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MicroRNA-31-3p Is Involved in Substance P (SP)-Associated Inflammation in Human Colonic Epithelial Cells and Experimental Colitis

2017; Elsevier BV; Volume: 188; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2017.10.023

ISSN

1525-2191

Autores

Kai Fang, Ivy Ka Man Law, David Padua, Aristea Sideri, Vanessa Huang, Christopher G. Kevil, Dimitrios Iliopoulos, Charalabos Pothoulakis,

Tópico(s)

Inflammation biomarkers and pathways

Resumo

Substance P (SP) mediates colitis. SP signaling regulates the expression of several miRNAs, including miR-31-3p, in human colonocytes. However, the role of miR-31-3p in colitis and the underlying mechanisms has not been elucidated. We performed real-time PCR analysis of miR-31-3p expression in human colonic epithelial cells overexpressing neurokinin-1 receptor (NCM460 NK-1R) in response to SP stimulation and in NCM460 cells after IL-6, IL8, tumor necrosis factor (TNF)-α, and interferon-γ exposure. Functions of miR-31-3p were tested in NCM460–NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis. Targets of miRNA-31-3p were confirmed by Western blot analysis and luciferase reporter assay. Jun N-terminal kinase inhibition decreased SP-induced miR-31-3p expression. miR-31-3p expression was increased in both TNBS- and DSS-induced colitis and human colonic biopsies from ulcerative colitis, compared with controls. Intracolonic administration of a miR-31-3p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic TNF-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) mRNA expression. Conversely, overexpression of miR-31-3p ameliorated the severity of DSS-induced colitis. Bioinformatic, luciferase reporter assay, and Western blot analyses identified RhoA as a target of miR-31-3p in NCM460 cells. Constitutive activation of RhoA led to increased expression of CCL2, IL6, TNF-α, and CXCL10 in NCM460–NK-1R cells on SP stimulation. Our results reveal a novel SP–miR-31-3p–RhoA pathway that protects from colitis. The use of miR-31-3p mimics may be a promising approach for colitis treatment. Substance P (SP) mediates colitis. SP signaling regulates the expression of several miRNAs, including miR-31-3p, in human colonocytes. However, the role of miR-31-3p in colitis and the underlying mechanisms has not been elucidated. We performed real-time PCR analysis of miR-31-3p expression in human colonic epithelial cells overexpressing neurokinin-1 receptor (NCM460 NK-1R) in response to SP stimulation and in NCM460 cells after IL-6, IL8, tumor necrosis factor (TNF)-α, and interferon-γ exposure. Functions of miR-31-3p were tested in NCM460–NK-1R cells and the trinitrobenzene sulfonic acid (TNBS) and dextran sodium sulfate (DSS) models of colitis. Targets of miRNA-31-3p were confirmed by Western blot analysis and luciferase reporter assay. Jun N-terminal kinase inhibition decreased SP-induced miR-31-3p expression. miR-31-3p expression was increased in both TNBS- and DSS-induced colitis and human colonic biopsies from ulcerative colitis, compared with controls. Intracolonic administration of a miR-31-3p chemical inhibitor exacerbated TNBS- and DSS-induced colitis and increased colonic TNF-α, CXCL10, and chemokine (C-C motif) ligand 2 (CCL2) mRNA expression. Conversely, overexpression of miR-31-3p ameliorated the severity of DSS-induced colitis. Bioinformatic, luciferase reporter assay, and Western blot analyses identified RhoA as a target of miR-31-3p in NCM460 cells. Constitutive activation of RhoA led to increased expression of CCL2, IL6, TNF-α, and CXCL10 in NCM460–NK-1R cells on SP stimulation. Our results reveal a novel SP–miR-31-3p–RhoA pathway that protects from colitis. The use of miR-31-3p mimics may be a promising approach for colitis treatment. miRNAs are short (19 to 22 nucleotides), single-stranded RNA molecules that negatively regulate gene expression by inducing mRNA degradation and/or inhibition of target mRNA translation.1Valencia-Sanchez M.A. Liu J. Hannon G.J. Parker R. Control of translation and mRNA degradation by miRNAs and siRNAs.Genes Dev. 2006; 20: 515-524Crossref PubMed Scopus (1728) Google Scholar Functional studies have demonstrated that miRNAs play a critical role in many pathologic conditions, including inflammation2Iliopoulos D. MicroRNA circuits regulate the cancer-inflammation link.Sci Signal. 2014; 7: pe8Crossref PubMed Scopus (19) Google Scholar and pathogenesis of colitis.3Nguyen H.T. Dalmasso G. Muller S. Carriere J. Seibold F. Darfeuille-Michaud A. Crohn's disease-associated adherent invasive Escherichia coli modulate levels of microRNAs in intestinal epithelial cells to reduce autophagy.Gastroenterology. 2014; 146: 508-519Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 4Koukos G. Polytarchou C. Kaplan J.L. Morley-Fletcher A. Gras-Miralles B. Kokkotou E. Baril-Dore M. Pothoulakis C. Winter H.S. Iliopoulos D. MicroRNA-124 regulates STAT3 expression and is down-regulated in colon tissues of pediatric patients with ulcerative colitis.Gastroenterology. 2013; 145: 842-852.e2Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 5Wu F. Zikusoka M. Trindade A. Dassopoulos T. Harris M.L. Bayless T.M. Brant S.R. Chakravarti S. Kwon J.H. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha.Gastroenterology. 2008; 135: 1624-1635.e24Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar Substance P (SP) is a neuropeptide expressed in the central nervous system and several peripheral organs, including the intestine.6Steinhoff M.S. von Mentzer B. Geppetti P. Pothoulakis C. Bunnett N.W. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease.Physiol Rev. 2014; 94: 265-301Crossref PubMed Scopus (378) Google Scholar SP plays a critical role in colitis pathophysiology by interacting with its high-affinity neurokinin-1 receptor (NK-1R)7Pothoulakis C. Castagliuolo I. LaMont J.T. Jaffer A. O'Keane J.C. Snider R.M. Leeman S.E. CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.Proc Natl Acad Sci U S A. 1994; 91: 947-951Crossref PubMed Scopus (255) Google Scholar, 8Stucchi A.F. Shebani K.O. Leeman S.E. Wang C.C. Reed K.L. Fruin A.B. Gower A.C. McClung J.P. Andry C.D. O'Brien M.J. Pothoulakis C. Becker J.M. A neurokinin 1 receptor antagonist reduces an ongoing ileal pouch inflammation and the response to a subsequent inflammatory stimulus.Am J Physiol Gastrointest Liver Physiol. 2003; 285: G1259-G1267Crossref PubMed Scopus (39) Google Scholar, 9Castagliuolo I. Riegler M. Pasha A. Nikulasson S. Lu B. Gerard C. Gerard N.P. Pothoulakis C. Neurokinin-1 (NK-1) receptor is required in Clostridium difficile- induced enteritis.J Clin Invest. 1998; 101: 1547-1550Crossref PubMed Scopus (160) Google Scholar and activating signaling pathways related to NF-κB10Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases.Biochem J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar, 11Karagiannides I. Kokkotou E. Tansky M. Tchkonia T. Giorgadze N. O'Brien M. Leeman S.E. Kirkland J.L. Pothoulakis C. Induction of colitis causes inflammatory responses in fat depots: evidence for substance P pathways in human mesenteric preadipocytes.Proc Natl Acad Sci U S A. 2006; 103: 5207-5212Crossref PubMed Scopus (75) Google Scholar, 12Lieb K. Fiebich B.L. Berger M. Bauer J. Schulze-Osthoff K. The neuropeptide substance P activates transcription factor NF-kappa B and kappa B-dependent gene expression in human astrocytoma cells.J Immunol. 1997; 159: 4952-4958Crossref PubMed Google Scholar and Jun N-terminal kinase (JNK)13Tansky M.F. Pothoulakis C. Leeman S.E. Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor.Proc Natl Acad Sci U S A. 2007; 104: 10691-10696Crossref PubMed Scopus (46) Google Scholar in different cell types, including colonocytes.10Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases.Biochem J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar, 13Tansky M.F. Pothoulakis C. Leeman S.E. Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor.Proc Natl Acad Sci U S A. 2007; 104: 10691-10696Crossref PubMed Scopus (46) Google Scholar, 14Koon H.W. Zhao D. Zhan Y. Moyer M.P. Pothoulakis C. Substance P mediates antiapoptotic responses in human colonocytes by Akt activation.Proc Natl Acad Sci U S A. 2007; 104: 2013-2018Crossref PubMed Scopus (90) Google Scholar SP is expressed in inflammatory cells in mice.15Joos G.F. Germonpre P.R. Pauwels R.A. Role of tachykinins in asthma.Allergy. 2000; 55: 321-337Crossref PubMed Scopus (177) Google Scholar In addition, SP and NK-1R expressions are increased in the colon of inflammatory bowel disease (IBD) patients,6Steinhoff M.S. von Mentzer B. Geppetti P. Pothoulakis C. Bunnett N.W. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease.Physiol Rev. 2014; 94: 265-301Crossref PubMed Scopus (378) Google Scholar, 16Goode T. O'Connell J. Anton P. Wong H. Reeve J. O'Sullivan G.C. Collins J.K. Shanahan F. Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localisation.Gut. 2000; 47: 387-396Crossref PubMed Scopus (119) Google Scholar, 17Koon H.W. Zhao D. Xu H. Bowe C. Moss A. Moyer M.P. Pothoulakis C. Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar further suggesting a critical role for the SP–NK-1R interactions in IBD pathogenesis. Along these lines, human colonic epithelial cells overexpressing NK-1R (NCM460–NK-1R) were selected to study SP–NK-1R signaling functions in colonic epithelial cells in vitro. Recently, microarray analysis indicated that among others miR-31-3p is induced by SP stimulation of NCM460–NK-1R cells.18Fang K. Sideri A. Law I.K. Bakirtzi K. Polytarchou C. Iliopoulos D. Pothoulakis C. Identification of a novel substance P (SP)-neurokinin-1 receptor (NK-1R) microRNA-221-5p inflammatory network in human colonic epithelial cells.Cell Mol Gastroenterol Hepatol. 2015; 1: 503-515Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar The sequence of miR-31-3p is identical in humans, cows, dogs, rats, and mice, suggesting that it may play an important physiological regulatory role.19Chang K.W. Kao S.Y. Wu Y.H. Tsai M.M. Tu H.F. Liu C.J. Lui M.T. Lin S.C. Passenger strand miRNA miR-31* regulates the phenotypes of oral cancer cells by targeting RhoA.Oral Oncol. 2013; 49: 27-33Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Recent studies demonstrated that miR-31-3p is associated with colorectal cancer,20Mlcochova J. Faltejskova-Vychytilova P. Ferracin M. Zagatti B. Radova L. Svoboda M. Nemecek R. John S. Kiss I. Vyzula R. Negrini M. Slaby O. MicroRNA expression profiling identifies miR-31-5p/3p as associated with time to progression in wild-type RAS metastatic colorectal cancer treated with cetuximab.Oncotarget. 2015; 6: 38695-38704Crossref PubMed Scopus (68) Google Scholar, 21Manceau G. Imbeaud S. Thiebaut R. Liebaert F. Fontaine K. Rousseau F. Genin B. Le Corre D. Didelot A. Vincent M. Bachet J.B. Chibaudel B. Bouche O. Landi B. Bibeau F. Leroy K. Penault-Llorca F. Van Laethem J.L. Demetter P. Tejpar S. Rossi S. Mosakhani N. Osterlund P. Ristamaki R. Sarhadi V. Knuutila S. Boige V. Andre T. Laurent-Puig P. Hsa-miR-31-3p expression is linked to progression-free survival in patients with KRAS wild-type metastatic colorectal cancer treated with anti-EGFR therapy.Clin Cancer Res. 2014; 20: 3338-3347Crossref PubMed Scopus (88) Google Scholar whereas SP–NK-1R interactions are involved in both IBD-associated colitis and colitis-associated cancer.6Steinhoff M.S. von Mentzer B. Geppetti P. Pothoulakis C. Bunnett N.W. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease.Physiol Rev. 2014; 94: 265-301Crossref PubMed Scopus (378) Google Scholar From these considerations, it was hypothesized that the SP-regulated miR-31-3p may be involved in the pathophysiology of colitis. To address this hypothesis, the mechanisms of regulation of miR-31-3p expression by SP and proinflammatory cytokines were studied, its signaling in human colonic epithelial cells examined, and its function in experimental colitis models determined. NCM460 human colonic epithelial cells overexpressing NK-1R (NCM460–NK-1R) were cultured as previously described10Zhao D. Kuhnt-Moore S. Zeng H. Pan A. Wu J.S. Simeonidis S. Moyer M.P. Pothoulakis C. Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves Rho family small GTPases.Biochem J. 2002; 368: 665-672Crossref PubMed Scopus (67) Google Scholar; cells were starved in serum-free medium overnight and then stimulated with 0.1 μmol/L SP concentration at the indicated times. The JNK inhibitor SP600125 (catalog number 8177) was from Cell Signaling Technology (Danvers, MA). SP (catalog number S6883) was purchased from Sigma-Aldrich (St. Louis, MO). IL-6 (catalog number 7270-IL-025), IL-8 (catalog number 618-IL-010), tumor necrosis factor (TNF)-α (catalog number 210-TA), and interferon-γ (catalog number 285-IF) were purchased from R&D Systems (Minneapolis, MN). Protease inhibitor cocktail (catalog number sc-29130) was purchased from Santa Cruz Biotechnology (Dallas, TX). Rabbit anti-RhoA (catalog number SC-26C4) and mouse anti–c-Jun (catalog number SC-74543) antibodies were purchased from Santa Cruz Biotechnology. Mouse anti–β-actin (catalog number A5316) was purchased from Sigma-Aldrich. RhoA L63 recombinant adenovirus (constitutively active, ADV-157) and null control recombinant adenovirus(ADV-001) were purchased from Cell Biolabs, Inc. (San Diego, CA). Rho activation assay kit (catalog number BK036) was purchased from Cytoskeleton, Inc. (Denver, CO). Human RhoA siRNA (catalog number SC-29471), c-Jun siRNA (catalog number SC-29223), and control siRNA (SC-37007) were purchased from Santa Cruz Biotechnology. Lipofectamine 2000 (catalog number 52758) was purchased from Life Technologies (Carlsbad, CA). Dextran sodium sulfate (DSS; catalog number 9011-18-1) was from Affymetrix (Canoga Park, CA). Trinitrobenzene sulfonic acid (TNBS) was from Sigma-Aldrich (catalog number 92822). Total RNAs from the colon tissues of patients with active ulcerative colitis (UC) (n = 14), active Crohn disease (CD; n = 15), and healthy individuals (n = 9 to 10) were purchased from Origene (Rockville, MD). These biopsy samples were obtained from accredited US-based medical institutions through strict institutional review board protocols and with fully documented patient consent. For miRNA in situ hybridization analysis, human biopsies were obtained from patients with UC or CD, and control colon samples were obtained from non-IBD patients without abnormalities at colonoscopy or clinical history of gastrointestinal disease. These tissues were obtained from the University of California Los Angeles Medical Center through institutional review board protocols and with fully documented patient consent. Inhibitors of miR-31-3p (catalog number 4464084), negative anti-miRNA controls (catalog number 4464076), a miR-31-3p mimic (catalog number 4464066), and mimic miRNA controls (catalog number 4464058) were purchased from Life Technologies. Mouse anti–miR-31-3p and its negative control (sequence of anti–miR-31-3p is 5′-AATATGTTGGCATAGC-3′, sequence of anti-miRNA control is 5′-ACGTCTATACGCCCA-3′) were purchased from Qiagen (Germantown, MD). Lipid-based siPORTNeoFX Transfection Agent (AM4511) was purchased from Ambion (Canoga Park, CA). For miR-31-3p silencing or overexpression, cells were transfected with antisense (as)-miR-31-3p or miR-31-3p mimic, respectively. Cells transfected with antisensecontrol miRNA, or miRNA-mimic control served as controls. For c-Jun or RhoA silencing, NCM460–NK-1R cells were transfected with the respective siRNA or control siRNA with the use of lipofectamine RNAiMAX from Life Technologies. High-capacity reverse transcription reagent for cDNA was from Applied Biosystems (catalog number 4368813; Foster City, CA). Real-time PCR primers were purchased from Life Technologies, except miR-31-3p primers that were purchased from Qiagen (catalog number 205415). Total RNA of the NCM460–NK-1R cells were isolated by using Trizol reagents, the RNA concentrations were determined by Nanodrop. Mouse serum RNA was isolated by using miRCURY RNA Isolation Kit-Biofluids (Qiagen; catalog number 300112) according to the manufacture's protocol. The miRNA template for real-time PCR analysis was prepared by using Qiagen reagents. RNU1A1 (Qiagen; catalog number 203909) expression was used as an internal control. The Ct value formula was used to calculate the relative expression of selected miRNAs, as was previously reported.22Fang K. Zhang S. Glawe J. Grisham M.B. Kevil C.G. Temporal genome expression profile analysis during t-cell-mediated colitis: identification of novel targets and pathways.Inflamm Bowel Dis. 2012; 18: 1411-1423Crossref PubMed Scopus (17) Google Scholar Conversion of the cDNA of RNA samples was performed as previously reported,18Fang K. Sideri A. Law I.K. Bakirtzi K. Polytarchou C. Iliopoulos D. Pothoulakis C. Identification of a novel substance P (SP)-neurokinin-1 receptor (NK-1R) microRNA-221-5p inflammatory network in human colonic epithelial cells.Cell Mol Gastroenterol Hepatol. 2015; 1: 503-515Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar and the levels of mRNA expression were determined by quantitative real-time PCR analysis. All of the primers were purchased from Thermo Fisher Scientific (Waltham, MA): human IL-6 (Hs00174131_m1); chemokine (C-C motif) ligand 2 (CCL2; Hs00234140_m1); IL-1B (Hs00173097_m1); TNF-α (Hs01113624_g1); CXCL10 (Hs01124251_g1); RhoA (Hs00357608_m1); mouse IL-6 (Mm00446190_m1); CCL2 (Mm00441242_m1); mouse TNF-α (Mm00443258-m1), and CXCL10 (Mm00445235-m1). Human 18S (Hs03928990_g1) or mouse 18S (Mm03928990_g1) gene expression was selected as an internal control. Human colonic epithelial cells seeded at a density of 6 × 104 cells per well in 6-well plates were incubated in complete medium overnight and then incubated with 200 pfu/cell of Ad-RhoA-L63 or null control recombinant adenovirus in serum-free medium for 1 hour, followed by incubation in compete medium for 24 hours, as previously reported.23Fang K. Fu W. Beardsley A.R. Sun X. Lisanti M.P. Liu J. Overexpression of caveolin-1 inhibits endothelial cell proliferation by arresting the cell cycle at G0/G1 phase.Cell Cycle. 2007; 6: 199-204Crossref PubMed Scopus (44) Google Scholar NCM460–NK-1R cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated with radiolabeled immunoprecipitation assay buffer that contained the protease inhibitors and sodium orthovanadate (Santa Cruz Biotechnology) for 5 minutes on ice. Equal amounts of cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blocked (PBS, 5% nonfat dry milk, 0.05% Tween-20) and probed with antibodies, followed by corresponding horseradish peroxidase-labeled secondary antibodies. Blots were developed with an enhanced chemiluminescence reagent from Thermo Fisher Scientific (catalog number 34080). The signal was quantified by using ImageJ software version 1.47 (NIH, Bethesda, MD). RhoA activity was measured according to the procedure provided in the Rho Activation Assay Biochem Kit (Cytoskeleton, Inc.; catalog number BK036) manual. Cells were washed with PBS and then incubated with lysis buffer for 10 minutes at 4°C. Cell lysates were centrifuged at 10,000 × g for 1 minute. An equal amount of rhotekin-RBD beads was incubated with the fresh equal amount of supernatants for 1 hour of rotation at 4°C, and the beads were then washed with wash buffer. Equal volumes of the samples were subjected then to Western blot analysis by using a monoclonal antibody against RhoA. To normalize the amount of protein of the samples, equal volumes of cells lysates were subjected to Western blot analysis by using a β-actin antibody. RhoA 3′ untranslated region (UTR) that contained the predicted binding sites and mutated sequence was chemically synthesized by GENEWIZ (South Plainfield, NJ). The wild-type and mutant of the RhoA 3′ UTR sequence were then subcloned into the luciferase reporter vector purchased from SwitchGear Genomics (catalog number 32011; Carlsbad, CA). Luciferase reporter constructs that contained the wild-type or mutant of 3′ UTR of RhoA were transfected to NCM460–NK-1R cells by using lipofectamine 2000 (Invitrogen, Carlsbad, CA) with miR-31-3p mimic or control mimic. Cell lysates were prepared 24 hours after transfection, and luciferase activity was measured by using the LightSwitch Luciferase Assay Kit (catalog number LS100) from SwitchGear Genomics according to the manufacturer's instructions. In situ hybridization was performed on mice colon tissue from C57BL/6J mice after treatment of DSS or TNBS, or human UC, or CD and control colon tissue, as was previously reported.24Law I.K. Bakirtzi K. Polytarchou C. Oikonomopoulos A. Hommes D. Iliopoulos D. Pothoulakis C. Neurotensin-regulated miR-133alpha is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis.Gut. 2015; 64: 1095-1104Crossref PubMed Scopus (37) Google Scholar The sequence of the probe specific for miR-31-3p (catalog number 39840-15) was as follows: 5′-GATGGCAATATGTTGGCATAGCA-3′. The hybridization was performed according to the manufacturer's instructions by using miRCURY LNA microRNA ISH Optimization Kit (formalin-fixed, paraffin-embedded; Qiagen). DSS, TNBS, and T-cell–transfer colitis were induced as previously described,22Fang K. Zhang S. Glawe J. Grisham M.B. Kevil C.G. Temporal genome expression profile analysis during t-cell-mediated colitis: identification of novel targets and pathways.Inflamm Bowel Dis. 2012; 18: 1411-1423Crossref PubMed Scopus (17) Google Scholar, 24Law I.K. Bakirtzi K. Polytarchou C. Oikonomopoulos A. Hommes D. Iliopoulos D. Pothoulakis C. Neurotensin-regulated miR-133alpha is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis.Gut. 2015; 64: 1095-1104Crossref PubMed Scopus (37) Google Scholar and animal studies were approved by the institutional animal care and use committee. NK-1R knockout (KO) mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and bred in the University of California Los Angeles animal facility. Total RNA from the colonic tissues of TNBS and T-cell–transfer colitis models were purified by use of the miReasy Mini Kit (Qiagen, Valencia, CA) and from the DSS model by use of the lithium chloride precipitation method.25Viennois E. Chen F. Laroui H. Baker M.T. Merlin D. Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA.BMC Res Notes. 2013; 6: 360Crossref PubMed Scopus (104) Google Scholar The body weight of DSS and TNBS colitis mice was monitored daily. Colon tissue sections were observed under 10× objective lens and scored for histopathologic analysis in a double-blinded manner (I.K.M.L. and D.P.) as previously reported.24Law I.K. Bakirtzi K. Polytarchou C. Oikonomopoulos A. Hommes D. Iliopoulos D. Pothoulakis C. Neurotensin-regulated miR-133alpha is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis.Gut. 2015; 64: 1095-1104Crossref PubMed Scopus (37) Google Scholar Six- to 8-week–old male C57BL/6J mice (n = 5 to 8 per group) were injected intracolonicaly with 80 mg/kg TNBS (Sigma-Aldrich) in 30% ethanol or 30% ethanol alone. Mice were sacrificed after 7 days by cervical dislocation, and colonic tissues were collected. To assess the function of miR-31-3p, mice were intracolonicaly administered with 80 mg/kg TNBS, followed by intracolonic administration of 40 μg of miRCURY LNA inhibitor directed against mmu-miR-31-3p or a control inhibitor on days 1, 3, and 5 by using a previously reported approach.24Law I.K. Bakirtzi K. Polytarchou C. Oikonomopoulos A. Hommes D. Iliopoulos D. Pothoulakis C. Neurotensin-regulated miR-133alpha is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis.Gut. 2015; 64: 1095-1104Crossref PubMed Scopus (37) Google Scholar All mice were sacrificed 7 days after TNBS administration. C57BL/6J mice received 2% DSS in the drinking water ad libitum for 5 days (n = 8 mice per group). On days 1, 3, and 5, mice were intracolonicaly injected with 40 μg of miRCURY LNA inhibitor against mmu-miR-31-3p (Qiagen) or control inhibitor as previously reported.24Law I.K. Bakirtzi K. Polytarchou C. Oikonomopoulos A. Hommes D. Iliopoulos D. Pothoulakis C. Neurotensin-regulated miR-133alpha is involved in proinflammatory signalling in human colonic epithelial cells and in experimental colitis.Gut. 2015; 64: 1095-1104Crossref PubMed Scopus (37) Google Scholar In separate studies, on days 1, 3, and 5, mice were intracolonicaly injected with 80 μg of miR-31-3p AgomiR (miR-31-3p mimic, with the sequence as follows: sense strand, 5′-UGCUAUGCCAACAUAUUGCCAU-3′; antisense strand, 5′-GGCAAUAUGUUGGCAUAGCAUU-3′); or control AgomiR (control mimic, with the sequence as follows: sense strand, 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense strand, 5′-ACGUGACACGUUCGGAGAATT-3′) (both from Bioland Scientific LLC, Paramount, CA). The AgomiR miR-31-3p is a chemically modified double-strand miR-31-3p mimic, having its antisense strand modified with two phosphorothioates at the 5′ end, four phosphorothioates at the 3′ end, a 3′ end cholesterol group and full-length nucleotide 2′-methoxy modification. Mice were sacrificed at day 6 by cervical dislocation, and colonic tissues were collected. A disease activity index (DAI) was calculated from measurements of mice weight loss, rectal bleeding, and stool consistency as previously reported.26Dai X. Chen X. Chen Q. Shi L. Liang H. Zhou Z. Liu Q. Pang W. Hou D. Wang C. Zen K. Yuan Y. Zhang C.Y. Xia L. MicroRNA-193a-3p reduces intestinal inflammation in response to microbiota via down-regulation of colonic PepT1.J Biol Chem. 2015; 290: 16099-16115Crossref PubMed Scopus (63) Google Scholar Male (Rag−/−) mice on C57BL/6 background aged 6 to 8 weeks were used as recipients of CD4CD45Rbhi T-cell transfer, as previously reported.22Fang K. Zhang S. Glawe J. Grisham M.B. Kevil C.G. Temporal genome expression profile analysis during t-cell-mediated colitis: identification of novel targets and pathways.Inflamm Bowel Dis. 2012; 18: 1411-1423Crossref PubMed Scopus (17) Google Scholar T-cell transfer mice were sacrificed at weeks 0 and 4 (n = 4 for each group). Harvested colon tissue was cut in half longitudinally with one-half put in RNA later and stored at −80°C and the other half fixed in 10% formaldehyde for histopathologic analysis. The t-test for two-group comparisons and analysis of variance for multiple group comparisons determined statistically significant differences between the experimental groups. For the DAI, polymorphonuclear neutrophil score, and histopathology score, the Kruskal-Wallis test was used to analyze differences between the groups. P < 0.05 was considered statistically significant. All results were expressed as means ± SD. To verify previous gene expression analysis results showing that SP treatment stimulates miR-31-3p expression in NCM460–NK-1R cells,18Fang K. Sideri A. Law I.K. Bakirtzi K. Polytarchou C. Iliopoulos D. Pothoulakis C. Identification of a novel substance P (SP)-neurokinin-1 receptor (NK-1R) microRNA-221-5p inflammatory network in human colonic epithelial cells.Cell Mol Gastroenterol Hepatol. 2015; 1: 503-515Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar 0.1 μmol/L SP was used to stimulate NCM460–NK-1R cells in serum-free medium for 0.5 and 6 hours, as previously described.14Koon H.W. Zhao D. Zhan Y. Moyer M.P. Pothoulakis C. Substance P mediates antiapoptotic responses in human colonocytes by Akt activation.Proc Natl Acad Sci U S A. 2007; 104: 2013-2018Crossref PubMed Scopus (90) Google Scholar, 27Koon H.W. Zhao D. Na X. Moyer M.P. Pothoulakis C. Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes.J Biol Chem. 2004; 279: 45519-45527Crossref PubMed Scopus (101) Google Scholar Because SP modulated the development of colitis by the release of proinflammatory cytokines,6Steinhoff M.S. von Mentzer B. Geppetti P. Pothoulakis C. Bunnett N.W. Tachykinins and their receptors: contributions to physiological control and the mechanisms of disease.Physiol Rev. 2014; 94: 265-301Crossref PubMed Scopus (378) Google Scholar the effects of 10 ng/mL of IL-6, IL-8, TNF-α, interferon-γ, and a cytokine cocktail that included 10 ng/mL of IL-6, IL-8, TNF-α, and interferon-γ were also examined on miR-31-3p expression. Total RNA was isolated and analysis was performed as previously reported.18Fang K. Sideri A. Law I.K. Bakirtzi K. Polytarchou C. Iliopoulos D. Pothoulakis C. Identification of a novel substance P (SP)-neurokinin-1 receptor (NK-1R) microRNA-221-5p inflammatory network in human colonic epithelial cells.Cell Mol Gastroenterol Hepatol. 2015; 1: 503-515Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Real-time PCR demonstrated that miR-31-3p expression in NCM460–NK-1R cells had a twofold increase at 6 hours after SP exposure, although no significant difference was observed 30 minutes after SP stimulation (n = 3) (Figure 1A). Stimulation of NCM460 cells with individual cytokines or the cyto

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