Xiao-2018-Long Noncoding RNA uc.173 Promotes R.pdf

Long Noncoding RNA uc.173 Promotes Renewal of the IntestinalMucosa by Inducing Degradation of MicroRNA 195Lan Xiao,1,2Jing Wu,1,2Jun-Yao Wang,1,2Hee Kyoung Chung,1,2Sudhakar Kalakonda,1,2Jaladanki N.Rao,1,2Myriam Gorospe,3and Jian-Ying Wang1,2,41Cell Biology Group,Department of Surgery,University of Maryland School of Medicine,Baltimore,Maryland;2BaltimoreVeterans Affairs Medical Center,Baltimore,Maryland;3Laboratory of Genetics and Genomics,National Institute on Aging-IRP,National Institutes of Health,Baltimore,Maryland;4Department of Pathology,University of Maryland School of Medicine,Baltimore,MarylandBACKGROUND AND AIMS:The mammalian intestinal epithe-lium self-renews rapidly and homeostasis is preserved viatightly controlled mechanisms.Long noncoding RNAs tran-scribed from ultraconserved regions(T-UCRs)control differentcell functions,but little is known about their role in maintainingthe integrity of the intestinal epithelium.We searched forT-UCRs that regulate growth of the intestinal mucosa andinvestigated the mechanism by which T-UCR uc.173 regulatesepithelial renewal.METHODS:C57BL/6J mice were deprivedof food for 48 hours in fasting experiments.Some mice weregiven intraperitoneal injections of a plasmid encoding LNA-anti-uc.173,to knock down endogenous uc.173.For studiesusing organoids,primary enterocytes were isolated from theintestine and transfected with the uc.173 transgene to increaseuc.173 levels.Intestinal epithelial cells(Caco-2 and IEC-6 lines)were transfected with LNA-anti-uc.173 or uc.173 transgene.Wequantified intestinal epithelial renewal based on BrdU incor-poration,villus height and crypt depth,and cell number.Theassociation of uc.173 with microRNA 195(miRNA195)wasdetermined by RNA pull-down assays.RESULTS:Genome-wideprofile analyses identified 21 T-UCRs,including uc.173,thatwere differentially expressed between intestinal mucosa offasted vs non-fasted mice.Increasing levels of uc.173 byexpression of a transgene increased growth of intestinalepithelial cells and organoids.Decreasing uc.173 levels by LNA-anti-uc.173 in mice reduced renewal of the intestinal epithe-lium.We found that uc.173 interacted directly with the primarytranscript of miRNA195,leading to miRNA195 degradation.CONCLUSIONS:In analyses of intestinal epithelial cells andmice,we identified uc.173 noncoding RNA that regulatesgrowth of the intestinal mucosa and stimulates intestinalepithelial renewal by reducing levels of miRNA195.Keywords:lncRNAs;IEC;Post-transcriptionalRegulation;Mouse Model.The epithelium of the mammalian intestinal mucosaundergoes rapid and constant renewal throughoutthe life of the organism,and acts as a physical barrier be-tween the luminal microbiota and the body.Intestinal stemand progenitor cells drive this process and replicateAbbreviations used in this paper:BrdU,bromodeoxyuridine;DFMO,D,L-a-difluoro-methylornithine;FISH,fluorescence in situ hybridization;IEC,intestinal epithelial cell;LNA,locked nucleic acid;lncRNA,longnoncoding RNAs;Luc,luciferase;microRNA,miRNA;PCR,polymerasechain reaction;pri-miRNA,primary miRNAs;Q,quantitative;RT,reversetranscription;SEM,standard error of the mean;T-UCR,transcribed fromUCR;UCR,ultraconserved region.Most current article 2018 by the AGA Institute0016-5085/$36.00https:/doi.org/10.1053/j.gastro.2017.10.009Gastroenterology 2018;154:599611BASIC ANDTRANSLATIONAL ATperpetuallyintheproliferativezonenearthebaseofthecrypts,while the newly divided cells differentiate to all the maturecell types as they migrate up along the crypt-villus axis toreplace lost cells.1,2The human small intestinal epitheliumgoes through approximately w1011mitoses daily,and thisrapid turnover rate is tightly controlled by numerous intra-cellularand extracellular factors atmultiplelevelstomaintaintissue homeostasis.3Food in the intestinal lumen is one of thestrongest stimulants of mucosal growth,whereas food star-vation in patients with total parenteral nutrition results inatrophy of the intestinal mucosa.4,5Intestinal mucosal growthand repair also require the supply of polyamines to thedividing cells in the crypts;by contrast,polyamine depletionrepresses mucosal renewal and delays healing.6,7In responseto stressful environments,the integrity of the intestinalepitheliumispreservedbyrapidandadaptivechangesingeneexpression patterns of intestinal epithelial cells(IECs),whichmodulate cell proliferation,migration,differentiation,andapoptosis.1,3Inhibition of intestinal mucosal growth occurscommonly in various pathologies and disrupts the epitheliumintegrity,leading to bacterial translocation and predisposingthe mucosa to destructive inflammation.8LongnoncodingRNAs(lncRNAs)aredefinedastranscribed RNAs spanning 200 nt in length that controlevery level of gene expression,including chromatin remodel-ing,transcriptional and post-transcriptional processes,andprotein metabolism.9The differential expression of lncRNAsduring disease progression suggests a significant role forlncRNAs in human pathologies.9,10Recently,several lines ofevidence indicate that the interplay between lncRNAs andmicroRNAs(miRNAs)can affect gene regulatory programsgoverning physiologic processes and human diseases.9,11Forexample,lncRNAs can be primary miRNAs(pri-miRNAs),function as molecular sponges for miRNAs,and/or controlpri-miRNA processing.1113Ultraconserved regions(UCRs)representthe conservedsequences ofthehumangenomethatare likely to be functional but do not encode proteins,and arethus named“dark matter”of the human genome.14RNAstranscribed from UCRs(T-UCRs)are a class of intriguinglncRNAsthatoriginatefromgenomicelementslocatedin bothintra-and intergenic regions with near-perfect evolutionaryconservation in many mammalian genomes.The striking evolutionary retention of UCRs suggests thatthey may have a key function in physiology and disease.Accordingly,the expression of several T-UCRs is altered invarious cancers,15,16and the uc.283A has been identified asbeing subject to epigenetic inactivation by CpG island hyper-methylation.17However,no reports so far have addressed theinvolvement of T-UCRs in the regulation of intestinal mucosagrowthandadaptation.Here,wepresentevidencethatT-UCRsareintimatelyimplicatedintheregulationofintestinalmucosarenewal through a novel network of functional interactionbetween lncRNAs and miRNAs.The expression patterns ofT-UCRs in the intestinal epithelium exhibited distinct signa-tures in response to food starvation,and the T-UCR uc.173stimulated growth of the small intestinal mucosa.Moreover,uc.173 interacted with and destabilized the pri-miR-195transcript.BecauseT-UCRsareabsolutelyconservedbetween orthologous regions of the human,rat,and mousegenomes,these findings provide a strong rationale for devel-oping therapeutic strategies directed at uc.173 and/or itsinteraction with miRNA195 to stimulate the regeneration ofthe intestinal mucosa and its adaptation in surgical intensivecare patients supported with total parenteral nutrition.MethodsChemicals and Cell CultureCaco-2 and IEC-6 cells were purchased from the AmericanType Culture Collection(Manassas,VA)and were maintained instandardcultureconditions.18Antibodiesrecognizingp53,JunD,HuR,AUF1,DROSHA,and GAPDH were obtained from SantaCruz Biotechnology(Santa Cruz,CA)and BD Biosciences(Sparks,MD).The secondary antibody conjugated to horse-radish peroxidase was obtained from Sigma(St.Louis,MO).Locked nucleic acid(LNA)-modified anti-uc.173 oligonucleo-tides that antagonize uc.173 and control LNA-scrambled oligo-nucleotideswerecustom-generatedbyExiqon(Vadbaek,Denmark).Pre-miR miRNA precursor of miRNA195(pre-miR-195)and anti-miR inhibitor of miRNA195(anti-miR-195)werepurchased from Ambion(Austin,TX).Biotin-labeled mature andpri-miR-195 were custom-made by Dharmacon(Lafayette,CO).Murine StudiesC57BL/6J mice(male and female,69 weeks old)werepurchased from The Jackson Laboratory(Bar Harbor,ME)andhoused in a pathogen-free animal facility at the Baltimore VAMedical Center(Baltimore,MD).All animal experiments wereconducted in accordance with National Institutes of Healthguidelines and were approved by the Institutional Animal CareandUseCommitteeofUniversityMarylandSchoolofMedicineandBaltimoreVAhospital.Animalsweredeprivedoffoodbutallowedfree access to tap water for 48 hours in the fasting model.Instudies of LNA-mediated uc.173 silencing,mice were injectedintraperitoneally with LNA-anti-uc.173(500 mg/100-g bodyEDITORS NOTESBACKGROUND AND CONTEXTRNAs transcribed from ultraconserved regions(T-UCRs)are a novel class of long noncoding RNAs.This studysearched for T-UCRs in the intestinal mucosa andinvestigated the mechanism by which T-UCR uc.173enhances the epithelial renewal.NEW FINDINGSTwenty-one T-UCRs,including uc.173,were differentiallyexpressed in response to food starvation.Increaseduc.173 stimulated growth of the small intestinal mucosaby destabilizing the primary microRNA 195.LIMITATIONSThis study fully elucidated the role of uc.173 only,whilethe functions of other T-UCRs in regulation of themucosal growth remain unknown.IMPACTThese findings provide a strong rationale for developingtherapeuticstostimulatetheregenerationoftheintestinal mucosa in intensive care patients supportedwith total parenteral nutrition.600Xiao et alGastroenterology Vol.154,No.3BASIC ANDTRANSLATIONAL ATwt/day)or control LNA-scrambled oligonucleotides for 4 consec-utive days.19On day 5,a 4-cm small intestinal segment taken0.5 cm distal to the ligament of Trietz was removed,and the mu-cosawasscrapedwithaglassslideforvariousmeasurements.20,21Intestinal Organoid CultureIsolation and culture of primary enterocytes were con-ducted following the method described previously.2,22Briefly,primary crypts were released from the small intestinal mucosain mice;isolated crypts were mixed with matrigel and culturedin Advanced Dulbeccos modified Eagle medium/F12 medium.The levels of DNA synthesis were measured by bromodeox-yuridine(BrdU)incorporation,and the growth of organoidswas examined by measuring surface area of organoid hori-zontal cross sections.Plasmid ConstructionAn expression vector containing a 284-bp fragment flankingthe human uc.173 locus under the control of pCMV promoter andthe full-length miRNA195 promoter(positions from-1500 to 1)luciferase(Luc)reporterconstructwereconstructedasdescribedpreviously.23Primer sequences for generating these constructsare provided in Supplementary Table 1.Transient transfectionswere performed using the Lipofectamine reagent following themanufacturers recommendations(Invitrogen,Grand Island,NY).Forty-eight hours after transfection using Lipofectamine,cellswere harvested for analysis.In studies to assay the activity of themiRNA195 promoter,the promoter constructs were transfectedinto cells along with phRL-null,a Renilla Luc control reportervector from Promega(Madison,WI),to monitor transfectionefficiencies.The Luc activity from individual constructs wasnormalized by Renilla-driven Luc activity in every experiment.Isolation of Nuclear and Cytoplasmic RNANuclearandcytoplasmicRNAfractionsfromCaco-2cells were isolated and purified with the SurePrep Nuclear/Cytoplasmic RNA Purification Kit from Fisher Bioreagents(FairLawn,NJ).To assess any cross-contamination between cyto-plasmic and nuclear fractions,the levels of b-tubulin(a specificcytoplasmic protein marker)and lamin B(a nuclear proteinmarker)were examined in the 2 fractions.20Quantitative Real-time Polymerase ChainReaction,T-UCR Microarray,andImmunoblotting AnalysesTotal RNA was isolated by using the RNeasy mini kit(Qia-gen,Valencia,CA)and used in reverse transcription(RT)andpolymerase chain reaction(PCR)amplification reactions asdescribed.23Quantitative(Q)-PCR analysis was performed us-ing Step-one-plus Systems with specific primers,probes,andsoftware(Applied Biosystems,Foster City,CA).For T-UCR array studies,total RNA was purified with amiRCURY RNA isolation kit,and lncRCURY LNA array of T-UCRprofiling services was performed by Arraystar(Rockville,MD).The levels of uc.173 were quantified by Q-PCR analysis by usingTaqman ncRNA assay.To examine protein levels,whole-cell lysates were preparedusing 2%sodium dodecyl sulfate,sonicated,and centrifuged.The supernatants were boiled and size-fractionated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).After transferring proteins onto nitrocellulose filters,the blotswere incubated with primary antibody;following incubationswith secondary antibody.Biotin-labeled RNA Pull-down AssaysBiotin-labeledpri-miR-195ormaturemiRNA195wastransfected,and 24 hours later,whole-cell lysates were collected,mixed with Streptavidin-Dynal beads,and incubated on a rotatorovernight.20After the beads were washed thoroughly,the beads-bound RNA was isolated and subjected to RT followed by Q-PCRanalysis.Input RNA was extracted and served as control.RNA-Fluorescence In Situ Hybridization AssayRNA-fluorescence in situ hybridization(FISH)assay wasperformed with the ISH optimization kit from Exiqon(Vedbaek,Denmark)as described.19,20The slides were dehydrated andhybridized with fluorescent LNA-probe,and then processedusing Zeiss(Munich,Germany)confocal microscope.Statistical AnalysisAll values were expressed as the means standard error ofthe mean(SEM).Unpaired,2-tailed Students t test was usedwhen indicated with P 2-fold down orup)observedinresultsdescribed in A.Values aremeansSEMofdatafrom 4 animals.*P .05comparedwithcontrols.(C)Basal levels of variousT-UCRs relative to uc.173inthemucosaasmeasured by RT-Q-PCRanalysis.(D)Levelsofmucosaluc.173inthesmallintestineasmeasured by RT-Q-PCRanalysis.Valuesaremeans SEM of data from6animals.*P.05comparedwithcontrols.(E)Levels of uc.173 in cellsgrowth-arrestedinG1phasebypolyaminedepletion.Caco-2cellswere exposed to DFMO(5mmol/L)aloneorDFMOplusputrescine(Put,10 mmol/L)for 4 days.*P .05 compared withcontrols or cells treatedwith DFMO plus Put.(F)Levelsofcytoplasmic(cyto)and nuclear uc.173,host gene UBE2B mRNA,andlncRNAHULCinCaco-2 cells.602Xiao et alGastroenterology Vol.154,No.3BASIC ANDTRANSLATIONAL ATpCMV promoter(Figure 2A,schematic);48 hours later,cellular uc.173 levels increased strongly and specifically(Figure 2A,right),but other T-UCRs such as uc.346 anduc.283 did not(data not shown).Importantly,ectopicexpression of uc.173 increased cell numbers compared withcells transfected with a control vector(Figure 2B).In anex vivo model,raising uc.173 levels by transfection with theuc.173 expression vector(Supplementary Figure 2A)alsostimulated the growth of intestinal organoids.As shown inFigure 2C,an intestinal organoid could be initiated from asingle proliferating cell,but by 8 days after culture,thestructures of organoids consisted of multiple cells in bothcontrol and uc.173-transfected organoids.Strikingly,how-ever,ectopic expression of uc.173 markedly activated DNAsynthesis in multiple cells,as determined by the increasedBrdU incorporation(Figure 2D,left),and augmented thesurface area of intestinal organoids(Figure 2D,right).Thenumbers of cells per