to be a potential target of pol-miR-n199-3p (Ning & Sun, 2020b).To elucidate the mechanism underlying SARM modulating V. anguillarum infection, we examined the regulatory role of candidate miRNAs on SARM.The sequence features were first analyzed.As shown in Figure 2A, the sequence of SARM 3'UTR was completely complementary to the seed sequence of pol-miR-n199-3p.To confirm the specific binding site between pol-miR-n199-3p and SARM 3'UTR, a mutated form of SARM 3'UTR (SARM-mut) was constructed (Figure 2A).The dual-luciferase reporter assay was then conducted using HEK293T cells to verify the interaction between pol-miR-n199-3p and SARM 3'UTR.Results showed that pol-miR-n199-3p significantly reduced luciferase activity when co-transfected with the wild-type SARM 3'UTR but not with the mutated type (Figure 2B).Moreover, no significant decrease in luciferase activity was observed when pol-miR-n199-3p was replaced by miR-NC or wild-type SARM 3'UTR alone (Figure 2B).Finally, to determine the regulatory effect of pol-miR-n199-3p on SARM expression, a flounder model with pol-miR-n199-3p overexpression was established.Elevated pol-miR-n199-3p expression was confirmed by RT-qPCR (Supplementary Figure S2).SARM expression was significantly suppressed in fish with pol-miR-n199-3p overexpression, but not in the control fish (Figure 2C).Thus, these results indicate that pol-miR-n199-3p represses SARM expression via specific interactions with its 3'UTR.

Pol-miR-n199-3p enhances host antibacterial immunity by suppressing SARM
In our previous study on miRNA expression profiles in V. anguillarum-infected flounder, we found that pol-miR-n199-3p is significantly up-regulated in response to V. anguillarum infection at 6, 12, and 24 hpi (Ning & Sun, 2020b).In the present study, the expression levels of pol-miR-n199-3p and SARM were shown to be significantly negatively correlated  3A).Thus, we subsequently explored whether pol-miR-n199-3p had any effect on SARM expression.Results showed that pol-miR-n199-3p significantly suppressed SARM expression in flounder (Figure 3B).Given that SARM exacerbated bacterial infection by suppressing inflammatory cytokines, we wondered whether pol-miR-n199-3p could affect SARM-mediated IL-1β and TNF-α.As shown in Figure 3C, D, overexpression of pol-miR-n199-3p markedly increased the expression of IL-1β and TNF-α.To examine the direct effects of pol-miR-n199-3p on bacterial infection, bacterial counts were determined in fish with or without pol-miR-n199-3p overexpression upon V. anguillarum challenge.Our findings indicated that the up-regulation of pol-miR-n199-3p contributed to the inhibition of bacterial disseminations in both the spleen and liver (Figure 3E, F).These observations suggest that pol-miR-n199-3p promotes host defense against bacterial infection via increasing the expression of SARMmediated inflammatory cytokines.

Pol-lnc78 regulates pol-miR-n199-3p expression via interaction
In our previous study on lncRNAs in flounder, we predicted that pol-lnc78 may target pol-miR-n199-3p (Ning & Sun, 2021a).To determine whether pol-lnc78 interacts with pol-miR-n199-3p, a dual-luciferase reporter assay was performed in HEK293T cells.The sequence of pol-lnc78 was first analyzed to identify the binding site for pol-miR-n199-3p (Figure 4A).Subsequently, both a wild-type pol-lnc78 reporter and mutated-type reporter with a mutated binding site were constructed.The dual-luciferase assay revealed that pol-miR-n199-3p strongly inhibited luciferase activity of the wild-type pol-lnc78 reporter but elicited no response in the mutated-type reporter (Figure 4B).To investigate the regulatory roles of pol-lnc78 on the target, a flounder model with pol-lnc78 overexpression was established using plasmid pCN3-lnc78 injection.After verification of increased pol-lnc78 expression at 3 d post-administration (Supplementary Figure S3), pol-miR-n199-3p expression was determined.As shown in Figure 4C, pol-lnc78 significantly reduced the expression of pol-miR-n199-3p in vivo.Together, these observations indicate that pol-lnc78 negatively regulates pol-miR-n199-3p expression through direct interaction.

Pol-lnc78 aggravates bacterial infection via suppression of pol-miR-n199-3p
In our previous research, we identified pol-lnc78 as a potential immune-related lncRNA engaged in V. anguillarum infection (Ning & Sun, 2021a).Here, validation of expression patterns indicated that pol-lnc78 was significantly down-regulated in the V. anguillarum-challenged flounder at 6, 12, and 24 h (Figure 5A).Correlation analysis showed that pol-lnc78 was significantly and negatively correlated (P<0.05) with pol-miR-n199-3p expression upon bacterial stimulation (Figure 5B).Moreover, in vivo overexpression of pol-lnc78 significantly decreased the expression of pol-miR-n199-3p in V. anguillarum-infected flounder (Figure 5C).Exploration of the potential regulatory effects of pol-lnc78 on inflammatory cytokines mediated by pol-miR-n199-3p during infection showed that overexpression of pol-lnc78 significantly repressed IL-1β and TNF-α expression in V. anguillaruminfected flounder (Figure 5D, E).An in vivo infection study was conducted using flounder with or without pol-lnc78 overexpression.Results indicated significantly increased bacterial loads in both the spleen and liver of fish overexpressing pol-lnc78 (Figure 5F, G).Collectively, these findings suggest that pol-lnc78 promotes bacterial dissemination by inhibiting pol-miR-n199-3p.

Pol-lnc78, as a ceRNA, sponges pol-miR-n199-3p to increase SARM expression
In our previous study on lncRNA-miRNA-mRNA networks in V. anguillarum-infected flounder, we predicted that the pol-lnc78pol-miR-n199-3p-SARM axis may be involved in immunerelated ceRNA networks (Ning & Sun, 2021a).In the current study, we validated the interaction between pol-lnc78 and pol-miR-n199-3p and the targeting of SARM by pol-miR-n199-3p.Subsequently, we explored whether pol-lnc78 could regulate SARM expression.In vivo analysis showed that overexpression of pol-lnc78 significantly up-regulated SARM expression (Figure 6A).Moreover, correlation analysis showed that the expression levels of pol-lnc78 and SARM

DISCUSSION
Bacterial diseases greatly impact the fish farming industry, with vibriosis, one of the most prevalent aquaculture diseases globally, causing substantial economic losses (Egidius, 1987).Among the causative agents of vibriosis, V. anguillarum is noteworthy due to its pathogenicity and ubiquitous existence in the water environment, leading to widespread infections in various aquatic animals, including Atlantic cod (Lokesh et al., 2012), orange-spotted grouper (Huang et al., 2014), tongue sole (Zhang et al., 2015), turbot (Gao et al., 2016), miiuy croaker (Chu et al., 2017), and flounder (Gao et al., 2013).The innate immune system acts as the first and central line of defense in teleost fish, with the TLR signaling pathway playing V. anguillarum for 6, 12, and 24 h were examined.hpi: Hours post infection.B: Pol-lnc78 and pol-miR-n199-3p were significantly and negatively correlated.Expression levels of pol-lnc78 and pol-miR-n199-3p in flounder stimulated with or without V. anguillarum for 6, 12, and 24 h were tested, then subjected to correlation analysis.r, correlation coefficient.C-E: Pol-lnc78 suppressed expression of pol-miR-n199-3p and its mediated inflammatory cytokines upon V. anguillarum stimulation.Flounder were administered with or without pol-lnc78 expression plasmid or administered with pCN3 vector for 2.5 d, then injected with V. anguillarum for another 12 h.Expression levels of pol-miR-n199-3p (C), IL-1β (D), and TNF-α (E) were examined.F, G: Pol-lnc78 promoted V. anguillarum dissemination.Flounder were administered with or without pol-lnc78 expression plasmid or administered with pCN3 vector for 2.   a crucial role in antibacterial responses.Among the five TIR adaptors, SARM is uniquely inhibitory and highly conserved (O'Neill & Bowie, 2007).In the present study, we observed a significant decline in the expression of flounder SARM during V. anguillarum infection.The inhibitory role of SARM on the TLR pathway also appears to be conserved across mammals.For example, in human HEK293T cells, overexpression of SARM significantly represses LPS-mediated up-regulation of inflammatory cytokines, such as IL-1β and TNF-α (Carlsson et al., 2016).Burkholderia pseudomallei infection can increase SARM expression in mice, resulting in the suppression of TRIF and interferon β (IFNβ) production (Pudla et al., 2011).Similarly, we found that flounder SARM significantly reduced the expression of inflammatory cytokines IL-1β and TNF-α upon V. anguillarum stimulation.Previous studies have shown divergence in the function of SARM between invertebrates and mammals.In worms and flies, SARM orthologs contribute to host resistance against microbial pathogens (Liberati et al., 2004;Monsanto-Hearne et al., 2017), while SARM -/-mice exhibit higher survival from LPS-stimulated sepsis due to increased IL-1β release (Carty et al., 2019).Here, we found that overexpression of SARM in lower vertebrates such as flounder significantly exacerbated bacterial dissemination, indicating the functional resemblance of fish SARM to that of mammals.
The molecular mechanism by which SARM regulates immune function remains enigmatic, even in mammals.Studies on invertebrate model animals have suggested a potential miRNA-mediated modulation of SARM (Finger et al., 2019;Monsanto-Hearne et al., 2017).In teleost fish, miRNAs are recognized as ubiquitous and versatile regulators, critical in immune responses.For instance, in the orange-spotted grouper, miR-122 modulates the immune response to Aeromonas hydrophila infection by targeting IL-15 (Liu et al., 2020); in miiuy croaker, miR-144 and miR-217 act as negative modulators of the inflammatory response against Vibrio harveyi infection (Chu et al., 2021a).Recent studies have also suggested that miRNAs are involved in the flounder response to V. anguillarum challenge (Ning & Sun, 2020b).In the present study, pol-miR-n199-3p was shown to target the 3' UTR of SARM, leading to a reduction in expression.These findings are consistent with the established mechanism of miRNA modulation, whereby miRNAs interact with the 3'UTR of the target gene, thus facilitating its degradation or suppressing its translation at the post-transcriptional level (Cannell et al., 2008).Accumulating evidence suggests that fish miRNAs can affect pathogens.For example, flounder pol-miR-novel_171 promotes gram-negative bacterial dissemination (Li et al., 2020), pol-miR-150 negatively regulates gram-positive bacterial infection (Sun et al., 2021), and miR-206 increases Mycobacterium marinum burden in zebrafish (Wright et al., 2021).Here, we found that pol-miR-n199-3p played a protective role against pathogen infection in flounder.Of note, the inhibition of V. anguillarum dissemination by pol-miR-n199-3p was attributable to the increase in SARM-mediated inflammatory cytokines.Moreover, we found that SARM was linked to the regulation of lncRNA.
Despite continued research on lncRNAs since the first lncRNA (Xist) was discovered in humans (Borsani et al., 1991), identification of lncRNAs in non-model species, especially fish, has only gained momentum in recent years.In our previous study, we systematically identified flounder lncRNAs through bioinformatics analysis, highlighting pol-lnc78 as an immune-related lncRNA (Ning & Sun, 2021a).In the current study, functional experiments confirmed the importance of pol-lnc78 in the host immune response to bacterial challenge, with pol-lnc78 shown to serve as a negative regulator.Recently, lncRNA-mediated ceRNA crosstalk has become a hotspot of research.In mammals, ceRNAs have been strongly implicated in the pathogenesis of cancers, providing crucial diagnostic biomarkers and therapeutic targets (Abdollahzadeh et al., 2019;Chen et al., 2017;Wang et al., 2017a).In lower vertebrates, limited research has been conducted on lncRNA-mediated ceRNA mechanisms, except for a few studies associated with miiuy croaker immunity.Specifically, in miiuy croaker, lncRNA IRL promotes the immune response against bacterial infection by counteracting the suppressive effects of miR-27c-3p on IRAK4 (Zheng et al., 2021a), while lncRNA AANCR increases MITA expression through competitively binding to miR-210 and regulates host antiviral responses (Chu et al., 2021b).Given this context, determining whether ceRNA crosstalk occurs widely among teleost fish is important.Here, we revealed that flounder pol-lnc78 acts as a ceRNA to increase SARM expression through sponging pol-miR-n199-3p, with this ceRNA network regulating host defense responses to bacterial infection.These results highlight the essential effects of lncRNAs in host immunity and lend further support to the ubiquity of ceRNA regulation in fish.

CONCLUSIONS
Our study identified SARM, pol-miR-n199-3p, and pol-lnc78 as key regulators in the response of flounder to V. anguillarum infection through modulation of inflammatory processes.Pol-miR-n199-3p repressed SARM expression by targeting its 3' UTR.Concurrently, pol-lnc78 acted as a ceRNA, counteracting the inhibitory effects of pol-miR-n199-3p on SARM.These observations provide new insights into the mechanisms of SARM, as well as the roles of ceRNA in lower vertebrates.

Figure 1
Figure 1 SARM acts as a negative regulator in host defense against Vibrio anguillarum infection A: Symptoms of flounder infected with V. anguillarum.Compared to control fish, V. anguillarum-infected flounder exhibited ulcerative skin (us) and fin hemorrhage (fh).Swollen spleen was observed in V. anguillarum-infected flounder compared to control fish.B: SARM expression decreased in response to V. anguillarum infection.SARM expression in flounder infected with or without (control) V. anguillarum for 6, 12, and 24 h was examined.C, D: SARM inhibited expression of inflammatory cytokines.Flounder were administered with SARM expression plasmid or pCN3 vector for 2.5 d, then injected with V. anguillarum for another 12 h.Expression levels of IL-1β (C) and TNF-α (D) were examined.E, F: SARM exacerbated V. anguillarum infection.Flounder were administered with or without (control) SARM expression plasmid for 2.5 d, then injected with V. anguillarum for another 12 h.Bacterial loads in spleen (E) and liver (F) were determined through plate count.Ctrl: Control; hpi, hours post infection.P-values were calculated with Student's t-test.* : P<0.05; ** : P<0.01.Error bars indicate SD.