Fabbri

Gellersen

Kajihara

Kosaka

Myatt

Nagaraja

Salamonsen

Thery

Vallhov

Zeng

English

PubMed

Kunal M. Shah

Jason Webber

Raffaella Carzaniga

Deborah M. Taylor

Luca Fusi

Aled Clayton

Jan J. Brosens

Geraldine Hartshorne

Mark Christian

Crossref

Journal of Molecular Cell Biology

Induction of microRNA resistance and secretion in differentiating human endometrial stromal cells

Shah, Kunal M.

Webber, Jason, Dr.

Carzaniga, Raffaella

Taylor, Deborah M.

Fusi, Luca

Clayton, Aled

Brosens, Jan J.

Hartshorne, Geraldine M.

Christian, Mark

Warwick Research Archives Portal Repository

Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair.

Cytokines and chemokines during human embryo implantation: roles in implantation and early placentation.

Deﬁnition of microRNAs that repress expression of the tumor suppressor gene FOXO1 in endometrial cancer.

Deletion of Dicer in somatic cells of the female reproductive tract causes sterility.

Differential expression of FOXO1 and FOXO3a confers resistance to oxidative cell death upon endometrial decidualization.

Essential and overlapping functions for mammalian Argonautes in microRNA silencing.

Exosomes containing glycoprotein 350 released by EBV-transformed B cells selectively target B cells through CD21 and block EBV infection in vitro.

Human villous trophoblasts express and secrete placenta-speciﬁc microRNAs into maternal circulation via exosomes.

Isolation and characterization of exosomes from cell culture supernatants and biological ﬂuids.

MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting

MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms.

The diversityofsexsteroidaction:theroleofmicro-RNAs andFOXOtranscriptionfactorsincyclingendometrium and cancer.

The expression proﬁle of micro-RNA in endometrium and endometriosis and the inﬂuence of ovarian steroids on their expression.

Unraveling the mystery of cancer by secretory microRNA: horizontal microRNA. Transfer between living cells.

Webber, Jason P.

Hartshorne, Geraldine

Online Research @ Cardiff

Letter to the EditorInduction of microRNA resistance and secretionin differentiating human endometrial stromal cellsDear Editor,A key event in the preparation forpregnancy is the differentiation of humanendometrial stromal cells (hESCs) intoepithelioid decidual cells. This process,termed decidualization, is initiated in thestromal cells of the superficial endometriallayer 9 days after ovulation. Upon decid-ualization, hESCs acquire the ability to resistoxidative stress, to modulate immuneresponses to fetal alloantigens, and tocontrol trophoblast invasion. Differentiatingprimary hESCs recapitulate many changesin gene expression observed upon decidua-lization in vivo with progesterone and cAMPsignaling pathways cooperating in hESCs atseveral levels [see review (Gellersen andBrosens, 2003)].MicroRNAs (miRNAs) are a diverse classof non-coding small RNAs, which post-transcriptionally regulate gene expressionby interacting with sites in the 3′UTRs ofmRNAs. miRNA synthesis begins with thetranscription of miRNA genes to produceprimary miRNAs (pri-miRNAs). In thenucleus, the pri-miRNAs are cleaved bythe Drosha/DGCR8 complex to produce70 nt pre-miRNAs. Exportin-5 transferspre-miRNAs to the cytoplasm where Dicerremoves the loop to give duplex maturemiRNA of 22 nt. Argonaute proteinsfacilitate the miRNA–mRNA interactionsand mediate silencing. In addition to theirdiverse roles in the parental cell, miRNAscan act as paracrine or endocrine signalsand are present in the plasma as well asin the conditioned medium of culturedcells (Kosaka and Ochiya, 2011).Endometrial miRNA expression is cycledependent and regulated by ovarian hor-mones (Pan et al., 2007). With miRNAmicroarray analysis we identified 16 signifi-cantly (P , 0.05) differentially expressedmiRNAs in primary hESCs that had beenvehicle treated or decidualized with 8-Br-cAMP and medroxyprogesterone acetate(C + M) for 8 days (Supplementary TableS1). Real-time qPCR was used to monitorthe expression of four miRNAs (miR-29b,miR-29c, miR-100, and miR-143) in inde-pendent primary hESC cultures treatedeither with vehicle or with C + M for 2, 4,or 8 days. These miRNAs were selected forstudy as they showed the most significantchanges in expression and were likely totarget genes important for decidual func-tion including DNA methyltransferase 3b(DNMT3B), Tribbles 2 (TRIB2) and prokine-ticin 1 (PROK1). Figure 1A shows thatthe expression of miR-29b, miR-29c,and miR-100 increases as expected by8 days of C + M treatment. In contrast,the levels of mature miR-143 graduallydeclined, which is also in keeping with thearray data. The changes in the pri-forms ofthe four miRNAs during decidualizationlargely mirrored the changes observed inmature miRNA levels (SupplementaryFigure S1), indicating that transcriptionalregulation of miRNA genes occurs duringdecidualization.Next, we examined whether key compo-nents of the miRNA synthesis pathway arealtered upon decidualization of hESCs.Parallel primary cultures treated withC + M for various time points were har-vested for total protein and RNA. Thelevels of Drosha, Dicer, Ago1 and Ago2were assessed at the protein and transcriptlevels (Figure 1B and SupplementaryFigure S2). Drosha levels modestlydecline in response to C + M treatment.Conversely, Dicer is up-regulated andagain this response seemed more pro-nounced at the protein level. Strikingly,C + M treatment profoundly inhibited theabundance of Ago1 and Ago2, especiallyat the later time points. Densitometric ana-lysis comparing three independent samplesfrom day 0 and day 8 confirmed the changesobserved (Figure 1B). siRNA-mediatedknockdown of Dicer did not block the induc-tion of the classical decidual marker genesprolactin (PRL) and insulin-like growthfactor binding protein 1 (IGFBP1) duringdecidualization (Supplementary Figure S3).The robust induction of these genes indi-cated that Dicer-depleted hESCs are stillable to differentiate upon treatment withC + M. This result is in keeping with thedata from the conditional Dicer KO mousewhere female mice lacking Dicer in thereproductive tract were infertile due todefective oviducts but exhibited histo-logically normal decidual responses(Nagaraja et al., 2008).Although Dicer knockdown did not blockhESC differentiation, we speculated thatregulated miRNAs target and modulate theexpression of specific decidual genes. An ex-perimentally validated target of the miR-29family is DNMT3B (Fabbri et al., 2007). Totest if DNMT3B expression is inhibitedupon decidualization as a consequence ofmiR-29b/c induction, primary cultureswere first transfected with anti-miR-29b/cand then treated with vehicle or C + M. Asshown in Figure 1C, DNMT3B expression isindeed down-regulated upon decidualiza-tion at the protein level. Furthermore, thetransfection of anti-miR-29b/c enhancedDNMT3B protein levels in undifferentiatedcells, although this response was entirelylost upon treatment with C + M. In decidua-lizing hESCs, the effect of anti-miR-29b/ctransfection on DNMT3B expression was in-distinguishable from transfection negativecontrol anti-miR or non-targeting anti-miR-100. Similar findings were also observedwith respect to DNMT3B mRNA levels, forTRIB2, a predicted target of both miR-29b/cand miR-100, and for PROK1, a predictedtarget of miR-100 (Supplementary Figure S4).We employed a reporter assay to monitorthe generic miRNA activity in bothdoi:10.1093/jmcb/mjs058 Journal of Molecular Cell Biology (2013), 5, 67–70 | 67Published online October 25, 2012# The Author (2012). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/),which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. at Cancer Research Wales Library on August 14, 2013http://jmcb.oxfordjournals.org/Downloaded from Figure 1 Decidualization leads to differential miRNA expression, miRNA pathway regulation and miRNA secretion. (A) Primary hESCs were vehicletreated (day 0) or treated with C + M for 2, 4, or 8 days. qPCR was performed for mature miR-29b, miR-29c, miR-100, and miR-143. miRNA levelswere normalized U6 snRNA levels. Data show the mean of n ¼ 3 and error bars denote the SEM. *P , 0.05. (B) Western blots of miRNA pathwaycomponents in hESCs treated with vehicle or C + M for 2, 4, and 8 days. GAPDH acted as a loading control. The graph shows densitometric ana-lysis of three independent blots comparing vehicle treated and day 8 C + M treated samples. Vehicle-treated levels were normalized to 1. *P ,0.05. (C) hESCs were transfected with negative control (NC) anti-miR or anti-miR-29b/c or anti-miR-100 and treated with vehicle or C + M for 2days before harvesting and western blotting for DNMT3B. Alpha-tubulin acted as a loading control. (D) hESCs in 24-well plates were transfectedwith pCMV Luc miR-30-(P) (300 ng/well), pcH110, 20 ng/well of miR-30, and 50 ng/well p3XFLAG-AGO2. hESCs were treated with vehicle or C +M for 3 days before harvesting. Luciferase activity levels were normalized to beta-galactosidase levels. Data represent fold change relative to thereporter-only sample. Each bar denotes the mean of four replicates and the error bars represent the SEM. *P , 0.05. (E) Cultured hESCs weretreated with vehicle (day 0) or C + M for 2, 4, and 8 days. For the final 2 days before harvesting, cells were incubated in serum-free medium.qPCR was used to profile expression of miR-29b, miR-29c, miR-100, and miR-143 in conditioned media. miRNA expression levels were normalizedto levels of spiked cel-miR-39. Data shows the mean of three replicates and the error bars represent the SEM. *P , 0.05. (F) Serum-free condi-tioned medium from decidualized hESCs was serially centrifuged and the supernatants were used immediately (pre-spin) or ultracentrifuged(postspin). qPCR was performed for miR-100. Unconditioned media was also tested in parallel. Bars show miR-100 levels normalized to68 | Journal of Molecular Cell Biology Shah et al. at Cancer Research Wales Library on August 14, 2013http://jmcb.oxfordjournals.org/Downloaded from undifferentiated and decidualized hESCs.Primary cultures were co-transfected withpCMV Luc miR-30-(P), a reporter constructcontaining eight miR-30-binding sites inthe 3′UTR (Zeng et al., 2003), and pSupermiR-30. The co-expression of pSupermiR-30 inhibited luciferase activity inboth vehicle and C + M treated cells(Figure 1D). However, miR-30-dependentrepression was significantly higher in un-differentiated compared with decidualizinghESCs. Furthermore, the co-expression ofAgo2 in decidualizing cells restored thelevel of miR-30-dependent repression tothat observed in undifferentiated cells.Together, the data show that down-regulation of Ago2 induces resistance toendogenous miRNA-dependent gene re-pression in decidualizing cells. Althoughprevious studies found that Ago-deficientmouse embryonic stem cells were moreapoptotic (Su et al., 2009), this is unlikelyto be the case for hESCs as C + M treat-ment renders hESCs more resistant toapoptosis (Kajihara et al., 2006).Therefore, the resistance to miRNA func-tion observed in decidualizing hESCs is un-likely to be a secondary effect of increasedapoptosis. During decidualization, thecAMP dependent up-regulation of FOXO1is important for cell cycle regulation andconferring resistance to oxidative stress,as well as the induction of differentiationmarkers such as IGFBP1 (Lam et al.,2012). It has also been documented inendometrial cancer that the miR-27family can reduce FOXO1 expression(Myatt et al., 2010). We observed a down-regulation of miR-27b upon decidualiza-tion (Supplementary Table S1) and thiscould contribute to the increase in FOXO1levels that are crucial for several decidualcell phenotypes, including resistance oxi-dative stress-induced apoptosis.Decidualizing hESCs acquire a secretoryphenotype (Salamonsen et al., 2007). Asdifferentiation also bestows resistanceto miRNA-mediated gene silencing, wespeculated that decidual miRNAs may beintended for export. To test this hypoth-esis, primary hESCs were decidualizedwith C + M for various time points. Thecultures were maintained in serum-freemedium for 48 h prior to harvesting. RNAwas then extracted from the conditionedmedium and miR-29b, miR-29c, miR-100,and miR-143 amplified by qPCR(Figure 1E). The abundance of the fourmiRNA species in the spent media wasuniformly higher by 8 days of C + Mtreatment. Notably, the rise in miR-143 se-cretion appears to be an early event in thedecidual process. This is particularly strik-ing as the cellular levels of both the pri-and mature forms of this miRNA declinein response to C + M treatment(Figure 1A and Supplementary Figure S1).The data suggest that miR-143 may bestored in undifferentiated hESCs andreadily released upon decidualization.For secretion, miRNA must be packagedin membrane-bound vesicles, known vari-ously as microvesicles, exosomes, and largemembranous vesicles. Ultracentrifugationof serum-free conditioned medium depletedmiR-100 levels in the supernatant butmiR-100 was abundant in the resultantpellet (Figure 1F and SupplementaryFigure S5A). Western blotting showedthat the ultracentrifugation pellet wasenriched in exosome markers TSG101and HSP90 (Supplementary Figure S5B).The enrichment of small RNA in the ultra-centrifugation pellets was confirmedusing an Agilent Bioanalyzer (SupplementaryFigure S5C).Ultracentrifugation at 100000 g is rou-tinely used to pellet exosomes (Theryet al., 2006), and transmission electronmicroscopy revealed that the pellets fromdecidual cell conditioned medium con-tained cup-shaped vesicles that measured,100 nm in diameter, indicative of exo-somes (Figure 1G). Conditioned mediumthat had been incubated with decidualizedhESCs for 4 or 8 days was analyzed usingthe Nanosight nanoparticle trackingsystem (Figure 1H). The size profiles of par-ticles in decidual cell conditioned mediawere typical of samples rich in exosomes(Vallhov et al., 2011), with a large propor-tion of particles having a diameter of100 nm or less. Larger particles are likelyto be aggregates of exosomes.We sought to test if human embryos andother cell types present at the feto-maternal interface could take up secreteddecidual miRNAs. We transfected hESCswith pSuperior cel-miR-39, whichexpresses a miRNA without mammalianorthologs, and treated the cells with C + Mfor 6 days. The conditioned medium wascollected and added to a number of rele-vant cell lines, including Ishikawa cells(representing endometrial epithelialcells), BeWo cells (a trophoblast cellline), and human umbilical vein endothe-lial cells (HUVECs). Human embryos andhESCs, decidualized or not, were alsotested as recipients. Where embryos wereused as recipients, the conditionedmedium was ultracentrifuged and thepellet was resuspended in embryo culturemedium. Following exposure to condi-tioned medium, cel-miR-39 was detectedin HUVECs, BeWo cells, and hESCs and,to a lesser extent, in Ishikawa cells(Figure 1I). Although we were able toamplify endogenous U6 snRNA in humanembryos, cel-miR-39 was not detectable.However, this finding must be consideredpreliminary in view of the presence of thezona pellucida in some of our culturedembryos, which could have preventeduptake, and those embryos with breachedzonas failed to develop into blastocysts.HUVECs contained the highest levels ofcel-miR-39, which were 1000-fold lessabundant than in the donor hESCs thathad been transfected with pSuperiorcel-miR-39. We did not observe a signifi-cant difference in cel-miR-39 uptakebetween undifferentiated and decidualizedhESCs. Thus, it appears likely that variouscells that make up the feto-maternal inter-face take up miRNAs secreted by decidua-lized hESCs.In summary, our results show that miRNAsignaling is profoundly altered during decid-ualization of hESCs in preparation for preg-nancy. The physiological relevance ofdiminished miRNA silencing in decidualizedhESCs may be a defence mechanism tospiked cel-miR-39 levels for triplicate samples. Error bars denote SEM. ND indicates not detected. (G) Transmission electron micrograph of vesi-cles obtained by ultracentrifugation of serum-free conditioned medium from decidualized hESCs. (H) Nanosight size profiling of particles in de-cidual cell conditioned media. (I) Uptake of cel-miR-39 by embryos and cells from conditioned medium of decidualized hESCs transfected withpSuperior cel-miR-39. Cel-miR-39 levels were normalized to endogenous U6 snRNA levels. Bars represent measurements for triplicates anderror bars denote SEM (see Supplementary Method).Induction of microRNA resistance in hESCs Journal of Molecular Cell Biology | 69 at Cancer Research Wales Library on August 14, 2013http://jmcb.oxfordjournals.org/Downloaded from protect from the silencing effects of tropho-blast-derived miRNAs. A recent study foundthat placenta-specific miRNAs were abun-dant in the circulation of pregnant womenand trophoblast cells secreted miRNAs inexosomes (Luo et al., 2009). Our data alsoprovide the first evidence that miRNAs canbe added to the growing number of factorsknown to be secreted by decidual cells andtheir uptake by other cells of the endomet-rium may have physiological consequencesthat remain to be explored.[Supplementary material is available atJournal of Molecular Cell Biology online.We thank Nick Dibb (Imperial CollegeLondon) for advice on the manuscript.This work was supported by BBSRC grant(BB/D52657X/1), the Genesis ResearchTrust, and the Biomedical Research Unit inReproductive Health, a joint initiativebetween the University Hospitals Coventryand Warwickshire NHS Trust and WarwickMedical School.]Kunal M. Shah1, Jason Webber2, RaffaellaCarzaniga3, Deborah M. Taylor4, Luca Fusi1, AledClayton2, Jan J. Brosens4, GeraldineHartshorne4, and Mark Christian1,*1Division of Surgery and Cancer, Institute ofReproductive and Developmental Biology,Imperial College London, HammersmithCampus, London W12 0NN, UK2Institute of Cancer and Genetics, School ofMedicine, Cardiff University, Velindre CancerCentre, Whitchurch, Cardiff CF14 2TL, UK3Electron Microscopy Centre, BiochemistryBuilding, Imperial College London, SouthKensington Campus, London SW7 2AZ, UK4Division of Reproductive Health, WarwickMedical School, University of Warwick,Coventry CV4 7AL, UK*Correspondence to: Mark Christian, E-mail:m.christian@imperial.ac.ukReferencesFabbri, M., Garzon, R., Cimmino, A., et al. (2007).MicroRNA-29 family reverts aberrant methylationin lung cancer by targeting DNA methyltrans-ferases 3A and 3B. Proc. Natl Acad. Sci. USA104, 15805–15810.Gellersen, B., and Brosens, J. (2003). Cyclic AMPand progesterone receptor cross-talk in humanendometrium: a decidualizing affair. J. 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Cell Biol. 3.22, 1-3, 22, 29.Vallhov, H., Gutzeit, C., Johansson, S.M., et al.(2011). Exosomes containing glycoprotein 350released by EBV-transformed B cells selectivelytarget B cells through CD21 and block EBV infec-tion in vitro. J. Immunol. 186, 73–82.Zeng, Y., Yi, R., and Cullen, B.R. (2003). MicroRNAsand small interfering RNAs can inhibit mRNA ex-pression by similar mechanisms. Proc. Natl Acad.Sci. USA 100, 9779–9784.70 | Journal of Molecular Cell Biology Shah et al. at Cancer Research Wales Library on August 14, 2013http://jmcb.oxfordjournals.org/Downloaded from 

Induction of microRNA resistance and secretion in differentiating human endometrial stromal cells [Letter]

https://orca.cardiff.ac.uk/id/eprint/50140/1/induction_of_microRNA%20full.pdf

Induction of microRNA resistance and secretion in differentiating human endometrial stromal cells

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