1、Nucleic Acids Research,20191doi:10.1093/nar/gkz199FOXP1 circular RNA sustains mesenchymal stem cellidentity via microRNA inhibitionAlessandro Cherubini1,Mario Barilani1,2,Riccardo L.Rossi3,Murtadhah M.K.Jalal4,6,Francesco Rusconi1,Giuseppe Buono1,Enrico Ragni1,Giovanna Cantarella2,5,Hamish A.R.W.Sim
2、pson4,Bruno P eault6,7andLorenza Lazzari1,*1Laboratory of Regenerative Medicine-Cell Factory,Department of Transfusion Medicine and Hematology,Fondazione IRCCS Ca Granda Ospedale Maggiore Policlinico,Milan,20122,Italy,2Department of Clinical Sciencesand Community Health,Universit a degli Studi di Mi
3、lano,Milan,20122,Italy,3Istituto Nazionale Genetica Molecolare“Romeo ed Enrica Invernizzi,Milan,20122 Italy,4Department of Orthopaedic Surgery,The Royal Infirmary ofEdinburgh,Edinburgh,EH16 4SA,UK,5Department of Otolaryngology,Fondazione IRCCS Ca Granda OspedaleMaggiore Policlinico,Milan,20122,Italy
4、,6MRC Centre for Regenerative Medicine,The University of Edinburgh,Edinburgh,EH16 4UU,UK and7Orthopaedic Hospital Research Centre,David Geffen School of Medicine,Universityof California at Los Angeles,California,90095,USAReceived January 25,2019;Revised March 11,2019;Editorial Decision March 13,2019
5、;Accepted March 18,2019ABSTRACTStem cell identity and plasticity are controlled bymaster regulatory genes and complex circuits alsoinvolving non-coding RNAs.Circular RNAs(circR-NAs)are a class of RNAs generated from protein-coding genes by backsplicing,resulting in stableRNA structures devoid of fre
6、e 5 and 3 ends.Lit-tle is known of the mechanisms of action of circR-NAs,let alone in stem cell biology.In this study,for the first time,we determined that a circRNA con-trols mesenchymal stem cell(MSC)identity and dif-ferentiation.High-throughput MSC expression profil-ing from different tissues rev
7、ealed a large number ofexpressed circRNAs.Among those,circFOXP1 wasenriched in MSCs compared to differentiated meso-dermal derivatives.Silencing of circFOXP1 dramati-cally impaired MSC differentiation in culture andinvivo.Furthermore,we demonstrated a direct inter-action between circFOXP1 and miR-17
8、3p/miR-1275p,which results in the modulation of non-canonicalWnt and EGFR pathways.Finally,we addressed theinterplay between canonical and non-canonical Wntpathways.Reprogramming to pluripotency of MSCsreduced circFOXP1 and non-canonical Wnt,whereascanonical Wnt was boosted.The opposing effect wasob
9、served during generation of MSCs from humanpluripotent stem cells.Our results provide unprece-dented evidence for a regulatory role for circFOXP1as a gatekeeper of pivotal stem cell molecular net-works.INTRODUCTIONHuman mesenchymal stem cells(MSCs)are multipotentcells that can be isolated from vario
10、us adult or perinataltissues and possess the ability to self-renew in culture anddifferentiate into mesodermal derivatives,including osteo-cytes,chondrocytes and adipocytes(13).Due to this dif-ferentiation potential and other properties to regenerateinjured tissues indirectly via growth factor secre
11、tion andimmunomodulation,MSCs hold promise for regenerativemedicine.In particular,MSCs are used in bone reconstruc-tion therapies and are frequently associated with biomate-rials or subjected to ex vivo stimuli to induce proper mat-uration into fully functional osteocytes and chondrocytes(4).The the
12、rapeutic efficacy of MSCs hinges upon a finecontrol of MSC lineage specification.Thus,it is crucial togain a deeper understanding of the molecular mechanismsthat regulate their differentiation(57).Factors involved infatedecisionandintermediateorfinalstagesofMSCdiffer-entiation,such as Wnt and transf
13、orming growth factor-?,have attracted increasing attention from the scientific com-munity(810).On the other hand,little is known aboutthe molecular mechanisms that regulate the maintenanceof human MSC identity and their uncommitted state.Eventhough roles for epidermal growth factor receptor(EGFR)and
14、 non-canonical Wnt signaling have been documented(1113),the integration of these signaling pathways withepigeneticregulators,suchasnon-codingRNAs(ncRNAs),*To whom correspondence should be addressed.Tel:+39 0255034053;Email:lorenza.lazzaripoliclinico.mi.itC?The Author(s)2019.Published by Oxford Unive
15、rsity Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License(http:/creativecommons.org/licenses/by-nc/4.0/),which permits non-commercial re-use,distribution,and reproduction in any medium,provide
16、d the original workis properly cited.For commercial re-use,please contact Downloaded from https:/ by guest on 03 April 20192 Nucleic Acids Research,2019is not understood.Therefore,the aim of this study was touncover molecular networks that sustain the undifferenti-ated state and self-renewal of MSCs
17、 in an epigenetic per-spective.Mammalian cells contain thousands of RNA moleculesthat do not code for proteins,but play key roles in the regu-lation of physiological processes(1416).Recent researchhas indicated that microRNAs(miRNAs)and long non-coding RNAs(lncRNAs)regulate the differentiation andce
18、ll fate decisions of MSCs(1719).Circular RNAs(circR-NAs)are a recently discovered class of ncRNAs.AlthoughtheexistenceofcircRNAswasproposedmorethan20yearsago,for many years they were thought to be functionlessbyproductsofmRNAsplicing(2022).Nevertheless,recentstudies have identified a large number of
19、 endogenous circR-NAs in various tissues,at different developmental stages,and in many organisms under diverse conditions of growthand stress,thus hinting at a relevant functional role of cir-cRNAs in cellular biology and pathophysiology(2325).CircRNAs may be generated from exons,introns orintron-co
20、ntainingexonsbyaback-splicingreactionthatco-valently links an upstream 3?-splice site to a downstream 5?-splice site,leading to a closed loop structure(26).This par-ticular conformation is reported to increase the stability ofcircRNA,compared to its linear counterpart.Even thoughthe mechanisms under
21、lying these events are not fully un-derstood,recent studies have demonstrated that the pres-ence of specific mammalian genomic features,such as re-verse complementary sequences in the flanking introns andthe activity of specific RNA-binding proteins,enhance cir-cRNA biogenesis(2730).Concerning the b
22、iological function of circRNAs,theirmechanisms of action have been largely unexplored.Ithas been proposed that some circRNAs play an impor-tant role in gene regulation by acting as competing endoge-nous RNAs(ceRNAs).For instance,miRNA spongingisa mechanism of action of ceRNA,as shown for SRY andCDR1
23、 as in neuronal tissues(24,31)and for HIPK3 in var-ious cancers(32).In addition,circRNAs can promote theexpression of their parental genes by regulating the RNAPol II transcription complex in the nucleus(33).Other mechanisms for circRNAs have been proposed,e.g.as hubs for protein interaction,as show
24、n for circ-Mbl,which interacts with the Mbl protein to compete for thesplicing of its linear counterpart(29).Furthermore,circ-Foxo3 has been demonstrated to regulate cell cycle progres-sion by forming ternary complexes with CDK2 and p21(34).Moreover,it has recently been shown that circRNAscan be tra
25、nslated efficiently into small truncated peptides,even though the molecular activity of this type of circRNA-derivedproteinisnotyetunderstood(35).Finally,highcon-servation of circRNAs across species and their tissue-anddevelopmental stage-specific expression suggest their rolein cell identity and fa
26、te determination during development(23,24,3639).In the current study,we report the unique role of a cir-cRNA originating from the FOXP1 gene(circFOXP1)inthe maintenance of MSC identity and regulation of differ-entiation.CircFOXP1 acts as a miRNA sponge targetingmiR-173p and miR-1275p,and promotes pr
27、oliferationand differentiation of MSCs,supporting the hypothesis ofcircRNAsasmajorplayersinstemcellfatedecision-makingprocesses.MATERIALS AND METHODSCell line,cell culture and treatmentsMSCs and HSFs were cultured in?-minimum essentialmedium supplemented with 20%fetal bovine serum(FBS;LifeTechnologi
28、es,cat.no.10270106)at37Cand5%CO2.MSCs were isolated from both cord blood and bone mar-row,asdescribedpreviously(4043).TheMSCidentitywasconfirmed by the immunophenotype profile(1):cells werepositive for the MSC surface antigens CD73,CD90 andCD105,and were negative for the hematopoietic markersCD45 an
29、d CD34(data not shown).The human materialwas obtained after informed consent from healthy donors,and the study was approved by the internal Ethics Com-mittee.All experiments were performed according to theamended Declaration of Helsinki.Adipo-,osteo-and chondrogenic differentiation ofMSCs was induce
30、d using hMSC Osteogenic Bullet Kit(Lonza,cat.no.PT-3002),hMSC Chondro Bullet Kit(Lonza,cat.no.PT-3003)andhMSCAdipogenicBulletKit(Lonza,cat.no.PT-3004)according to the manufacturer sinstructions.DifferentiationwasconfirmedbystainingwithOilRedO(SigmaAldrich,cat.no.O1391)foradipogenesis,Alizarin Red(Si
31、gma Aldrich,cat.no.13022-3)for osteo-genesis and Alcian Blue(Sigma Aldrich,cat.no.A5268)forchondrogenesis.HEK-293T cells were obtained from the American TypeCultureCollection(ATCC)andwereculturedinDulbecco smodified Eagle medium supplemented with 10%FBS.Allcells were routinely tested for mycoplasma
32、using the My-coAlert Mycoplasma Detection kit(Lonza,cat.no.LT07).Human induced pluripotent stem cells(hiPSCs)derivedfrom MSCs were generated and characterized by uSTEMcompany(uSTEM,Padova,Italy).uSTEM also generatedBJ fibroblasts-hiPSCs used to obtain hiPSC-derived MSCs,following the protocol descri
33、bed elsewhere(44).Transcription was blocked by adding 1?g/ml actino-mycin D(Sigma Aldrich,cat.no.A9415)or dimethylsulphoxide(Sigma Aldrich,cat.no 472301)as a control tothe cell culture medium.Microarray analysisSample preparation and microarray hybridization wereperformed according to the manufactur
34、er s protocol(Ar-raystar).Briefly,circRNAs were treated with Rnase R toremove linear RNAs.Then,each sample was amplified andtranscribed into fluorescent cRNA using the random prim-ing method with a Super RNA Labeling Kit(Arraystar).The labeled cRNA was hybridized onto an Arraystar Hu-man Circular RN
35、A Microarray(Arraystar V1.0).The ar-raywasscannedwiththeAgilentScannerG2505C,andrawdata were extracted by Agilent Feature Extraction software(version 11.0.1.1).Identification of circRNAs followed cir-cBASE database nomenclature(45).Downloaded from https:/ by guest on 03 April 2019Nucleic Acids Resea
36、rch,2019 3RNA isolation,reverse transcription and qRT-PCR analysisThe total RNA from cell lines was isolated using TRI-zol reagent(Ambion,cat.no.15596026).For RNA isola-tion from adipo,osteo and cartilage healthy tissues,sam-ples were disrupted using Tissue Tearor(Biospec Prod-ucts).RNA concentratio
37、n and quality were verified using aNanoDropND-1000spectrophotometer(NanoDropTech-nologies).For the quantitative real time PCR(qRT-PCR)assay,cDNAwassynthesizedfrom1?goftotalRNAwithSuper-Script IV VILO(Invitrogen,cat.no.11756500).The cDNAwas diluted 10-fold and 1?l used as template for qRT-PCRanalysis
38、usingSYBRSelectMasterMixforCFX(LifeTechnologies,cat.no.4472937)on a CFX96 thermal cycler(BioRad)followingthemanufacturer sinstruction.Accord-ing to the MIQE guidelines(46),amplification efficiency,correlation coefficient and slope of standard curves gener-ated using serial dilutions for each primer
39、pair were evalu-ated(Supplementary Table S1).The relative expression lev-els of the selected targets were determined using the?Ctmethod and normalized,where not differentially specified,toACTBmRNAlevels.InthecaseofmiRNA,themiScriptII RT Kit(Qiagen,cat.no.218161)and miScript SYBRGreenPCRKit(Qiagen,ca
40、t.no.218073)wereused,accord-ing to the manufacturer s protocols.qRT-PCR products were resolved on 2%agarose gel.The DNA products were gel purified using the Wizard SVClean-Up system(Promega,cat.no.A9281),according tothe manufacturer s protocol,and submitted to Sanger se-quencing for sequence validat
41、ion.All primers used in thisstudy,listed in Supplementary Table S2,were designed us-ing Primer 3 software.DNA construct and mutagenesisTo obtain the expression of circFOXP1,the genomic re-gion with the Alu sequence was amplified using Q5 High-Fidelity DNA Polymerase(NEB,cat.no.M0491).Thepolymerase c
42、hain reaction(PCR)products were insertedinto the pEYFP-C1 vector.The luciferase reporter wasconstructed by subcloning the circFOXP1 fragment di-rectly downstream of the Renilla luciferase cassette intothe psiCHECK2 vector.Mutation of each miRNA-bindingsite was performed using the Q5 Site-Directed Mu
43、tagenesisKit(NEB,cat.no.E0554S).For the CRISPR/Cas9 assay,gRNA cloning vectors were constructed using pUC-gRNAcloning vector.All constructs were verified by sequencing.All primers used are listed in Supplementary Table S2.RNase R treatment of total RNAA 2?g sample of total RNA was treated with 3 U/?
44、g ofRnase R(Epicentre Biotechnologies,cat.no.RNR07250)or water as a control(Mock)for 20 min at 37C.DigestedRNAwassubsequentlypurifiedusinganRNeasyMinEluteCleanup Kit(Qiagen,cat.no.74204).Nuclear and cytoplasmic fractionationNuclear/cytoplasmic fractionation was performed as fol-lows.Cellswerewashedw
45、ithcoldphosphate-bufferedsaline(PBS),harvested by scraping and centrifuged for 5 min at500g.Cellpelletswerelysedin5(v/v)ice-coldBufferA(10mMTrisHCl,pH7.4,10mMNaCl,3mMMgCl2,10%glycerol,1 mM ethylenediaminetetraacetic acid(EDTA),0.5 mM dithiothreitol(DDT)and protease inhibitor cock-tail)for 10 min at
46、4C.Lysates were cleared by centrifuga-tionfor5 minat 1200 g and 4C,and thesupernatantwascollected and saved as the cytoplasmic fraction on ice.Thecrude nuclear pellet was resuspended in 250?l of Buffer B(10 mM TrisHCl,pH 7.5,15 mM KCl,30 mM NaCl,10%glycerol,1 mM EDTA,0.3%sodium deoxycholate,0.5%IGEP
47、AL,0.5 mM DTT,and 1 protease inhibitor cock-tail)andcentrifugedfor5minat1200gand4C.Thepel-let was resuspended in 5 (v/v)ice-cold Buffer F(10 mMTrisHCl,pH 7.0,100 mM NaCl,30 mM Na Pyrophos-phate(Na4P2O7),50 mM NaF,5 mM ZnCl2,1%Triton X-100 and 1 protease inhibitor cocktail),incubated for 10min on ice
48、 and then centrifuged for 5 min at 12 000 g and4C.The nuclear supernatant was collected on ice.RNA FISHIn situ hybridization was performed using biotinylatedDNA oligonucleotides(probes)specific for the circ-FOXP1 sequence or negative control(SupplementaryTable S2).MSCs at 8090%confluence were fixed
49、in4%paraformaldehyde for 20 min.After prehybridization(PBS/0.5%Triton X-100),cells were incubated with probesin hybridization buffer(40%formamide,10%dextran sul-fate,4 saline-sodium citrate buffer,10 mM DDT and1 mg/ml yeast transfer RNA)at 37C overnight.Signalswere detected using a tyramide-conjugat
50、ed Alexa 568 flu-orochrome tyramide signal amplification kit(Invitrogen,cat.no.T20934).Nuclei were counterstained with 4?,6-diamidino-2-phenylindole(Invitrogen,cat.no.D1306).Im-ages were acquired using a Leica TCS SP2 confocal micro-scope,with an HCX PL APO IBD.BL 63 /1.4 objective.Oligonucleotide t
