The long noncoding RNA NARL regulates immune responses via microRNA-mediated NOD1 downregulation in teleost fish
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100414
ISSN1083-351X
AutoresWeiwei Zheng, Qing Chu, Tianjun Xu,
Tópico(s)interferon and immune responses
ResumoIncreasing evidence shows that the long noncoding RNA (lncRNA) is a major regulator and participates in the regulation of various physiological and pathological processes, such as cell proliferation, differentiation, metastasis, and apoptosis. Unlike mammals, however, the study of lncRNA in lower invertebrates is just beginning and the extent of lncRNA-mediate regulation remains unclear. Here, we for the first time identify an lncRNA, termed nucleotide oligomerization domain 1 (NOD1) antibacterial and antiviral-related lncRNA (NARL), as a key regulator for innate immunity in teleost fish. We found that NOD1 plays an important role in the antibacterial and antiviral process in fish and that the microRNA miR-217-5p inhibits NOD1 expression and thus weakens the NF-κB and the IRF3-driven signaling pathway. Furthermore, our results indicated that NARL functions as a competing endogenous RNA (ceRNA) for miR-217-5p to regulate protein abundance of NOD1; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA NARL can competitive adsorption miR-217-5p to regulate the miR-217-5p/NOD1 axis is widespread in teleost fish. Taken together, our results reveal that NARL in fish is a critical positive regulator of innate immune responses to viral and bacterial infection by suppressing a feedback to NOD1-NF-κB/IRF3-mediated signaling. Increasing evidence shows that the long noncoding RNA (lncRNA) is a major regulator and participates in the regulation of various physiological and pathological processes, such as cell proliferation, differentiation, metastasis, and apoptosis. Unlike mammals, however, the study of lncRNA in lower invertebrates is just beginning and the extent of lncRNA-mediate regulation remains unclear. Here, we for the first time identify an lncRNA, termed nucleotide oligomerization domain 1 (NOD1) antibacterial and antiviral-related lncRNA (NARL), as a key regulator for innate immunity in teleost fish. We found that NOD1 plays an important role in the antibacterial and antiviral process in fish and that the microRNA miR-217-5p inhibits NOD1 expression and thus weakens the NF-κB and the IRF3-driven signaling pathway. Furthermore, our results indicated that NARL functions as a competing endogenous RNA (ceRNA) for miR-217-5p to regulate protein abundance of NOD1; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA NARL can competitive adsorption miR-217-5p to regulate the miR-217-5p/NOD1 axis is widespread in teleost fish. Taken together, our results reveal that NARL in fish is a critical positive regulator of innate immune responses to viral and bacterial infection by suppressing a feedback to NOD1-NF-κB/IRF3-mediated signaling. Innate immunity is the first line of host defense and mainly composes of pattern recognition receptors (PRRs) and their signal pathways. Nucleotide-binding oligomerization domain-like receptors (NLRs) are part of the cytosolic PRRs and play key roles in response to various bacterial infections (1Girardin S.E. Jéhanno M. Mengin-Lecreulx D. Sansonetti P.J. Alzari P.M. Philpott D.J. Identification of the critical residues involved in peptidoglycan detection by Nod1.J. Biol. Chem. 2005; 280: 38648-38656Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). As the members of the NLR family, nucleotide oligomerization domain (NOD) 1 and NOD2 are important for epithelial and myeloid cell signaling in response to cytosolic bacterial peptidoglycans, muramyl dipeptide (MDP) and G-D-glutamyl-mesodiaminopimelic acid (iE-DAP) (2Caruso R. Warner N. Inohara N. Núñez G. NOD1 and NOD2: Signaling, host defense, and inflammatory disease.Immunity. 2014; 41: 898-908Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 3Philpott D.J. Sorbara M.T. Robertson S.J. Croitoru K. Girardin S.E. NOD proteins: Regulators of inflammation in health and disease.Nat. Rev. Immunol. 2014; 14: 9-23Crossref PubMed Scopus (368) Google Scholar). NOD1 can monitor bacterial intrusions in cells and promotes the activation of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways through RIP2 kinase (4Fritz T. Niederreiter L. Adolph T. Blumberg R.S. Kaser A. Crohn's disease: NOD2, autophagy and ER stress converge.Gut. 2011; 60: 1580-1588Crossref PubMed Scopus (146) Google Scholar, 5Park J.-H. Kim Y.-G. McDonald C. Kanneganti T.-D. Hasegawa M. Body-Malapel M. Inohara N. Nunez G. RICK/RIP2 mediates innate immune responses induced through Nod1 and Nod2 but not TLRs.J. Immunol. 2007; 178: 2380-2386Crossref PubMed Scopus (361) Google Scholar). In lower vertebrates, increasing pieces of evidence show that NOD1 is widespread in fish and participates in the regulation of antibacterial immune response after pathogen infection (6Li J. Gao Y. Xu T. Comparative genomic and evolution of vertebrate NOD1 and NOD2 genes and their immune response in miiuy croaker.Fish. Shellfish Immunol. 2015; 46: 387-397Crossref PubMed Scopus (27) Google Scholar, 7Park S.B. Hikima J.-I. Suzuki Y. Ohtani M. Nho S.W. Cha I.S. Jang H.B. Kondo H. Hirono I. Aoki T. Molecular cloning and functional analysis of nucleotide-binding oligomerization domain 1 (NOD1) in olive flounder, Paralichthys olivaceus.Dev. Comp. Immunol. 2012; 36: 680-687Crossref PubMed Scopus (39) Google Scholar, 8Swain B. Basu M. Samanta M. NOD1 and NOD2 receptors in mrigal (Cirrhinus mrigala): Inductive expression and downstream signalling in ligand stimulation and bacterial infections.J. Biosci. 2013; 38: 533-548Crossref PubMed Scopus (33) Google Scholar, 9Bi D. Gao Y. Chu Q. Cui J. Xu T. NOD1 is the innate immune receptor for iE-DAP and can activate NF-κB pathway in teleost fish.Dev. Comp. Immunol. 2017; 76: 238-246Crossref PubMed Scopus (25) Google Scholar, 10Bi D. Wang Y. Gao Y. Li X. Chu Q. Cui J. Xu T. Recognition of lipopolysaccharide and activation of NF-κB by cytosolic sensor NOD1 in teleost fish.Front. Immunol. 2018; 9: 1413Crossref PubMed Scopus (28) Google Scholar). Recently, researchers have demonstrated that fish NOD1 plays an important role in recognizing lipopolysaccharides (LPS) and iE-DAP, as well as initiating downstream antiviral signaling pathways by identifying RNA virus (9Bi D. Gao Y. Chu Q. Cui J. Xu T. NOD1 is the innate immune receptor for iE-DAP and can activate NF-κB pathway in teleost fish.Dev. Comp. Immunol. 2017; 76: 238-246Crossref PubMed Scopus (25) Google Scholar, 10Bi D. Wang Y. Gao Y. Li X. Chu Q. Cui J. Xu T. Recognition of lipopolysaccharide and activation of NF-κB by cytosolic sensor NOD1 in teleost fish.Front. Immunol. 2018; 9: 1413Crossref PubMed Scopus (28) Google Scholar, 11Wu X.M. Zhang J. Li P.W. Hu Y.W. Cao L. Ouyang S. Bi Y.H. Nie P. Chang M.X. NOD1 promotes antiviral signaling by binding viral RNA and regulating the interaction of MDA5 and MAVS.J. Immunol. 2020; 204: 2216-2231Crossref PubMed Scopus (13) Google Scholar). Unlike mammals, TLR4, as the central protein that recognizes LPS, does not exist in most fish. Therefore, it was found that fish NOD1 can recognize LPS and iE-DAP and eventually causes an inflammatory response that could elucidate the resistance of fish against bacterial infections. Recently, a series of regulatory factors involved in the regulation of the NOD1 signaling pathway has been found. CENTB1 and E3 Ligase RNF34 regulate the NOD1 signaling pathway through directly regulating the expression of NOD1 (12Yamamoto-Furusho J.K. Barnich N. Xavier R. Hisamatsu T. Podolsky D.K. Centaurin β1 down-regulates nucleotide-binding oligomerization domains 1-and 2-dependent NF-κB activation.J. Biol. Chem. 2006; 281: 36060-36070Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 13Zhang R. Zhao J. Song Y. Wang X. Wang L. Xu J. Song C. Liu F. The E3 ligase RNF34 is a novel negative regulator of the NOD1 pathway.Cell. Physiol. Biochem. 2014; 33: 1954-1962Crossref PubMed Scopus (13) Google Scholar); the molecular chaperone HSP90 regulates the NOD1 signal pathway by regulating the stability of NOD1 (14Hahn J.-S. Regulation of Nod1 by Hsp90 chaperone complex.FEBS Lett. 2005; 579: 4513-4519Crossref PubMed Scopus (60) Google Scholar); it was also found that SGT1 can act as a positive regulator of the NOD1 signaling pathway (15da Silva Correia J. Miranda Y. Leonard N. Ulevitch R. SGT1 is essential for Nod1 activation.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 6764-6769Crossref PubMed Scopus (111) Google Scholar). In addition to protein regulatory factors, microRNA (miRNA) has also been found to play an important role in the NOD1 signaling pathway in recent years. For example, PPARγ-regulated miR-125a could target NOD1 and regulate NOD1-mediated angiogenesis (16Kang H. Park Y. Lee A. Seo H. Kim M.J. Choi J. Jo H.-N. Jeong H.-N. Cho J.G. Chang W. Negative regulation of NOD1 mediated angiogenesis by PPARγ-regulated miR-125a.Biochem. Biophys. Res. Commun. 2017; 482: 28-34Crossref PubMed Scopus (9) Google Scholar). Since NOD1 is an important intracellular receptor that recognizes LPS in fish, the elucidation of the regulatory mechanism of the NOD1 signaling pathway in fish is even more important. Our laboratory had found for the first time that miR-144 and miR-217-5p can target and negatively regulate the expression of NOD1 to attenuate the NOD1-mediated inflammatory response in fish (17Chu Q. Bi D. Zheng W. Xu T. MicroRNA negatively regulates NF-κB-mediated immune responses by targeting NOD1 in the teleost fish Miichthys miiuy.Sci. China Life Sci. 2020; https://doi.org/10.1007/s11427-020-1777-yCrossref Scopus (4) Google Scholar). The long noncoding RNA (lncRNA) is defined as RNA with noncoding potential and larger than 200 nucleotides length (18Hu G. Cabrera A. Kono M. Mok S. Chaal B.K. Haase S. Engelberg K. Cheemadan S. Spielmann T. Preiser P.R. Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum.Nat. Biotechnol. 2010; 28: 91-98Crossref PubMed Scopus (143) Google Scholar, 19Djebali S. Davis C.A. Merkel A. Dobin A. Lassmann T. Mortazavi A. Tanzer A. Lagarde J. Lin W. Schlesinger F. Landscape of transcription in human cells.Nature. 2012; 489: 101-108Crossref PubMed Scopus (3112) Google Scholar). An increasing number of studies have shown that lncRNA is a major regulator and participates in the regulation of cell proliferation, differentiation, metastasis, and apoptosis (20Esteller M. Non-coding RNAs in human disease.Nat. Rev. Genet. 2011; 12: 861-874Crossref PubMed Scopus (2942) Google Scholar, 21Mercer T.R. Dinger M.E. Mattick J.S. Long non-coding RNAs: Insights into functions.Nat. Rev. Genet. 2009; 10: 155-159Crossref PubMed Scopus (3891) Google Scholar). Recent studies also show that lncRNAs can play the role of competitive endogenous RNAs (ceRNA) by adsorbing miRNA, thus regulating the binding of miRNA and mRNA. Until now, the function of lncRNAs has only been widely studied in mouse and human, such as lncRNA LINC01133, MIAT, or UCA1, all can be used as ceRNA to competitively bind miRNA to regulate mRNA (22Yang X.-Z. Cheng T.-T. He Q.-J. Lei Z.-Y. Chi J. Tang Z. Liao Q.-X. Zhang H. Zeng L.-S. Cui S.-Z. LINC01133 as ceRNA inhibits gastric cancer progression by sponging miR-106a-3p to regulate APC expression and the Wnt/β-catenin pathway.Mol. Cancer. 2018; 17: 126Crossref PubMed Scopus (127) Google Scholar, 23Zhu X. Yuan Y. Rao S. Wang P. LncRNA MIAT enhances cardiac hypertrophy partly through sponging miR-150.Eur. Rev. Med. Pharmacol. Sci. 2016; 20: 3653PubMed Google Scholar, 24Nie W. Ge H.-J. Yang X.-Q. Sun X. Huang H. Tao X. Chen W.-S. Li B. LncRNA-UCA1 exerts oncogenic functions in non-small cell lung cancer by targeting miR-193a-3p.Cancer Lett. 2016; 371: 99-106Crossref PubMed Scopus (300) Google Scholar). However, the function mechanism of lncRNA in lower vertebrates is still poorly understood. Teleost is a representative population of lower vertebrates and an important part of early vertebrate evolution, so it is considered an excellent biological model in immunology research. The teleost fish not only has a very important significance in biological evolution and immunity but also its status in the aquatic industry is not to be underestimated. However, artificially cultivated fishes are extremely vulnerable to various pathogens, especially Gram-negative bacteria and RNA virus, such as Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, and Siniperca chuatsi rhabdovirus (SCRV) (25Luis A.L.I. Campos E.N.V. de Oliveira J.L. Guilger-Casagrande M. de Lima R. Castanha R.F. de Castro V.L. Fraceto L.F. Zein nanoparticles impregnated with eugenol and garlic essential oils for treating fish pathogens.ACS. Omega. 2020; 5: 15557-15566Crossref PubMed Scopus (3) Google Scholar, 26Chamaillard M. Hashimoto M. Horie Y. Masumoto J. Qiu S. Saab L. Ogura Y. Kawasaki A. Fukase K. Kusumoto S. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid.Nat. Immunol. 2003; 4: 702-707Crossref PubMed Scopus (961) Google Scholar). The mechanism that induces fish disease and how to prevent and solve these fish diseases are problems that need to be discovered and solved urgently. LPS and iE-DAP are pathogenic components carried by various Gram-negative bacteria and can induce an inflammatory response in the host (27Hoang M. Potter J.A. Gysler S.M. Han C.S. Guller S. Norwitz E.R. Abrahams V.M. Human fetal membranes generate distinct cytokine profiles in response to bacterial toll-like receptor and Nod-like receptor agonists.Biol. Reprod. 2014; 90: 31-39Crossref PubMed Scopus (65) Google Scholar, 28Chu Q. Sun Y. Cui J. Xu T. MicroRNA-3570 modulates the NF-κB pathway in teleost fish by targeting MyD88.J. Immunol. 2017; 198: 3274-3282Crossref PubMed Scopus (47) Google Scholar). At present, the pathogenic mechanism of Gram-negative bacteria and RNA virus has been relatively comprehensive in mammals. However, the research process of teleost fish is different from that of mammals and has great limitations and shortcomings as the slow development of research technology and difficulty in obtaining materials. In this study, we firstly identified a ceRNA regulatory loop involved in innate immune responses in teleost fish. Our previous work has revealed that miR-217-5p can negatively regulate NOD1 and suppress the antibacterial immune response in miiuy croaker (Miichthys miiuy) (17Chu Q. Bi D. Zheng W. Xu T. MicroRNA negatively regulates NF-κB-mediated immune responses by targeting NOD1 in the teleost fish Miichthys miiuy.Sci. China Life Sci. 2020; https://doi.org/10.1007/s11427-020-1777-yCrossref Scopus (4) Google Scholar). Herein, we further suggest that miR-217-5p can negatively regulate NOD1 and can suppress the antiviral response in miiuy croaker. Furthermore, we found that an lncRNA, named NARL, can act as a ceRNA for miR-217-5p to facilitate NOD1 expression, thus modulating innate immune responses. Our research not only contributes to the understanding of the ceRNA network mechanism in teleost fish but also provides a reference for the importance of lncRNA in the innate immune response in lower vertebrates. Increasing pieces of studies have shown that lncRNA can play a regulatory role as a spongy body adsorbing miRNA and interfere with the host's immune signaling pathways through the sponge mechanism (29Fu T. Ji K. Jin L. Zhang J. Wu X. Ji X. Fan B. Jia Z. Wang A. Liu J. ASB16-AS1 up-regulated and phosphorylated TRIM37 to activate NF-κB pathway and promote proliferation, stemness, and cisplatin resistance of gastric cancer.J. Gastric. Cancer. 2020; 24: 45-59Crossref Scopus (9) Google Scholar). We treated miiuy croaker with LPS and SCRV to identify whereas lncRNAs are potentially involved in the regulation of infection, and then the expression of lncRNAs in the spleen tissues of the treated group and the untreated group was analyzed by RNA-seq data. In total, 8942 distinct lncRNA candidates were found. The number of diverse length distribution of lncRNAs was different, and the length of most lncRNAs was less than 500 nucleotides (Fig. 1A). As shown in Figure 1B, we found that a highly expressed lncRNA (named NARL according to the participating function) is copresent in the differentially expressed genes obtained after LPS stimulation and SCRV infection (Table S2). To characterize the complete sequence of NARL, the single-molecule full-length transcript sequencing was used and demonstrated that the length of NARL was 1992 base pairs (bp) and NRAL locates on miiuy croaker, N. diacanthus, and L. crocea chromosome 10, between gene EIF4E and gene lingo2a, and consists of only one exon (Fig. 1C). Then, we validated that NARL was mainly distributed in the cytoplasm by cytoplasm/nucleus fraction assay both in MIC cells and in MKC cells (Fig. 1D). Consistent with NARL being an ncRNA, no putative protein is conserved in all species, and the CPC (coding potential calculator) computational algorithm predicts that NARL has a very low coding potential (Fig. 1E). We first detected the NARL expression under different LPS or SCRV stimulation times to verify the results of RNA-seq. As shown in Figure 2A, we found that the expression of NARL was upregulated after LPS and SCRV stimulation. Two small interfering RNAs against NARL (si-NARL-1 and si-NARL-2) and the overexpression plasmid of NARL were constructed to explore the biological functions of NARL. As expected, both two siRNAs can inhibit the expression of NARL, and si-NARL-1 could induce higher inhibitory efficiency (Fig. 2B), so we chose si-NARL-1 to continue the follow-up experiment. And the NARL overexpression plasmid could significantly increase NARL expression levels (Fig. 2D). Inflammatory cytokines and IFN-stimulated genes (ISGs) play an important role in the innate immune response; thus, we focused on investigating the function of NARL in regulating the expression of inflammatory cytokines and ISGs genes. As shown in Figure 2C, knockdown or overexpression NARL can significantly inhibit or promote the expression levels of TNF-α, IL-8, and IL-1β upon LPS stimulation. Then, knockdown or overexpression NARL also can significantly inhibit or promote the expression levels of TNF-α, MX1, and ISG15 upon SCRV infection (Fig. 2E). Furthermore, cell apoptosis analysis showed that NARL knockdown significantly increased the proportion of apoptotic cells (Fig. 2F). We conducted EdU assays and ATP activity assay to examine cell proliferation and cell viability for further explore the role of NARL in innate immunity. As shown in Figure 2, G and H, the results showed that knockdown of NARL considerably decreased the percentages of EdU-positive cells and cell viability, whereas it greatly increased when NARL was overexpressed. Previous studies have indicated that the low concentration of LPS can stimulate cell proliferative activity (30Zhu S. Lu Y. Dexmedetomidine suppressed the biological behavior of HK-2 cells treated with LPS by down-regulating ALKBH5.Inflammation. 2020; 43: 2256-2263Crossref PubMed Scopus (5) Google Scholar). These results suggested that NARL can promote the proliferation and viability of miiuy croaker cell lines. To summarize, the data suggest that NARL plays as a positive modulator in regulating inflammatory responses, as well as cell proliferation and cell viability. The changes in cell proliferation and cell apoptosis are a result of the regulation effect of NARL on innate immune responses. In other words, NARL can positively regulate the antibacterial responses or antiviral response and upregulate the expression of inflammatory cytokines or antiviral genes, reducing the attack of pathogen to cells, promoting cell proliferation, and reducing cell apoptosis. Many cytoplasmic lncRNAs have been reported to act as ceRNAs by competitively binding miRNAs (29Fu T. Ji K. Jin L. Zhang J. Wu X. Ji X. Fan B. Jia Z. Wang A. Liu J. ASB16-AS1 up-regulated and phosphorylated TRIM37 to activate NF-κB pathway and promote proliferation, stemness, and cisplatin resistance of gastric cancer.J. Gastric. Cancer. 2020; 24: 45-59Crossref Scopus (9) Google Scholar, 31Li C. Gao Q. Wang M. Xin H. LncRNA SNHG1 contributes to the regulation of acute myeloid leukemia cell growth by modulating miR-489-3p/SOX12/Wnt/β-catenin signaling.J. Cell. Physiol. 2020; 236: 653-663Crossref PubMed Scopus (2) Google Scholar). Through the cytoplasm/nucleus fraction assay experiments, we have been able to determine that NARL is mainly distributed in the cytoplasm. Therefore, to further confirm whether NARL acts as a ceRNA, we first inserted the Argonaute-2 (Ago2) sequence into the pcDNA3.1-flag vector and obtained the Ago2-flag expression plasmid. We next construct the RIP assay with Ago2-flag in MIC cells. As shown in Figure 3A, NARL is significantly enriched in Ago2-containing micro-ribonucleoprotein complexes, which suggests that the miRNAs can directly bind to NARL. Then, we used Targetscan and miRanda softwares to predict miRNAs that can potentially target NARL and that the five microRNAs (including miR-217-5p, miR-103-5p, miR-143-5p, miR-221-5p, and miR-92-5p) were predicted to have a high probability of combining to NARL. Then, we used qRT-PCR to detect these five miRNAs' expressions in MIC cells when overexpressing NARL expression plasmid, and the result shows that the expression of miR-217-5p is significantly downregulated compared with the other four miRNAs (Fig. 3B). Meanwhile, we found that the expression of miR-217-5p significantly increased when the NARL was silenced (Fig. 3C). To test whether NARL can affect miR-217-5p activity, we constructed miR-217-5p sensor. The miR-217-5p sensor was constructed by inserting two copies of perfectly matched miR-217-5p fragments into the psiCHECK-2 vector (Fig. 3D). Then, we transfected with the miR-217-5p sensor into cells, along with miR-217-5p, control vector, or NARL overexpression plasmid, suggesting that NARL specifically sponged miR-217-5p, thereby preventing it from inhibiting luciferase activity (Fig. 3E). Taken together, these data suggest that NARL could regulate miR-217-5p expression and activity, and NARL may function as a sponge of miR-217-5p. We analyzed the sequence of NARL and found a binding site for miR-217-5p to investigate whether NARL can directly interact with miR-217-5p. Then, we constructed the NARL luciferase plasmid and the mutant plasmid with the mutated miR-217-5p binding site (Fig. 4A). The luciferase assay shows that miR-217-5p mimics and pre-miR-217 plasmid can both significantly inhibit the wild-type NARL luciferase plasmid activity, but have no effect on the mutated form (Fig. 4B). In addition, we inserted the wild-type or the mutant-type fragment of NARL into the mVenus-C1 vector and cotransfected with miR-217-5p to confirm that whether miR-217-5p can inhibit the levels of GFP. As shown in Figure 4C, miR-217-5p can significantly inhibit the levels of GFP, but cannot inhibit the level of the mutant type of NARL-GFP. To extend the findings, the western blotting assay has been conducted to examine the expression level of GFP protein (Fig. 4D). These results indicate that NARL may interact with miR-217-5p through the predicted miR-217-5p bind site. Through the previous RIP experiment, we have known that NARL could directly bind to miRNAs (Fig. 3A). We thus further tested the ability of NARL to bind to miR-217-5p. To this end, the Ago2 RIP assays are performed in MIC cells by cotransfecting Ago2-flag and miR-217-5p. The results from qRT-PCR analysis suggested that NARL and miR-217-5p were efficiently pulled down by Ago2-flag (Fig. 4E). For further confirming the direct interaction between NARL and miR-217-5p, we performed biotin-avidin pull-down experiments to examine whether NARL can be pulled down by MIC cells that were transfected with biotinylated NARL or a mutated form, then harvested for pull-down assay. The result showed that miR-217-5p could pull down by biotinylated wild-type NARL (Fig. 4F). Then we constructed plasmids that recognize lncRNAs by MS2 protein. We insert the MS2-12X fragment into the pcDNA3.1, pcDNA3.1-NARL, and the mutated type of NARL plasmids (pcDNA3.1-NARL-mut). Additionally, we constructed a fusion expression plasmid of GFP and MS2 to produce MS2-GFP fusion protein (pcDNA3.1-MS2-GFP), and the protein can bind to MS2-12X and also GFP antibody. Therefore, if miRNAs can bind to NARL, miR-217-5p can be pulled down by the GFP-MS2-12X complex. Analysis of qPCR results showed that the miR-217-5p enrichment of NARL was significantly higher than that of mutant NARL and empty plasmid (Fig. 4G). In summary, these results indicated that NARL can directly bind to miR-217-5p, and NARL may act as a sponge of miR-217-5p. To explore the role of miR-217-5p against the innate immune response, miR-217-5p and miR-217-5p inhibitors were transfected into MIC cells and stimulated by LPS, respectively. The results showed that certain inflammatory cytokines, including TNF-α, IL-8, and IL-1β, are significantly decreased or increased by the introduction of miR-217-5p mimics or miR-217-5p inhibitor upon LPS stimulation. Furthermore, we found that miR-217-5p or miR-217-5p inhibitor also could significantly decrease or elevated ISGs genes respectively, including MX1 and ISG15 (Fig. 5B). Here, we find that the effect of the miR-217-5p inhibitor on the expression of some of the innate immune response genes is not robust, but there are significant differences; we think that this may be related to the inhibitory efficiency of miRNA inhibitor, and background level of miR-217-5p is not high in cells. Then, NOD1-3'UTR plasmid, miR-217-5p, and reporter genes were cotransfected into EPC cells for further verifying that NOD1 can be regulated by miR-217-5p. The results showed that miR-217-5p could inhibit the NF-κB, IL-8, IL-1β, and IRF3 report genes' luciferase activity by inhibiting NOD1 (Fig. 5C). Furthermore, we tested the effects of miR-217-5p and miR-217-5p-i on MIC cell proliferation, the results showed that miR-217-5p inhibited cell proliferation and miR-217-5p-i promoted cell proliferation under LPS or SCRV stimulation (Fig. 5, D and E). Combined with our experimental results, we can think that an appropriate immune response or appropriate increase of cytokines will lead to cell proliferation and increase of cell viability. And excessive immune response or overexpression of some cytokines and genes will reduce cell proliferation and reduce cell activity. miR-217-5p regulates the expression of cytokines by inhibiting the expression of NOD1, thus affecting cell proliferation and cell viability. In summary, miR-217-5p plays an important regulatory role in inflammatory immune responses. Given that NARL interacts with miR-217-5p and miR-217-5p targets and regulates NOD1. Thus, we then tested whether NARL is able to regulate NOD1. NOD1 protein expression is significantly increased when NARL is overexpressed in MIC cells (left panel of Fig. 6A), while the NOD1 protein expression was significantly decreased through knockdown NARL (right panel of Fig. 6A). NARL expression plasmid or si-NARL is transfected into MIC cells and NOD1 expression levels were detected by qRT-PCR. The results show that NARL can indeed promote NOD1 expression while silencing NARL would significantly reduce NOD1 expression (Fig. 6B). These results indicated that miR-217-5p can inhibit NOD1 luciferase activity, while NARL can reverse the inhibition effect of miR-217-5p on NOD1 (Fig. 6C). Then, the MIC cells were cotransfected with miR-217-5p and NARL, and the western blotting assay showed that NARL could indeed reverse the inhibitory effect of miR-217-5p on NOD1 protein expression (Fig. 6D). Then, the NOD1 plasmid with full-length 3'UTR, miR-217-5p, NARL plasmid, and various reporter gene plasmids were cotransfected into EPC cells. The results showed that NARL could reverse the negative effect of miR-217-5p on the luciferase activities of NF-κB, IL-8, and IL-1β report genes (Fig. 6E). Moreover, we attempted to explore the effect of the NARL/miR-217-5p regulatory loop on cell proliferation. The results indicated that overexpression of NARL could counteract the negative effect of miR-217-5p on cell proliferation upon LPS or SCRV stimulation (Fig. 6F). We cotransfected miR-217-5p-i and si-NARL into MIC cells to further prove the results that NARL regulates NOD1 by modulating miR-217-5p. And the results show that miR-217-5p-i can reverse the negative effect of si-NARL on the NOD1 (Fig. 6G). Collectively, these data demonstrated that NARL serves as a ceRNA for miR-217-5p to regulate NOD1 expression. To address the generality of our findings, we first examined the sequence alignment of pre-miR-217 from different vertebrate species. Interestingly, as shown in Figure 7A, mature miR-217-5p displayed high conservation from mammals to fish. Further, the miR-217-5p binding site in NOD1 3'UTR also displayed high conservation from mammals to fish (Fig. 7B). To obtain the direct evidence that miR-217-5p could target NOD1 3'UTR across fish species, luciferase report plasmids were generated by cloning NOD1-3'UTR of L. crocea and N. diacanthus into pmirGLO vector, within the mutant devoid of miR-217-5p binding site as a control (Fig. 7C). Strikingly, miR-217-5p mimics were sufficient to decrease luciferase activities when respectively cotransfected with the wild types of L. crocea and N. diacanthus NOD1-3'UTR, whereas it shows no effect on the luciferase activity of cells transfected with their mutant-types (Fig. 7D). These results indicate that miR-217-5p can target the NOD1 gene in other fish species, which verifies that miR-217-5p is highly conserved among different species, and its function is also conserved to some extent. Additionally, we also verified the findings that miR-217-5p regulating lncRNA NARL a
Referência(s)