miR-24 Is Elevated in Ulcerative Colitis Patients and Regulates Intestinal Epithelial Barrier Function
2019; Elsevier BV; Volume: 189; Issue: 9 Linguagem: Inglês
10.1016/j.ajpath.2019.05.018
ISSN1525-2191
AutoresArtin Soroosh, Carl R. Rankin, Christos Polytarchou, Zulfiqar A. Lokhandwala, Ami Patel, Lin Chang, Charalabos Pothoulakis, Dimitrios Iliopoulos, David Padua,
Tópico(s)Caveolin-1 and cellular processes
ResumoInflammatory bowel disease is characterized by high levels of inflammation and loss of barrier integrity in the colon. The intestinal barrier is a dynamic network of proteins that encircle intestinal epithelial cells. miRNAs regulate protein-coding genes. In this study, miR-24 was found to be elevated in colonic biopsies and blood samples from ulcerative colitis (UC) patients compared with healthy controls. In the colon of UC patients, miR-24 is localized to intestinal epithelial cells, which prompted an investigation of intestinal epithelial barrier function. Two intestinal epithelial cell lines were used to study the effect of miR-24 overexpression on barrier integrity. Overexpression of miR-24 in both cell lines led to diminished transepithelial electrical resistance and increased dextran flux, suggesting an effect on barrier integrity. Overexpression of miR-24 did not induce apoptosis or affect cell proliferation, suggesting that the effect of miR-24 on barrier function was due to an effect on cell–cell junctions. Although the tight junctions in cells overexpressing miR-24 appeared normal, miR-24 overexpression led to a decrease in the tight junction–associated protein cingulin. Loss of cingulin compromised barrier formation; cingulin levels negatively correlated with disease severity in UC patients. Together, these data suggest that miR-24 is a significant regulator of intestinal barrier that may be important in the pathogenesis of UC. Inflammatory bowel disease is characterized by high levels of inflammation and loss of barrier integrity in the colon. The intestinal barrier is a dynamic network of proteins that encircle intestinal epithelial cells. miRNAs regulate protein-coding genes. In this study, miR-24 was found to be elevated in colonic biopsies and blood samples from ulcerative colitis (UC) patients compared with healthy controls. In the colon of UC patients, miR-24 is localized to intestinal epithelial cells, which prompted an investigation of intestinal epithelial barrier function. Two intestinal epithelial cell lines were used to study the effect of miR-24 overexpression on barrier integrity. Overexpression of miR-24 in both cell lines led to diminished transepithelial electrical resistance and increased dextran flux, suggesting an effect on barrier integrity. Overexpression of miR-24 did not induce apoptosis or affect cell proliferation, suggesting that the effect of miR-24 on barrier function was due to an effect on cell–cell junctions. Although the tight junctions in cells overexpressing miR-24 appeared normal, miR-24 overexpression led to a decrease in the tight junction–associated protein cingulin. Loss of cingulin compromised barrier formation; cingulin levels negatively correlated with disease severity in UC patients. Together, these data suggest that miR-24 is a significant regulator of intestinal barrier that may be important in the pathogenesis of UC. Inflammatory bowel disease (IBD) is comprised broadly of two subcategories: Crohn disease (CD) and ulcerative colitis (UC). CD and UC are chronic inflammatory diseases of the gastrointestinal tract, currently affecting >1.6 million Americans.1Kappelman M.D. Rifas-Shiman S.L. Porter C.Q. Ollendorf D.A. Sandler R.S. Galanko J.A. Finkelstein J.A. Direct health care costs of Crohn's disease and ulcerative colitis in US children and adults.Gastroenterology. 2008; 135: 1907-1913Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar With a complex etiology and limited lasting medical treatments, IBD patients may experience a lifetime of significant symptoms, which include severe diarrhea, bleeding, and vomiting. One pathophysiological feature of IBD patients is a loss of the intestinal barrier, even in areas that contain an intact epithelium.2Schmitz H. Barmeyer C. Fromm M. Runkel N. Foss H.D. Bentzel C.J. Riecken E.O. Schulzke J.D. Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis.Gastroenterology. 1999; 116: 301-309Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 3Pastorelli L. De Salvo C. Mercado J.R. Vecchi M. Pizarro T.T. Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics.Front Immunol. 2013; 4: 280Crossref PubMed Scopus (314) Google Scholar The intestinal barrier comprises a single layer of epithelial cells bound together by proteins that traverse the plasma membrane. This selectively permeable barrier compartmentalizes bacteria and other toxins to the lumen while allowing ions, nutrients, and water to be absorbed. Loss of the intestinal barrier leads to the exposure of luminal components to the underlying tissue. A dysfunctional barrier combined with an aberrant immune response are thought to be major risk factors for IBD.4Vindigni S.M. Zisman T.L. Suskind D.L. Damman C.J. The intestinal microbiome, barrier function, and immune system in inflammatory bowel disease: a tripartite pathophysiological circuit with implications for new therapeutic directions.Therap Adv Gastroenterol. 2016; 9: 606-625Crossref PubMed Scopus (116) Google Scholar The protein networks enabling intestinal barrier function are known as cell-cell junctions, which include the tight junction and adherens junction. Cell-cell junctions can confer strength or pore-forming abilities to the barrier.5Laukoetter M.G. Bruewer M. Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex.Curr Opin Gastroenterol. 2006; 22: 85-89Crossref PubMed Scopus (203) Google Scholar A major transmembrane protein in the adherens junction is E-cadherin, whereas tight junction transmembrane proteins include the junctional adhesion molecule family and claudin family of proteins.5Laukoetter M.G. Bruewer M. Nusrat A. Regulation of the intestinal epithelial barrier by the apical junctional complex.Curr Opin Gastroenterol. 2006; 22: 85-89Crossref PubMed Scopus (203) Google Scholar These transmembrane proteins are connected to cytosolic adaptor proteins, which include the zona occludens protein family and cingulin, which, in turn, connect to the actin cytoskeleton of the cell.6Matter K. Balda M.S. Signalling to and from tight junctions.Nat Rev Mol Cell Biol. 2003; 4: 225-236Crossref PubMed Scopus (714) Google Scholar This connection between cells to the underlying cellular cytoskeleton confers rigidity and strength to the junction.7Bachir A.I. Horwitz A.R. Nelson W.J. Bianchini J.M. Actin-based adhesion modules mediate cell interactions with the extracellular matrix and neighboring cells.Cold Spring Harb Perspect Biol. 2017; 9 (a023234)Crossref PubMed Scopus (82) Google Scholar However, junctional complexes are also highly dynamic.8Capaldo C.T. Nusrat A. Claudin switching: physiological plasticity of the tight junction.Semin Cell Dev Biol. 2015; 42: 22-29Crossref PubMed Scopus (71) Google Scholar To remove damaged proteins, these complexes are continually internalized and recycled back to the membrane or degraded.9Stamatovic S.M. Johnson A.M. Sladojevic N. Keep R.F. Andjelkovic A.V. Endocytosis of tight junction proteins and the regulation of degradation and recycling.Ann N Y Acad Sci. 2017; 1397: 54-65Crossref PubMed Scopus (58) Google Scholar To replace degraded proteins, junctional proteins are constantly synthesized by intestinal epithelial cells. When any of these aspects of the junctional dynamics are dysregulated, barrier dysfunction is likely to occur. One of the pathways the intestinal epithelium uses to regulate the levels of tight junction proteins is by using miRNAs.10Ikemura K. Iwamoto T. Okuda M. MicroRNAs as regulators of drug transporters, drug-metabolizing enzymes, and tight junctions: implication for intestinal barrier function.Pharmacol Ther. 2014; 143: 217-224Crossref PubMed Scopus (45) Google Scholar miRNAs are small noncoding RNAs that repress gene expression by binding to complementary sequences on mRNAs. Multiple studies have assayed alterations in miRNAs in IBD patients using unbiased methods.11Kalla R. Ventham N.T. Kennedy N.A. Quintana J.F. Nimmo E.R. Buck A.H. Satsangi J. MicroRNAs: new players in IBD.Gut. 2015; 64: 504-517Crossref PubMed Scopus (187) Google Scholar A subset of these miRNAs, including miR-223 and miR-301a, regulate intestinal epithelial barrier function.12Wang H. Chao K. Ng S.C. Bai A.H. Yu Q. Yu J. Li M. Cui Y. Chen M. Hu J.F. Zhang S. Pro-inflammatory miR-223 mediates the cross-talk between the IL23 pathway and the intestinal barrier in inflammatory bowel disease.Genome Biol. 2016; 17: 58Crossref PubMed Scopus (115) Google Scholar, 13He C. Yu T. Shi Y. Ma C. Yang W. Fang L. Sun M. Wu W. Xiao F. Guo F. Chen M. Yang H. Qian J. Cong Y. Liu Z. MicroRNA 301A promotes intestinal inflammation and colitis-associated cancer development by inhibiting BTG1.Gastroenterology. 2017; 152: 1434-1448.e15Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar One miRNA consistently shown to be elevated in IBD patients is miR-24, yet the possible role for miR-24 in IBD is unclear. Two studies have demonstrated 2- and 17-fold changes in miR-24 in colonic biopsies from actively inflamed UC patients compared with controls.14Coskun M. Bjerrum J.T. Seidelin J.B. Troelsen J.T. Olsen J. Nielsen O.H. miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic biomarkers in ulcerative colitis.World J Gastroenterol. 2013; 19: 4289-4299Crossref PubMed Scopus (69) Google Scholar, 15Wu 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 In addition, two studies have observed a twofold increase in blood-associated miR-24 in UC active patients compared with controls.16Iborra M. Bernuzzi F. Correale C. Vetrano S. Fiorino G. Beltran B. Marabita F. Locati M. Spinelli A. Nos P. Invernizzi P. Danese S. Identification of serum and tissue micro-RNA expression profiles in different stages of inflammatory bowel disease.Clin Exp Immunol. 2013; 173: 250-258Crossref PubMed Scopus (99) Google Scholar, 17Krissansen G.W. Yang Y. McQueen F.M. Leung E. Peek D. Chan Y.C. Print C. Dalbeth N. Williams M. Fraser A.G. Overexpression of miR-595 and miR-1246 in the sera of patients with active forms of inflammatory bowel disease.Inflamm Bowel Dis. 2015; 21: 520-530Crossref PubMed Scopus (39) Google Scholar In the results presented herein, using semiquantitative real-time PCR, miR-24 levels were found to be elevated in biopsies and whole blood from UC actively inflamed patients. To better define a possible role for miR-24 in the pathogenesis IBD, it was determined that miR-24 was expressed by intestinal epithelial cells in UC patients. When overexpressed in vitro, intestinal barriers in target epithelial cells failed to establish. Although cells overexpressing miR-24 grew normally, the protein levels of the tight junction adaptor protein cingulin were significantly reduced. When cingulin was down-regulated in intestinal epithelial cells, barrier formation was impaired. Therefore, targeting the miR-24–cingulin axis may represent one pathway to strengthen the intestinal barrier and reduce inflammation in UC patients. Colonic tissue biopsies and blood used in the miRNA analyses were obtained from the University of California Los Angeles Center for Inflammatory Bowel Diseases and the G Oppenheimer Center for Neurobiology of Stress and Resilience under the following institutional review board–approved protocols: numbers 12-00420, 13-000537, and 11-000199. Colon tissue biopsies from patients who met diagnostic Rome III criteria18Longstreth G.F. Thompson W.G. Chey W.D. Houghton L.A. Mearin F. Spiller R.C. Functional bowel disorders.Gastroenterology. 2006; 130: 1480-1491Abstract Full Text Full Text PDF PubMed Scopus (3894) Google Scholar for IBS and healthy control subjects were obtained during flexible sigmoidoscopy (at 30 cm) after tap water enemas. Colonic tissue used for the mild and severe inflammation, on the basis of pathology report analysis, was obtained from the University of California Los Angeles Center for Inflammatory Bowel Diseases under institutional review board–approved protocol 18-000209. Specimens were flash frozen in liquid nitrogen, and RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA). A Ficoll gradient (Roche, Basel, Switzerland) was used to isolate peripheral blood mononuclear cells from whole blood, according to the manufacturer's instructions. All University of California Los Angeles samples were obtained after a written informed consent was provided. Human UC patient colonic tissue used for the microarray analysis was obtained from Origene (Rockville, MD). Diagnoses were confirmed by an independent set of pathologists associated with Origene. Microarray Origene samples were analyzed, as previously described.19Padua D. Mahurkar-Joshi S. Law I.K. Polytarchou C. Vu J.P. Pisegna J.R. Shih D. Iliopoulos D. Pothoulakis C. A long noncoding RNA signature for ulcerative colitis identifies IFNG-AS1 as an enhancer of inflammation.Am J Physiol Gastrointest Liver Physiol. 2016; 311: G446-G457Crossref PubMed Scopus (73) Google Scholar Samples were obtained through institutional review board protocols and with documented patient consent, all from accredited US-based medical institutions. The microarray data are accessible through the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo; accession number GSE77013). Caco-2 and T84 cells (ATCC, Manassas, VA) were grown at 37°C with 5% CO2. The cells were grown in Dulbecco's modified Eagle's medium (Corning, Corning, NY) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO) and 1% penicillin-streptomycin (Corning). To subculture cells, confluent monolayers were washed with phosphate-buffered saline (PBS) and incubated with 0.25% trypsin with 1 mmol/L EDTA (Thermo Fisher Scientific). Suspended cells were centrifuged at 233 × g for 5 minutes. Both Caco-2 and T84 cells were split at a 1:5 ratio. Transepithelial electrical resistance (TEER) was used as a measurement of barrier integrity. A total of 100,000 cells were plated on 6.5-mm transwell inserts that had 0.4-μm pores (Thermo Fisher Scientific). A TC20 Automated cell counter was used to determine cell numbers (Bio-Rad Laboratories, Hercules, CA). Each subsequent day, TEER was measured using a dual electrode connected to an epithelial volt/ohm meter (World Precision Instruments, Sarasota, FL). On the last day of the experiment, dextran flux was measured. A solution containing 50 μg fluorescein isothiocyanate–labeled 4-kDa dextran and 50 μg of AlexaFluor 555–labeled 10-kDa dextran (Thermo Fisher Scientific), diluted in Hanks' balanced salt solution, was used (Corning). The dextran solution (100 μL) was added to the top of the transwell; and every 2 hours, for 6 total hours, 50 μL of the media in the bottom of the transwell was sampled. A Synergy HT plate reader was used to measure fluorescence (BioTek Instruments, Winooski, VT). To obtain absolute amount of dextran flux, a standard curve was used. Cells were transfected during plating, and all transfection reactions were performed in Opti-MEM media (Gibco). Seventy micromolar lipofectamine RNAiMax reagent was used, according to the manufacturer (Invitrogen, Carlsbad, CA). For overexpression experiments, both an miR-24 precursor and an miRNA precursor negative control were used at the concentration of 50 nmol/L (PM10737 and AM17110, respectively; Ambion, Austin, TX). For inhibition experiments, a chemically modified20Soroosh A. Koutsioumpa M. Pothoulakis C. Iliopoulos D. Functional role and therapeutic targeting of microRNAs in inflammatory bowel disease.Am J Physiol Gastrointest Liver Physiol. 2018; 314: G256-G262Crossref PubMed Scopus (41) Google Scholar miR-24 antisense oligonucleotide and negative control were used at the concentration of 50 nmol/L (YC10201383-FZA and YC10202119-FZA; Qiagen, Hilden, Germany). For siRNA experiments, both the cingulin siRNA and negative control were used at the concentration of 50 nmol/L (S33237 and 4390843, respectively; Ambion). To produce miR-24 overexpression of 15-fold, 50 pmol/L control precursor and miR-24 precursors were used. An miRNeasy kit (Qiagen) was used to extract and purify mRNA and miRNA from tissue or cells grown on transwells, according to the manufacturer's instructions. To harvest, transwell cells were first washed twice with PBS and then the membrane was excised. The membrane was then submerged in Qiazol before storing lysed cells at −80°C (Qiagen). All other purification steps were performed according to the manufacturer's instructions. To generate cDNA from mRNA, an iScript cDNA synthesis kit was used (Bio-Rad Laboratories). mRNA (500 ng) was reverse transcribed into cDNA, according to the manufacturer's instructions. A miRCURY LNA RT kit (Qiagen) was used to generate cDNA from 100 ng of miRNA, according to the manufacturer's instructions. A CFX384 real-time PCR system was used to amplify and detect SYBR Green–mediated signal (Bio-Rad Laboratories). To measure miR-24, a miRCURY LNA miRNA PCR assay primer was used (YP00204260; Qiagen). The housekeeping primers for miRNA experiments were U6 small nuclear RNA and 5S rRNA (YP00203907 and YP00203906, respectively; Qiagen). Cingulin (CGN), E-cadherin (CDH1), and claudin-7 (CLDN7) primers were purchased from Integrated DNA Technologies (Coralville, IA). β-Actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as housekeeping genes for mRNA analysis. The primer sequences are as follows: CGN, 5′-GCAACAAGGAGCTCCAGAAC-3′ (forward) and 5′-CCCTGGACATGTTTCAGCTT-3′ (reverse); CDH1, 5′-GGATTGCAAATTCCTGCCATTC-3′ (forward) and 5′-AACGTTGTCCCGGGTGTCA-3′ (reverse); CLDN7, 5′-GGATGATGAGCTGCAAAATG-3′ (forward) and 5′-CACCAGGGAGACCACCATTA-3′ (reverse); ACTB, 5′-CCCAGCACAATGAAGATCAA-3′ (forward) and 5′-ACATCTGCTGGAAGGTGGAC-3′ (reverse); GAPDH, 5′-ATGTTCGTCATGGGTGTGAA-3′ (forward) and 5′-GGTGCTAAGCAGTTGGTGGT-3′ (reverse); PLEKHA7, 5′-TAAAGACAGCCGAGAAGAAG-3′ (forward) and 5′-TGTCGGCACTGAAGTAGTAG-3′ (reverse); CLDN2, 5′-TGGCCTCTCTTGGCCTCCAACTTGT-3′ (forward) and 5′-TTGACCAGGCCTTGGAGAGCTC-3′ (reverse); CLDN3, 5′-CATCACGTCGCAGAACATCT-3′ (forward) and 5′-AGCAGCGAGTCGTACACCTT-3′ (reverse); and KRT8, 5′-CGAGGATATTGCCAACCGCAG-3′ (forward) and 5′-CCTCAATCTCAGCCTGGAGCC-3′ (reverse). The comparative Ct method was used to calculate fold change relative to the housekeeping genes.21Schmittgen T.D. Livak K.J. Analyzing real-time PCR data by the comparative C(T) method.Nat Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (17280) Google Scholar Confluent monolayers were washed twice with PBS and then the membrane was excised. Membranes were then submerged in 1× Lammeli sample buffer (Bioland Scientific, Paramount, CA) supplemented with 10% 2-mercaptoethanol (Sigma). Samples were left on ice for 30 minutes before slowly passing lysates through a 25-gauge needle. Lysates were then incubated at 95°C for 5 minutes, and then 10 μL of each sample was added to a 4% to 20% SDS–containing polyacrylamide gel (Bio-Rad Laboratories). A Trans-Blot Turbo system (Bio-Rad Laboratories) was used to transfer proteins onto a polyvinylidene difluoride membrane. Membranes were then blocked for 1 hour at room temperature in 5% milk in PBS with 0.01% Tween-20. Membranes were then incubated with primary antibody diluted in 5% bovine serum albumin overnight at 4°C. After five 10-minute washes with PBS with 0.01% Tween-20, the membrane was then incubated in secondary antibody diluted in 5% milk in PBS with 0.01% Tween-20 for 1 hour at room temperature. After five 10-minute washes with PBS with 0.01% Tween-20, protein was visualized using a Clarity enhanced chemiluminescence kit (Bio-Rad Laboratories) and ChemiDoc Touch Imager (Bio-Rad Laboratories). The cingulin (117796) and claudin-2 (53032) antibodies were from Abcam (Cambridge, UK). The claudin-7 (349100) and E-cadherin (clone 4A2C7) antibodies were from Invitrogen. The claudin-3 (SAB4500435) and PLEKHA7 (HPA038610) antibodies were from Sigma-Aldrich (St. Louis, MO). The GAPDH antibody was from Cell Signaling Technologies (Danvers, MA; clone 14C10). Horseradish peroxidase–conjugated secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA). To measure cell growth, 50,000 cells were plated in 24-well plates in duplicate. Each day after seeding, cells were trypsinized and a TC20 Automated Cell Counter (Bio-Rad Laboratories) was used to count the cells. To measure cell proliferation, 10,000 cells were plated in 96-well plates and a bromodeoxyuridine assay kit was performed according to the manufacturer (Cell Signaling). To measure apoptosis, a Caspase GLO 3/7 assay (Promega, Madison, WI) was used using the manufacturer's instructions. Briefly, cells grown on transwells were washed twice with PBS, and the membrane was excised and submerged in the buffer provided by the kit. After 15 minutes of incubation with shaking, a BioTek plate reader was used to measure luminescence from 50 μL of lysate. As a positive control, staurosporine (Tocris, Bristol, UK) was added to the bottom of the transwell at a concentration of 2 μmol/L for 8 hours before conducting the assay. Lysis buffer was used to measure background luminescence. As an alternate method to measure apoptosis, a Click-it terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) kit (Invitrogen) was performed on cells at the end point of TEER experiments, according to the manufacturer. DAPI (Invitrogen) was used as the nuclei stain. Staurosporine was used as a positive control for TUNEL assays and was used at the concentration of 2 μmol/L for a total of 3 hours for Caco-2 cells or 16 hours for T84 cells. Cells grown on transwells were washed twice with PBS and then fixed in 100% cold methanol for 20 minutes at −20°C. After two subsequent washes with PBS, transwells were blocked in 5% bovine serum albumin in PBS for 1 hour at room temperature. The transwell membranes were then excised from the transwell and incubated with mouse anti–zona occludens protein 1 (339100; Invitrogen) overnight at 4°C in a humidified container. Claudin-2, claudin-3, and claudin-7 antibodies were the same as used for Western blot analyses. The following day, after three washes in PBS, the transwell was incubated with a goat anti-mouse fluorescently labeled 488 antibody for 1 hour at room temperature (Invitrogen). After three washes with PBS, nuclei were stained with DAPI for 1 minute (Invitrogen), and Prolong Gold (Invitrogen) was used to mount membranes onto slides. Slides were imaged using an upright microscope (Zeiss, Oberkochen, Germany). A double-DIG labeled miRCURY detection probe for miR-24 (YD00617308-BCG; Qiagen) was used, as previously described.22Polytarchou C. Hommes D.W. Palumbo T. Hatziapostolou M. Koutsioumpa M. Koukos G. van der Meulen-de Jong A.E. Oikonomopoulos A. van Deen W.K. Vorvis C. Serebrennikova O.B. Birli E. Choi J. Chang L. Anton P.A. Tsichlis P.N. Pothoulakis C. Verspaget H.W. Iliopoulos D. MicroRNA214 is associated with progression of ulcerative colitis, and inhibition reduces development of colitis and colitis-associated cancer in mice.Gastroenterology. 2015; 149: 981-992.e11Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Statistical differences between groups were evaluated using an unpaired two-tailed t-test. Graphing was completed using GraphPad Prism version 6 (GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered statistically significant. Multiple studies have seen that miR-24 is elevated in the serum and colon of active inflamed ulcerative colitis (UC-active) patients.14Coskun M. Bjerrum J.T. Seidelin J.B. Troelsen J.T. Olsen J. Nielsen O.H. miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic biomarkers in ulcerative colitis.World J Gastroenterol. 2013; 19: 4289-4299Crossref PubMed Scopus (69) Google Scholar, 15Wu 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, 16Iborra M. Bernuzzi F. Correale C. Vetrano S. Fiorino G. Beltran B. Marabita F. Locati M. Spinelli A. Nos P. Invernizzi P. Danese S. Identification of serum and tissue micro-RNA expression profiles in different stages of inflammatory bowel disease.Clin Exp Immunol. 2013; 173: 250-258Crossref PubMed Scopus (99) Google Scholar, 17Krissansen G.W. Yang Y. McQueen F.M. Leung E. Peek D. Chan Y.C. Print C. Dalbeth N. Williams M. Fraser A.G. Overexpression of miR-595 and miR-1246 in the sera of patients with active forms of inflammatory bowel disease.Inflamm Bowel Dis. 2015; 21: 520-530Crossref PubMed Scopus (39) Google Scholar Although many studies have analyzed the effects of miR-24 on oncogenesis, few studies have profiled the function of miR-24 in the context of IBD. To validate these microarrays and RNA-sequencing studies, human colonic biopsies were obtained from patients undergoing colonoscopy procedures and real-time PCR was performed. Healthy controls were compared with UC-active patients, UC-inactive patients, IBS patients, and CD patients. It was observed that miR-24 levels were elevated sevenfold in UC-active patients (P < 0.0001) compared with the other groups, who had similar levels of miR-24 (Figure 1A). In UC-active patients, the elevated levels of miR-24 do not appear to be exclusive to colonic tissue as miR-24 was also elevated fourfold in the blood of UC-active patients compared with healthy controls and CD patients (Figure 1B). To determine the subset of cells that express miR-24, in situ hybridization was performed on colonic sections from UC patients and on healthy controls. It was observed that miR-24 localized to intestinal epithelial crypts in the colon (Figure 1C). These results indicate that in UC patients, miR-24 is specifically elevated in the intestinal epithelium and this elevation persists in the bloodstream. Given the localization of miR-24 to the colonic epithelium, which functions to generate a selectively permeable barrier that lines the gut, the effects of miR-24 on barrier formation were tested. If miR-24 regulates the barrier and miR-24 is altered in IBD, this alteration could contribute to the pathogenesis of IBD. Two barrier-forming human cell lines of intestinal epithelial origin were used, Caco-2 and T84 cells. As miR-24 is elevated in UC-active patients (Figure 1, A and B), an miR-24 mimic was transfected into Caco-2 and T84 cells. A 40-fold increase or greater in miR-24 was observed in both cell lines compared with control mimic–treated cells (Figure 2A). After plating both control or miR-24–overexpressing cells in transwells, two methods to test barrier function were then performed. As tight junctions form a barrier against ion flow, an electrode can be used to measure TEER (Figure 2B). Another method to test barrier function is a dextran flux assay. In this assay, fluorescently labeled dextran is added to the upper chamber of the transwell and the amount of dextran in the lower chamber is measured bihourly (Figure 2B). On every experiment performed, control cells gained TEER at an exponential rate. However, cells overexpressing miR-24 had diminished TEER at all time points (Figure 2C). To test if these alterations in barrier function occur at lower levels of overexpression, oligonucleotides were diluted to obtain a 15-fold overexpression of miR-24. Overexpression was still sufficient to impair barrier function (Supplemental Figure S1, A and B). For both T84 and Caco-2 cells, miR-24 overexpression significantly increased the amount of 4- and 10-kDa dextran flux at all time points (Figure 2, D and E). In control Caco-2 cells, some dextran fluxed over time, whereas T84 cells were almost completely impermeable to dextran flux when a high TEER was established (Figure 2, D and E). These data suggest that miR-24 regulates the formation of the intestinal epithelial barrier. To test if miR-24 inhibition could accelerate barrier formation, antisense miR-24 oligonucleotides were added to the cells during barrier formation. Although antisense miR-24 oligonucleotides caused a dramatic decrease in miR-24 in both Caco-2 and T84 cells, no alterations were observed in the establishment of barrier function, as measured by TEER (Figure 2, F and G). To understand the potential mechanism for the disruption of barrier function in cells with elevated miR-24, assays for apoptosis were performed. It would be detrimental to the establishment of a barrier if apoptosis is increased. As a first measurement of apoptosis, a caspase 3/7 activity assay was performed on Caco-2 and T84 cells with altered TEERs. Overexpression of miR-24 in Caco-2 cells reduced apoptosis, as demonstrated by decreased caspase 3/7 activity. In T84 cells, no difference in caspase 3/7 activity was observed between the two groups (Figure 3A). As a positive control, cells were treated with staurosporine, which induces apoptosis in caspase-independent and caspase-dependent mechanisms,23Belmokhtar C.A. Hillion J. Segal-Bendirdjian E. Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms.Oncogene. 2001; 20: 3354-3362Crossref PubMed Scopus (328) Google Scholar as demonstrated by a dramatic increase in caspase 3/7 activity (Figure 3A). As an alternate method to measure apoptosis, a TUNEL assay was performed on transwell inserts from Caco-2 and T84 cells. Both control miR
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