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The NF-κB−Responsive Long Noncoding RNA FIRRE Regulates Posttranscriptional Regulation of Inflammatory Gene Expression through Interacting with hnRNPU Yajing Lu, Xu Liu, Minghong Xie, Mingjia Liu, Mengling Ye, Mingxuan Li, Xian-Ming Chen, Xiaoqing Li and Rui Zhou

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http://www.jimmunol.org/content/suppl/2017/10/07/jimmunol.170009 1.DCSupplemental

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J Immunol published online 9 October 2017 http://www.jimmunol.org/content/early/2017/10/07/jimmun ol.1700091

Published October 9, 2017, doi:10.4049/jimmunol.1700091 The Journal of Immunology

The NF-kB–Responsive Long Noncoding RNA FIRRE Regulates Posttranscriptional Regulation of Inflammatory Gene Expression through Interacting with hnRNPU Yajing Lu,*,†,1 Xu Liu,*,1 Minghong Xie,* Mingjia Liu,* Mengling Ye,* Mingxuan Li,* Xian-Ming Chen,‡ Xiaoqing Li,x and Rui Zhou*

T

he innate immune system acts as a first line of defense against invading pathogens (1). Epithelial cells and macrophages have been recognized as important players in the defense against pathogen infection (2). These cells are equipped with several defense mechanisms to guard against infection by pathogens, which express a variety of pathogen pattern recognition receptors, such as TLRs, to recognize pathogens and pathogen-associated molecular patterns (3, 4). Upon specific microbial recognition, these receptors activate downstream signaling pathways including NF-ĸB to induce transcription of inflammatory genes (5–7). However, inflammation is a double-edged sword, as *Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei 430071, China; †Department of Endocrinology, Institute of Geriatric Medicine, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430077, China; ‡Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE 68178; and xCenter for Stem Cell Research and Application, Union Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China 1

Y.L. and X. Liu contributed equally to this work.

ORCIDs: 0000-0001-8361-6510 (X. Liu); 0000-0002-9927-5236 (R.Z.). Received for publication January 18, 2017. Accepted for publication September 11, 2017. This work was supported by the National Natural Science Foundation of China (Grants 31300744 and 81373132) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant 20130141120011). Address correspondence and reprint requests to Dr. Xiaoqing Li or Dr. Rui Zhou, Center for Stem Cell Research and Application, Union Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China (X. Li) or Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, 185 Donghu Road, Hubei 430071, China (R.Z.). E-mail addresses: [email protected] (X. Li) or [email protected] (R.Z.) The online version of this article contains supplemental material. Abbreviations used in this article: ARE, adenylate-uridylate–rich element; ChIP, chromatin immunoprecipitation; Ct, cycle threshold; FISH, fluorescence in situ hybridization; lncRNA, long noncoding RNA; mFIRRE, mouse FIRRE; PMPM, primary mouse peritoneal macrophage; RIP, RNA immunoprecipitation; shRNA, short hairpin RNA; siRNA, small interfering RNA; 39UTR, 39-untranslated region. This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles. Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700091

when excessive it can exacerbate tissue damage and cause chronic inflammatory diseases (8, 9). Therefore, the innate immune system has developed complicated self-regulatory systems to control excessive inflammation, for example, the expression of inflammatory genes is tightly regulated (3). The coordinated expression of inflammatory genes involves multiple steps that determine the rates of gene transcription, translation, and mRNA decay (10–12). Although transcription is an essential first step in the regulation of inflammatory gene expression, posttranscriptional regulation of translation and mRNA decay is key to control protein synthesis (13). The 39-untranslated region (39UTR) of mRNA represents an important element in the posttranscriptional regulation of inflammatory genes (14). Long noncoding RNAs (lncRNAs) are a newly identified class of ncRNAs (.200 nt) (15). Evidence to date indicates that lncRNAs may function as regulators in diverse biological processes, such as embryonic development, cell differentiation, and tumor metastasis (15–18). It is clear that lncRNAs are important regulators of gene expression, can be induced in innate immune cells, and act as key regulators of the inflammatory response (19). Indeed, lncRNAs have been associated with various inflammatory diseases (20–22). A panel of lncRNAs has been reported to be differentially regulated in macrophages after stimulation by ligands for TLRs (23). Several lncRNAs, such as lncRNA-Cox2 and lncRNA-Tnfaip3, have been shown to mediate both the activation and repression of distinct classes of inflammatory genes in murine macrophage cell lines (24, 25). However, lncRNA sequences are usually not as conserved as protein-coding genes (20). Most studies focused on immune-relative lncRNAs in mice, and the functions of lncRNAs in innate immunity in humans are largely unexplored (18, 26). Mechanistically, lncRNAs regulate gene transcription through their association in the nucleus with specific chromatin modification factors, such as the polycomb repressive complex 2 and heterogeneous nuclear ribonucleoproteins (hnRNPs) (27– 29). Other lncRNAs have been reported to impact on the splicing, stability, or translation of host mRNAs through posttranscriptional mechanisms (30). Nevertheless, the potential role of lncRNAs in posttranscriptional regulation of inflammatory genes is still unclear.

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Long noncoding RNAs, a newly identified class of noncoding RNAs, are important regulators of gene expression in innate immunity. We report in this study that the transcription of FIRRE, a conserved long noncoding RNA between humans and mice, is controlled by NF-kB signaling in macrophages and intestinal epithelial cells. Functionally, FIRRE appears to positively regulate the expression of several inflammatory genes in macrophages or intestinal epithelial cells in response to LPS stimulation via posttranscriptional mechanisms. Specifically, FIRRE physically interacts with heterogeneous nuclear ribonucleoproteins U, regulating the stability of mRNAs of selected inflammatory genes through targeting the AU-rich elements of their mRNAs in cells following LPS stimulation. Therefore, our data indicate a new regulatory role for NF-kB–responsive FIRRE in the posttranscriptional regulation of inflammatory genes in the innate immune system. The Journal of Immunology, 2017, 199: 000–000.

2 Functional intergenic repeating RNA element (FIRRE) is a newly identified lncRNA that can anchor the inactive X chromosome through maintaining H3K27me3 methylation (31). FIRRE can function as a nuclear-organization factor and influence the higher-order nuclear architecture across chromosomes through interacting with hnRNPU (32). However, the potential function of FIRRE in innate immunity is largely unclear. Previous studies showed that hnRNPU can be induced by TLR stimulation and positively regulates expression of selected genes by stabilizing their mRNAs (14). In this study, we demonstrate that FIRRE is a conserved lncRNA between humans and mice and its transcription is controlled by the NF-ĸB signaling in macrophages and intestinal epithelial cells. FIRRE can positively regulate the expression of several inflammatory genes at the posttranscriptional level through interacting with hnRNPU. Therefore, our data indicate a new regulatory role for FIRRE in the posttranscriptional regulation of inflammatory genes in the innate immune system.

FIRRE MODULATES INFLAMMATORY RESPONSES nuclear and cytoplasmic fractions for real-time PCR or Western blot. The Abs of hnRNPU (ab20666; Abcam) and GAPDH (Sigma-Aldrich) were used for blotting.

RNA immunoprecipitation Formaldehyde cross-linking RNA immunoprecipitation (RIP) was performed as described previously (34). Briefly, SW80 cells in culture were treated with 1.0% formaldehyde at 37˚C for 10 min. Then 250 ml/ml glycine was added to quench the cross-linking reactions. The cells were harvested, and nuclei pellets were collected. The purified nuclei lysates were immunoprecipitated using 2 mg anti-hnRNPU or IgG at 4˚C with rotation overnight. After incubation, protein G agarose beads were added. After 4 h of incubation at 4˚C, the immunoprecipitates were centrifuged and washed six times with RIP wash buffer. Then RNA was isolated from the immunoprecipitates. The presence of RNA was measured by real-time PCR. Gene-specific PCR primer pairs are listed in Supplemental Table I.

Chromatin immunoprecipitation

Plasmids and small interfering RNAs

Northern blot

Materials and Methods Cell lines and reagents

For plasmid constructs, the full-length sequence of human FIRRE was amplified using the primers depicted in Supplemental Table I. PCR products were cloned into the HindIII and XhoI sites of the pcDNA3.1 (+) vector. The target sequence for FIRRE knockdown assay is 59-GCCAAACCAAGAAGGGTTAGC-39 as FIRRE short hairpin RNA 1 (shRNA1); and 59-GACTGTACCTGGCTTGCAAAC-39 as FIRRE shRNA2. Two small interfering RNAs (siRNA) for mouse FIRRE (mFIRRE) were used to knockdown mouse FIRRE in RAW264.7 cells: mFIRRE-siRNA-1 (sense sequence: 59-GUCAGAAGCAUGCAAUGCUGTT-39) and mFIRREsiRNA-2 (sense sequence: 59-GCUCUUGUAAGGUAUGCUU-39). The sense sequence of siRNA for human hnRNPU was 59-GAUGAACACUUCGAUGACATT-39, and the nonspecific scrambled sequence 59-UUCUCCGAACGUGUCACGUUU-39 was synthesized by Genema (Shanghai, China). The pCMV-p65 expression plasmid was a kind gift from Dr. A.D. Badley (Mayo Clinic); pFRT-hnRNPU was obtained from Addgene.

Real-time PCR RNA was extracted using TRI-reagent and treaded with DNA-Free Kit (Applied Biosystems) to remove any remaining DNA. Comparative realtime PCR was performed by using the SYBR Green PCR Master Mix (Applied Biosystems). The presence of RNA was measured by real-time PCR using the StepOnePlus real-time detection system (Applied Biosystems). Specific primers for mRNAs are listed in Supplemental Table I. All reactions were run in triplicate. The cycle threshold (Ct) values were analyzed using the comparative Ct (DCt) method and the amount of target was obtained by normalizing to the endogenous reference and relative to the control (nontreated cells).

Nuclear and cytoplasmic extracts and Western blot A total of 5 3 106 cells were harvested in PBS and resuspended in 0.5 ml resuspension buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.2% N-octylglucoside, protease inhibitor mixture, RNase inhibitor, pH 7.9) for 10 min, incubated on ice. Homogenization was performed by applying 15 strokes with a 1 ml needle on ice. The cytoplasmic fraction of the supernatant was calculated after centrifugation (400 3 g 15 min). The pellet was resuspended in 0.2 ml PBS, 0.2 ml nuclear isolation buffer (40 mM Tris-HCl, 20 mM MgCl2, 4% Triton X-100, 1.28 M sucrose, pH 7.5), and 0.2 ml RNase-free H2O, followed by 20 min incubation on ice to clean out the residual cytoplasmic fraction. The nuclear fraction of the pellet was calculated after centrifugation. RNA and proteins were extracted from

Total RNAs (20 mg) harvested with TRI-reagent were run on a 15% Tris/ Borate/EDTA (90 mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3) urea gel (Invitrogen), and transferred to a Nytran nylon transfer membrane (Ambion). DIG-probes for FIRRE were hybridized using UltraHyb reagents (Ambion) according to the manufacturer’s instructions with GAPDH blotted as a control. The primers used for the DIG-labeling probe preparation are 59-CAAGATGGGTCCTGGCACT-39 and 59-CCTAACTACCTCTATTGGCTAT-39 for FIRRE and 59-CTCAAGACCTTGGGCTGGGACTG-39 and 59-AACTGGTTGAGCACAGGGTACTT-39 for GAPDH.

Luciferase reporter constructs and luciferase assay Luciferase assay was performed as described previously (36). Promoters of FIRRE were amplified by PCR from human genomic DNA, the PCR products were cloned into the SaII and XhoI sites of pGL3 Basic Vector (Promega). Mutations were introduced into the NF-kB binding sites using the DpnI restriction enzyme assay. For the luciferase reporter plasmids, sequences containing full-length VCAM1-39-UTR and IL-12p40 39-UTR were amplified from U937 cells by PCR and inserted into the pMIRREPORT Luciferase vector (Ambion). To construct luciferase reporter plasmids without adenylate-uridylate–rich elements (AREs) (VCAM1-39UTR and IL-12p40 39-UTR), the ARE region was deleted by PCRmediated truncation. All constructs were confirmed by sequencing. All the primers used are listed in Supplemental Table I. SW480 cells were cotransfected with the mixture of indicated luciferase reporter plasmid, pRL-TK-Renilla-luciferase, and the indicated amounts of pCMV-p65/FIRRE/hnRNPU constructs using Lipofectamine 2000 transfection reagent (Invitrogen). Total amounts of plasmid DNA were equalized via an empty control vector. After 48 h, the cells were then exposed to LPS for 4 h in the presence or absence of SC-514, followed by assessment of luciferase activity. Luciferase activities were measured with a DualLuciferase Reporter Assay System (Promega), according to the manufacturer’s instructions. Data are normalized for transfection efficiency by dividing Firefly luciferase activity with that of Renilla luciferase.

ELISA For the measurement of VCAM1 production by SW480, cells were transfected with FIRRE-shRNA1/hnRNPU-siRNA or pCDNA3.1-FIRRE/ pFRT-hnRNPU in six-well culture plates for 48 h. Cells were then collected with lysis buffer. The level of VCAM1 in the cell lyses was determined using a Quantikine ELISA kit (R&D Systems).

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Human macrophage cell line U937 was a gift from Dr. H.B. Shu (Wuhan University). Human intestinal epithelial cells SW480 and mouse macrophages RAW264.7 were obtained from the American Type Culture Collection. Primary mouse peritoneal macrophages (PMPMs) were isolated from male mice (C57BL/6J, 4–6 wk old) (Hubei Research Center of Laboratory Animals, Wuhan) and cultured as previously reported (33). SC-514 (100 mM; Sigma-Aldrich), a potent IKK-2 inhibitor, was used to inhibit NF-kB activation. LPS (Sigma-Aldrich) was used at a final concentration of 1 mg/ml. Actinomycin D (10 mg/ml) was purchased from Thermo Fisher Scientific (Pittsburgh, PA).

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (35, 36). U937 cells were fixed with 1% formaldehyde for 10 min. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M. Then cells were collected in ice-cold PBS, and resuspended in an SDS lysis buffer. Genomic DNA was then sheared to lengths ranging from 200 to 1000 bp by sonication. One percent of the cell extracts was taken as input, and the rest of the extracts were incubated with either anti-p65 Ab (Santa Cruz Biotechnology), or control IgG overnight at 4˚C, followed by precipitation with protein G-agarose beads. The immunoprecipitates were sequentially washed once with a low-salt buffer, once with a high-salt buffer, once with a LiCl buffer, and twice with a Tris buffer. The DNA-protein complex was eluted, and proteins were then digested with proteinase K for 1 h at 45˚C. The DNA was detected by realtime quantitative PCR analysis. ChIP primers for the NF-kB binding site regions of the FIRRE gene promoter are listed in Supplemental Table I.

The Journal of Immunology mRNA stability SW480 cells were transfected with FIRRE-shRNAs or the control shRNA for 24 h and exposed to LPS (1 mg/ml) and actinomycin D (10 mg/ml; Sigma-Aldrich). Cells were collected 0.5, 1, 2, and 4 h after LPS and actinomycin D treatment. Each sample was run in triplicate. The relative abundance of each mRNA was measured by real-time PCR, calculated using the ΔΔCt method, and normalized to GAPDH, and the half-lives of the RNAs were calculated as previously reported (37).

Fluorescence in situ hybridization

Chemotaxis assay SW480 and Jurkat cells were cocultured as previously reported (37). Briefly, SW480 cells were seeded at 1 3 105 cells per well in 24-well plates, transfected with pCDNA3.1-FIRRE or pFRT-hnRNPU for 48 h, and then exposed to LPS for 8 h in the presence or absence of a neutralizing Ab to VCAM1 (BD). SW480 cells were then cocultured with Jurkat cells grown in 8 nm membrane inserts (2 3 105). After 2.5 h of coculture, Jurkat cells that migrated through the insert membrane into the SW480 medium pool were collected. The number of Jurkat cells was counted under a microscope. Normal rabbit IgG (Santa Cruz) was used as a negative control.

LPS injection in vivo and immunohistochemistry for hnRNPU Male C57BL/6J mice (4–6 wk old, 18–20 g weight) were used for the study, with the approval of the Animal Care and Use Committee of Wuhan University. Animals received treatment of LPS (15 mg/kg bodyweight, i.p.) injection for 48 h, as previously reported (33). Five animals from each group were sacrificed and intestine tissues obtained for immunohistochemistry. An Ab to hnRNPU (ab20666; Abcam) was used.

Statistical analysis All data are presented as mean 6 SD of three experiments. Analysis was performed using a Student t test or the ANOVA test when appropriate. A p value ,0.05 was considered significant.

Results LPS stimulation increases the expression of selected lncRNAs in human macrophages and intestinal epithelial cells According to results from previous studies on the identification of lncRNAs in murine macrophage response to a TLR4 ligand (LPS) stimulation, we analyzed a large number of human and mouse homologous lncRNAs in the University of California Santa Cruz (http://genome.ucsc.edu/) and LNCipedia (http://lncipedia.org/) databases (26). These conserved lncRNAs in mice and humans were selected for further investigation. A total of more than 30 specific primers were designed for selected lncRNAs (primers listed in Supplemental Table I). We measured the expression levels of those lncRNAs in human macrophages (U937) and human intestinal epithelial cells (SW480) following stimulation with LPS (1 mg/ml) for 4 and 8 h. Real-time PCR analysis was performed to assess the levels of selected lncRNAs. Of these lncRNAs

expressed in U937 and SW480 cells, expression of AK093555, AK126635, AK128461, AK130580, AK024940, FIRRE, and TMC3-AS1 were significantly (p , 0.05) increased following LPS stimulation compared with non-LPS–treated control (Fig. 1). The expression levels of other selected lncRNAs, such as RP11446E9.2 and LOC392452, showed no change in cells following LPS stimulation (Fig. 1). Expression of IL-8 (an NF-ĸB–controlled gene) was measured for positive control in cells following LPS stimulation. FIRRE is a novel NF-kB–controlled lncRNA To test the role of NF-kB in LPS-altered lncRNA expression, we measured the levels of selected lncRNAs in U937 and SW480 cells in response to LPS for 4 and 8 h in the presence or absence of a specific IKK2 inhibitor, SC-514, which inhibits p65-associated transcriptional activation of the NF-kB pathway (39). We found that the expression of AK130580 and FIRRE was significantly increased in cells treated with LPS; SC-514 blocked the LPS-induced increase of AK130580 and FIRRE in the LPS-treated cells (Fig. 2A, Supplemental Fig. 1). The effects of SC-514 on the expression levels of IL-8 and other selected lncRNAs are shown in Supplemental Fig. 1. Considering the higher expression levels of FIRRE in both cell lines (with DCt values of 9.6 and 11 by real-time PCR in U937 and SW480 cells, respectively, versus DCt values of 13 and 16 for AK13058), we chose FIRRE for further study. First, we analyzed the kinetics of FIRRE alteration following LPS stimulation; U937 cells were exposed to LPS for various periods of time and expression was quantified by real-time PCR. The expression level of FIRRE started to increase after LPS treatment for 4 h and continued to increase until 48 h following LPS stimulation (Fig. 2B). Increased expression of FIRRE was also confirmed by Northern blot in U937 cells after LPS treatment for 4 h (Fig. 2C). Inhibition of FIRRE induction by SC-514 in LPS-stimulated cells suggests the involvement of transcriptional regulation by NF-kB components, in particular, the p65 component. To test this possibility, we analyzed the promoter of FIRRE gene and identified two potential NF-kB–binding sites in the promoter region of the FIRRE gene by MOTIF (http://motif.genome.jp/) database searches. Increased binding of p65 to the binding site at 21720, but not the putative binding site at 2308, in the promoter element of the FIRRE gene in U937 cells following LPS stimulation was demonstrated by ChIP analysis using specific primers for each putative binding site and negative control region of the FIRRE promoter (Fig. 2D). The p65 binding site in the promoter region of the IL-8 gene acted as a positive control. To further test the potential transactivation of the FIRRE gene by the p65 subunit, we generated luciferase reporter constructs covering the potential NF-kB binding sites, as well as various deletions and mutations, of the putative promoters of the FIRRE gene. LPS stimulation increased luciferase activity in cells transfected with the luciferase constructs that encompass the two putative NF-kB binding sites, compared with cells transfected with the pGL3-basic construct (Fig. 2E, 2F). SC-514 significantly inhibited LPS-induced increase of luciferase activity in SW480 cells transfected with the construct that encodes two putative NF-kB binding sites (Fig. 2E). Moreover, as shown in Fig. 2F, LPS stimulation increased luciferase activity in SW480 cells transfected with the luciferase constructs that encompass the putative NF-kB binding site at 21720 but not the putative binding site at 2308. A mutant of the NF-kB binding site at 21720 blocked LPS-induced luciferase activity (Fig. 2F). P65-associated transactivation of the FIRRE promoter was also confirmed by the upregulation of luciferase activity after p65 overexpression in SW480 cells (Fig. 2F). Together, these data demonstrate that p65 binding to the promoter element of the

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The fluorescence in situ hybridization (FISH) analysis was performed using the Fluorescent In Situ Hybridization Kit (C10910; RiboBio). The probes for FIRRE were designed and obtained from RiboBio (lncRNA FISH Probe Mix, C10920), and the sense strands of probes for FIRRE and a probe to 18s RNA were also provided as the matched negative control and the positive control, respectively. All probes were labeled with Cy3 and hybridization was performed according to the manufacturer’s instructions and as previously reported (38). Briefly, SW480 cells were cultured in 35 mm confocal dishes and stimulated with LPS for 4 h. The cells were washed with 13 PBS twice, fixed (10 min with 4% formaldehyde), and permeabilized with 70% ethanol. Prior to the hybridization, the cells were incubated with prehybridization solution at 37˚C for 30 min. Cells were then hybridized with the probes for FIRRE, 18s RNA, or the negative control (0.5 ng/ml final) in hybridization solution at 37˚C overnight. After hybridization, the cells were washed in washing buffer (43 SSC, 0.1% Tween-20) at 37˚C for 10 min twice (with the addition of DAPI in the second wash) and then in 13 PBS twice. The imaging was done immediately after with 13 PBS as the mounting medium using Leica LCS-SP8-STED (Medical Research Center for Structural Biology, Wuhan University).

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4 FIRRE gene mediates FIRRE upregulation in response to LPS stimulation, implicating that the transcription of FIRRE is controlled by the NF-kB signaling. FIRRE regulates the expression of several inflammatory genes

FIGURE 1. LPS stimulation increases the expression of selected lncRNAs in human macrophages and intestinal epithelial cells. Alterations of selected lncRNA expression in U937 (A) and SW480 (B) cells after exposure to LPS (1 mg/ml) for 4 and 8 h as assessed by real-time PCR. IL-8 as a positive control of LPS-induced gene. Data represent mean 6 SEM from three independent experiments. *p , 0.05 versus nontreated cells.

FIRRE interacts with hnRNPU and coregulates the expression of VCAM1 and IL12p40 genes Using U2 as a negative control, we conducted formaldehyde crosslinking RIP analysis and confirmed the direct physical association between FIRRE and hnRNPU in U937 and SW480 cells (Fig. 4A). hnRNPU is an RNA-binding protein and enhances the expression of specific genes by regulating mRNA stability (14). Consistent with the results of previous studies (40), a significant increase of hnRNPU at both RNA and protein levels was detected in U937 cells following LPS stimulation, and it reached a peak after treatment with LPS for 12 h (Fig. 4B). We then investigated whether hnRNPU is involved in FIRRE-modulated expression of the inflammatory genes in cells in response to LPS stimulation. First, we assessed the intracellular distribution of FIRRE and hnRNPU in cells following LPS stimulation. SW480 cells were exposed to LPS for 12 h, and FIRRE and hnRNPU levels were measured in the nuclear and cytoplasmic extracts. FIRRE and hnRNPU were present in both the nucleus and the cytoplasm. An increased RNA level of FIRRE and hnRNPU were detected in the nuclear and cytoplasmic extracts in LPS-treated cells (Fig. 4C). In contrast, the protein content of hnRNPU was increased in the cytoplasm after LPS treatment, but not in the nucleus (Fig. 4D). RNA-FISH analysis demonstrated that FIRRE was localized both in the cytoplasm and nucleus in SW480 cells and LPS stimulation increased its expression levels (Fig. 4E). Notably, our data revealed a strongly cytoplasmic distribution of FIRRE, which is different from the results previously reported in HEK293s, HeLa, and Patski cells (32), suggesting a cell-specific expression pattern of FIRRE. Next, we measured the expression of VCAM1 and IL12p40 in SW480 cells transfected with hnRNPU-siRNA and FIRRE-shRNA1. The efficiency of hnRNPU siRNA was tested at the mRNA and protein levels in SW480 cells (Supplemental Fig.

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To clarify the potential role of LPS-induced FIRRE in regulating the expression of inflammatory genes, we designed two independent shRNAs to knockdown FIRRE expression, and constructed a plasmid to overexpress FIRRE (pCDNA3.1-FIRRE). SW480 cells were transfected with FIRRE shRNAs or full-length FIRRE for 24 h, followed by LPS stimulation for 4 h. Both shRNA1 and shRNA2 to FIRRE significantly decreased FIRRE expression levels in the nonstimulated and LPS-stimulated SW480 cells, whereas the nonspecific control shRNA showed no effect (Fig. 3A). Then we investigated the effects of gain- or loss-of-function of FIRRE on the expression of selected inflammatory genes, including IL12p40, G-CSF, TNF-a, IL1B, VCAM1, CXCL10, IL-8, RANTES (Ccl5), MCP-1, IL-1A, ICSF-1, and IL-6. Knockdown of FIRRE decreased mRNA levels of IL-12p40, G-CSF, TNF-a, IL-1B, and VCAM1, and increased expression of IL-6 in the nonstimulated and LPSstimulated SW480 cells (Fig. 3A). In contrast, overexpression of FIRRE increased expression of IL-12p40, VCAM1, IL-1A, and ICSF1, and decreased mRNA levels of CXCL10, IL-8, IL-1B, RANTES, and IL-6 in SW480 cells (Fig. 3B). Taken together, these results suggest that FIRRE may participate in the regulation of many inflammatory genes. Previous studies indicated that FIRRE interacts with the nuclear-matrix factor hnRNPU in HEK293 cells (32). Given the consistent effects of FIRRE gain- or loss-of-function on the basal and LPS-induced expression of VCAM1 and IL12p40 in SW480 cells, we then focused on these two genes to address whether FIRRE modulates inflammatory gene expression through its interaction with hnRNPU.

FIRRE MODULATES INFLAMMATORY RESPONSES

The Journal of Immunology

5

2A). Knockdown of hnRNPU decreased both the mRNA and protein levels of VCAM1 (Fig. 4F, 4G). Furthermore, the expression of VCAM1 was even lower in both the FIRRE and hnRNPU-infected group compared with one of FIRRE/hnRNPUinfected group in SW480 cells (Fig. 4F, 4G). In contrast, overexpression of both FIRRE and hnRNPU aggravated the expression of VCAM1, compared with overexpression either FIRRE or hnRNPU alone in SW480 cells (Fig. 4F, 4G). Similar effects of knockdown of either FIRRE or hnRNPU, or their combination, on the expression of IL12p40 gene were observed in SW480 cells (Supplemental Fig. 2B). These findings demonstrate that FIRRE

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FIGURE 2. FIRRE is a novel NF-kB–controlled lncRNA. (A) FIRRE expression in U937 and SW480 cells following LPS stimulation for 4 and 8 h in the presence or absence of SC-514 as assessed by real-time PCR. Data presented as fold changes in the ratio of FIRRE levels to GAPDH. (B) U937 cells were exposed to LPS (1 mg/ml) for up to 48 h, followed by real-time PCR analysis for FIRRE mRNA. (C) U937 cells were exposed to LPS (1 mg/ml) for 4 h by Northern blot for FIRRE. (D) LPS induced the promoter element binding of p65 to FIRRE gene. U937 cells were exposed to LPS for 4 h, followed by ChIP assay using anti-p65 and PCR primers covering two binding sites and negative control region of the FIRRE promoter. An NF-kB binding site of the IL-8 promoter was analyzed for positive control. (E) FIRRE promoter luciferase reporter assay. The 2 kb upstream of the FIRRE was cloned and inserted into the pGL3-Basic luciferase construct. SC514 attenuated LPS-triggered luciferase activity in SW480 cells transfected with the luciferase construct. The pGL3-Basic was also measured for control. (F) SW480 cells were transfected with various luciferase reporter constructs including the potential p65 binding sites of the FIRRE promoter. The transfected cells were exposed to LPS for 4 h. Dual-luciferase reporter assay detection of the relative luciferase activities and presented as the ratio of the activity of the test construct with the pRL-TK luciferase reporter construct. SW480 cells were cotransfected with the pCMV-p65 to overexpress p65 and the luciferase reporter constructs for 24 h followed by measurement of luciferase activity. Data represent mean 6 SEM from three independent experiments. *p , 0.05 t test versus the nonstimulated cells or empty pCMV vector control, #p , 0.05 t test versus LPS-stimulated cells.

can interact with hnRNPU and coregulate the expression of VCAM1 and IL12p40 in SW480 cells. FIRRE regulates the RNA stabilization of VCAM1 and IL12p40 through targeting their AREs with hnRNPU Previous studies indicated that hnRNPU enhances mRNA stability (14). Given that FIRRE can interact with hnRNPU and coregulate the expression of VCAM1 and IL12p40, we questioned whether FIRRE regulates the mRNA stability of VCAM1 and IL12p40. To test this possibility, SW480 cells were transfected with FIRRE shRNAs for 24 h, and then treated with actinomycin D in the

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FIRRE MODULATES INFLAMMATORY RESPONSES

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FIGURE 3. FIRRE regulates the expression of several inflammatory genes. (A and B) Real-time PCR detection of expression of FIRRE, and some inflammation-related genes in SW480 cells transfected with the FIRRE-shRNAs or pCDNA3.1-FIRRE for 24 h and cultured for an additional 4 h in the presence or absence of LPS. Data represent mean 6 SEM from three independent experiments. *p , 0.05 t test versus the nonstimulated cells, #p , 0.05 t test versus LPS-stimulated cells.

presence of LPS for additional 2 h, followed by measurement of VCAM1, IL12p40, RANTES (ccl5), and actin mRNA levels by real-time PCR. Knockdown of FIRRE showed a significant decrease in VCAM1 and IL12p40 mRNA stability in SW480 cells (Fig. 5A). The RNA stability of RANTES, which is not regulated by FIRRE, and actin, which contains no ARE, showed no change in cells treated with the FIRRE shRNAs (Supplemental Fig. 2C). It is well known that AREs are one of the most common determinants of mRNA stability in mammalian cells (41). We asked whether FIRRE regulates mRNA stability through targeting AREs in general. First, we used two constructs containing the luciferase cDNA fused to the 39UTR (pcDNA3-luc-ARE) and the 39UTR with deletion of ARE (pcDNA3-luc-DARE) as previously reported

(37). SW480 cells were transfected with these constructs with pcDNA3.1-FIRRE (overexpression of FIRRE) or FIRRE-shRNA1 by assessment of luciferase activity 24 h after transfection. The transfection efficiency for the luciferase plasmids in SW480 cells was around 50% and, therefore, luciferase activity was normalized to the expression of the control Renilla luciferase construct. Overexpression FIRRE in SW480 cells resulted in a significant increase in luciferase activity of pcDNA3-luc-ARE, compared with that in the control (Supplemental Fig. 3A). In cells transfected with the construct deletion of the ARE nonamer (pcDNA3-luc-DARE), the associated luciferase activity was not changed compared with the control (Supplemental Fig. 3A). In contrast, FIRRE-shRNA1 decreased the luciferase activity of pcDNA3-luc-ARE and did not

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FIGURE 4. FIRRE interacts with hnRNPU and coregulates the expression of VCAM1 and IL12p40 genes. (A) RIP detection of the interaction between FIRRE with hnRNPU in SW480 and U937 cells by using anti-hnRNPU Ab, with U2 as a negative control. (B) U937 cells were exposed to LPS for up to 48 h, followed by real-time PCR for hnRNPU. (C) Nuclear versus cytoplasmic (Cyto) distribution of FIRRE and hnRNPU in SW480 cells following LPS stimulation. U2 RNA (a nuclear RNA) and RPS14 (a cytoplasmic RNA) were used as control. (D) SW480 cells were exposed to LPS for 12 h, and nuclear and cytoplasmic extracts were isolated, followed by Western blot analysis for hnRNPU. GAPDH and H3 were also blotted for control. (E) RNA-FISH detection of the expression of FIRRE in SW480 cells follow LPS stimulated by using RNA probe (red). Nuclei were detected with DAPI (blue). The matched negative control probes and the probe to 18s RNA were used as the negative and positive control, respectively (scale bar, 10 mm). (F) Effects of knockdown/overexpression of FIRRE and hnRNPU on VCAM1 mRNA levels in SW480 cells. Cells were transfected with the FIRRE-shRNA1/hnRNPUsiRNA or the full-length of FIRRE/hnRNPU for 48 h, followed by real-time PCR analysis. (G) Effects of knockdown/overexpression of FIRRE and hnRNPU on VCAM1 protein levels in SW480 cells. Cells were transfected with the FIRRE-shRNA1/hnRNPU-siRNA or the (Figure legend continues)

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change the luciferase activity of pcDNA3-luc-DARE compared with the control (Supplemental Fig. 3A). Moreover, we also tested whether hnRNPU regulates mRNA stability through targeting AREs in general. Overexpression of hnRNPU in SW480 cells

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FIGURE 5. FIRRE regulates the RNA stabilization of VCAM1 and IL12p40 genes through targeting their AREs with hnRNPU. (A) SW480 cells were transfected with FIRREshRNAs for 24 h. Actinomycin D (Act D) was then added and cells were collected for realtime PCR analysis. The stability of mRNAs was calculated, presented as the relative amount of mRNA to control. Quantification of VCAM1 and IL12p40 mRNA levels from three independent experiments were shown. *p , 0.05 t test versus the control cells. (B) SW480 cells were cotransfected with the luciferase construct containing the ARE in fulllength VCAM1 39UTR (pMIR-VCAM1-ARE) or the VCAM1 39UTR with deletion of ARE (pMIR-VCAM1-DARE), and FIRRE-shRNA/ hnRNPU-siRNA or pCDNA3.1-FIRRE/pFRThnRNPU for 48 h, followed by luciferase analysis. (C) RIP detection of the effects of FIRRE on the interaction of hnRNPU to 39UTR of VCAM1 in SW480 cells. (D) SW480 cells were transfected with the pMIR-VCAM1-ARE/ pMIR-IL12p40-ARE or pMIR-VCAM1-DARE/ pMIR-IL12p40-DARE, and stimulated by LPS for 4 h, followed by luciferase analysis. *p , 0.05 t test versus the control or nontreated cells, #p , 0.05 ANOVA test versus transfected with either FIRRE or hnRNPU.

resulted in a significant increase in luciferase activity of pcDNA3luc-ARE without impacting on that of pcDNA3-luc-DARE. Knockdown of hnRNPU decreased the luciferase activity of pcDNA3-lucARE (Supplemental Fig. 3A).

full-length of FIRRE/hnRNPU for 48 h, followed by ELISA analysis. Data represent mean 6 SEM from three independent experiments. *p , 0.05 t test versus the control, #p , 0.05 ANOVA test versus transfected with either FIRRE or hnRNPU.

The Journal of Immunology

cells. After 2.5 h of incubation, the Jurkat cells that migrated through the membrane into the SW480 medium pool were counted. Overexpression of both FIRRE and hnRNPU in SW480 cells caused a more significant increase of Jurkat cell migration than overexpression of only FIRRE or hnRNPU in SW480 cells (Fig. 6). Neutralizing Ab to VCAM1 showed a significant inhibitory effect of Jurkat cell migration even though both FIRRE and hnRNPU were overexpressed in SW480 cells (Fig. 6). These results indicate that FIRRE and hnRNPU coregulate the chemotaxis effects of VCAM1-expressing SW480 cells to Jurkat cells. FIRRE is conserved between humans and mice According to the results from previous studies (44), we noted that FIRRE has a conserved intergenic ortholog location on the X chromosome between humans and mice (Fig. 7A). To investigate whether LPS induces mFIRRE transcription through the NF-ĸB signaling pathway, we investigated the kinetics of mFIRRE expression in mouse macrophage cells (RAW264.7) following LPS stimulation. The expression of mFIRRE started to increase after LPS treatment for 4 h and reached a peak at 12 h, but continued to decrease slowly until 48 h following LPS stimulation (Fig. 7B). We also found an increased expression of mFIRRE in PMPMs treated with LPS for 4 h (Fig. 7C). When LPS (15 mg/kg body weight) was injected into the abdominal cavity of C57/BJ mice, a significant increase in the level of mFIRRE was detected in the PMPMs and intestinal tissues isolated 48 h after LPS injection (Fig. 7D). Furthermore, we treated RAW264.7 cells with SC-514 after LPS stimulation for 4 and 8 h. Expression levels of mFIRRE were significantly increased in cells exposed to LPS, and SC-514 blocked LPS-induced increase of mFIRRE in the LPS-treated cells (Fig. 7E). To investigate the regulatory effects of mFIRRE on inflammatory gene expression, we designed two different siRNAs to knockdown mFIRRE. Both siRNAs significantly decreased mFIRRE expression levels in the nonstimulated and LPS-stimulated RAW264.7 cells (Fig. 7F). Consistent with the effects of FIRRE in human macrophages and intestinal epithelial cells, knockdown of mFIRRE decreased mRNA expression levels of IL12p40 andVCAM1, and increased mRNA expression of IL6 in RAW264.7 cells (Fig. 7F). Moreover, we detected that mFIRRE interacts with hnRNPU in

FIRRE and hnRNPU coregulate chemotaxis effects of VCAM1 to Jurkat cells As an adhesion molecule, VCAM-1 expressed on the surface of epithelial cells tethers VLA-4–positive lymphocytes to epithelial cells, which is important in mucosal inflammation (42). We then tested the role of VCAM1-induced chemotaxis activity in intestinal epithelial cells. As previously reported (37), we chose Jurkat cells to coculture with intestinal epithelial cells. JKT is a cancer cell line changed from the original T lymphocytes; it expresses VLA-4 and has previously been used for chemotaxis assay (43). SW480 cells were treated with LPS after FIRRE/hnRNPU overexpression transfection for 24 h, and then in the presence or absence of a neutralizing Ab to VCAM1. Jurkat cells were grown in Transwell culture 8 nm membrane inserts cocultured with SW480

FIGURE 6. FIRRE and hnRNPU coregulate chemotaxis effects of VCAM1 to Jurkat cells. SW480 cells were transfected with pCDNA3.1FIRRE /pFRT-hnRNPU for 48 h, and then in the presence or absence of a neutralizing Ab to VCAM1. SW480 cells were then cocultured with Jurkat cells. Number of Jurkat cells transmigrated in cell chambers after 2.5 h was counted. Normal rabbit IgG was used as a negative control. *p , 0.05 t test versus the control, #p , 0.05 ANOVA test versus transfected with either FIRRE or hnRNPU.

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Because FIRRE can interact with hnRNPU and regulates the expression of VCAM1 and IL12p40, we predicted that it may regulate the stabilization of VCAM1and IL12p40 mRNA with hnRNPU by targeting their AREs. We constructed a Firefly luciferase reporter pMIR containing the 39UTR of VCAM1 (pMIRVCAM1-ARE) and IL12p40 (pMIR-IL12p40-ARE), and the mutant reporters by deleting the core ARE nucleotides (pMIRVCAM1-DARE, pMIR-IL12p40-DARE). SW480 cells were transfected with FIRRE-shRNAs and hnRNPU-siRNA together with pMIR-VCAM1-ARE/pMIR-IL12p40-ARE or pMIR-VCAM1DARE/pMIR-IL12p40-DARE for 48 h, followed by assessment of luciferase activity. Knockdown of both FIRRE and hnRNPU in SW480 cells showed a further decrease in luciferase activity of pMIRVCAM1-ARE/pMIR-IL12p40-ARE than cells with the knockdown of either FIRRE or hnRNPU alone (Fig. 5B, Supplemental Fig. 3B). Inhibition of associated luciferase activity was not detected in cells transfected with the ARE-deleted construct (pMIR-VCAM1DARE, pMIR-IL12p40-DARE) compared with the control (Fig. 5B, Supplemental Fig. 3B). Complementarily, overexpression of both FIRRE and hnRNPU in SW480 cells showed a further increase in luciferase activity of pMIR-VCAM1-ARE/pMIR-IL12p40-ARE than cells with overexpression of either FIRRE or hnRNPU alone (Fig. 5B, Supplemental Fig. 3B). SW480 cells transfected with the ARE-deleted construct (pMIR-VCAM1-DARE, pMIR-IL12p40DARE) showed no significant change in luciferase activity (Fig. 5B, Supplemental Fig. 3B). Furthermore, we found that FIRRE impacts the interaction of hnRNPU with VCAM1 mRNA 39UTR in a positive manner by RNA precipitation. The enrichment of VCAM1 mRNA 39UTR by an Ab to hnRNPU decreased significantly more in FIRRE knockdown cells than in control (Fig. 5C). In contrast, overexpression of FIRRE facilitated the interaction of hnRNPU to 39UTR of VCAM1 (Fig. 5C). The result suggests that FIRRE could promote the interaction of hnRNPU with VCAM1 mRNA 39UTR. Given the upregulation of FIRRE after LPS stimulation and the impact of FIRRE on VCAM1 and IL12p40 mRNA stability through targeting ARE, we speculated that LPS treatment should affect ARE-mediated posttranscription of VCAM1 and IL12p40. To test this possibility, SW480 cells were transfected with pMIR-VCAM1-ARE/pMIR-IL12p40-ARE or pMIR-VCAM1DARE/pMIR-IL12p40-DARE, and then exposed to LPS stimulation for 4 h. LPS treatment increased the luciferase activity in SW480 cells transfected with the constructs containing VCAM1 and IL12p40 39UTR with ARE, but did not alter the luciferase activity in cells transfected with constructs containing deletion of ARE (Fig. 5D). Taken together, the above data suggest that FIRRE regulates the mRNA stabilization of VCAM1 and IL12p40 through targeting ARE with hnRNPU in cells following LPS stimulation.

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FIGURE 7. FIRRE is conserved between humans and mice. (A) Location of FIRRE is conserved among human and mouse genomes. (B) RAW264.7 cells were exposed to LPS (1 mg/ml) for up to 48 h, followed by real-time PCR for FIRRE. (C) Expression of mFIRRE in PMPMs treated with LPS for 4 h. (D) Induction of mFIRRE in PMPMs and intestine tissue isolated after LPS peritoneal injection (48 h). (E) Expression of mFIRRE in RAW264.7 cells following LPS stimulation for 4 and 8 h in the presence or absence of SC-514 as assessed by real-time PCR. (F) Quantitative RT-PCR detection of expression of mFIRRE, and some inflammation-related genes in RAW264.7 cells transfected with mFIRRE-siRNAs, followed by LPS stimulation for 4 h. (G) RIP detection of the interaction between mFIRRE with hnRNPU in RAW264.7 cells by using anti-hnRNPU Ab, with IgG as a (Figure legend continues)

The Journal of Immunology RAW264.7 cells (Fig. 7G). Consistent with previous studies (40), a significant increase in mRNA expression of hnRNPU was detected in RAW264.7 cells following LPS stimulation (Fig. 7H), and for 48 h immunohistochemistry detected a significant increase of hnRNPU protein content in intestine tissue from mice after LPS i.p. injection (Fig. 7I). These results suggest that FIRRE is a conserved lncRNA among humans and mice, and may play an important regulatory role in the posttranscriptional regulation of inflammatory genes in the innate immune system.

Discussion

(24). Recent studies also suggest that lncRNAs may impact on the stability of host mRNAs through posttranscriptional mechanisms (30). However, how lncRNAs modulate inflammatory genes through posttranscriptional mechanisms is largely unknown. The data of our study indicate that FIRRE regulates the mRNA stability of several inflammatory genes, such as VCAM-1 and IL12p40. The coordinated expression of inflammatory genes involves both transcriptional and posttranscriptional mechanisms. A central part of posttranscriptional modulation of inflammatory gene expression is mediated by the regulation of mRNA stability (49). Evidence from in vitro and in vivo studies indicates that the posttranscriptional control of inflammatory transcripts is strongly dependent on ARE-mediated mechanisms (50–52). Several RNA-binding proteins recognize AREs within the 39UTRs of mRNAs and control their half-life in the cytoplasm (49). hnRNPU is an RNA binding protein and known as one of the most important mRNA stabilizing proteins (14). Some of these hnRNPU-regulated mRNAs code proteins with important immune functions, such as TNF-a, IL-6, and IL-1b, through the ARE motif. Meanwhile, hnRNPU also can interact with lncRNAs, including FIRRE. Hacisuleyman et al. (32) reported that hnRNPU is required for the regulation and maintenance of the proximal localization of the FIRRE locus and its trans-chromosomal localization sites. To our knowledge, data from our current study for the first time support that FIRRE may act as a scaffold molecule to help hnRNPU bind to AREs within the 39UTRs of inflammatory genes, representing a new mechanism showing that FIRRE regulates posttranscriptional mRNA stabilization by interacting with hnRNPU. However, how FIRRE directs the assembly of hnRNPU into AREs is unclear and merits further investigation. Evolutionary conservation is widely used as an indicator of the functional significance of newly discovered genes (53). Most (.95%) of lncRNAs appear to show little evolutionary sequence conservation and have been suggested to be nonfunctional (54). However, several lncRNAs have shown conservation of their functionality across species (55). For example, XIST is required for X inactivation in both human and mouse cells (56, 57). Local repeats have been reported to be enriched in several well-studied functional lncRNAs, such as XIST and FIRRE. Hacisuleyman et al. (32) showed that FIRRE has a unique 156 bp repeating RNA domain that occurs 16 and 8 times in mouse and human transcripts, respectively, with 96% sequence identity within species and 68% across species. Functionally, sequence duplications can endow a FIRRE with multiple platforms for the binding of hnRNPU. In our study, human and murine FIRRE are both transcriptionally induced by the NF-kB signaling pathway and regulate the same inflammatory response genes through interacting with hnRNPU. The similarities between human and murine FIRRE suggest that FIRRE may be an important regulator in innate immunity. In conclusion, our study provides evidence that FIRRE regulates the mRNA stability of inflammatory genes through interacting with hnRNPU by targeting their AREs in macrophages and intestinal epithelial cells. Posttranscriptional regulation of inflammatory genes by FIRRE may represent a new mechanism by which innate immune cells maintain homeostasis during inflammation, relevant to fine-tuning of cellular inflammatory responses in general.

negative control. (H) RAW264.7 cells were exposed to LPS for up to 48 h, followed by real-time PCR for hnRNPU. (I) Increase of hnRNPU protein staining was detected by immunohistochemistry in intestine tissue from mice following LPS i.p. injection (scale bar, 10 mm). *p , 0.05 t test versus the control or nontreated cells, #p , 0.05 t test versus LPS-stimulated cells.

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TLR/NF-kB signaling is critical to the fine-tuning of cell reactions to extracellular stimuli, including epithelial defense responses against pathogen infection (2). The role for lncRNAs in the regulation of TLR/NF-kB signaling–mediated inflammatory responses is largely unclear (18). In this study, we identified a subset of lncRNA genes as TLR4/NF-kB–responsive genes in human macrophage cells and intestinal epithelial cells. One such TLR4/NF-kB–responsive lncRNA is FIRRE, a previously identified X-inactivation lncRNA (31). Our analysis revealed that FIRRE is a novel NF-kB–dependent lncRNA and binding of the NF-kB subunit p65 to its promoter region (21720) activates its transcription. Functionally, FIRRE appears to mediate both the activation and repression of some inflammatory genes in macrophages and intestinal epithelial cells. Previous studies showed that FIRRE interacts with the nuclear-matrix factor hnRNPU (32). We confirmed the physical interaction of FIRRE and hnRNPU by RIP analysis in macrophage cells and intestinal epithelial cells upon TLR4 ligation. FIRRE and hnRNPU coregulate the expression of VCAM1 and IL12p40 through targeting ARE with hnRNPU. These findings demonstrate that FIRRE specifically promotes the expression of selected inflammatory genes by enhancing their mRNA stability through interaction with hnRNPU. Similar to many other RNA molecules, lncRNAs are initially transcribed by Poly II in the nucleus and can be regulated by transcriptional factors (45). NF-kB signaling can regulate both the activation and inhibition of lncRNA expression (46, 47). It was first described in 2013 in mouse bone marrow–derived dendritic cells that transcription of lncRNA genes in response to TLR signaling is activated in an NF-kB–dependent manner (26). Since then, a subset of lncRNA genes has been identified as NF-kB dependent. The NF-kB family of transcription factors consists of five members: p50, p52, p65 (RelA), c-Rel, and RelB. P65, c-Rel, and RelB contain the transcription activation domain and usually activate gene transcription (7). In this study, we demonstrated that promoter binding of the NF-kB p65 subunit is required for transactivation of the FIRRE in macrophages and intestinal epithelial cells following LPS stimulation. There is emerging evidence that lncRNAs are important regulators of innate immune responses (18). LncRNAs may control many aspects of innate immunity such as regulation of inflammatory mediators, differentiation, and cell migration through their interaction with various RNA-binding proteins (16, 47, 48). Most studies demonstrate that lncRNAs regulate inflammatory genes via transcription mechanisms. For example, lncRNA-COX2 regulates the transcription of late-primary response genes in innate immune cells through modulation of epigenetic chromatin remodeling

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Disclosures The authors have no financial conflicts of interest.

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