a novel osteomirs expression signature for osteoblast...

Research ArticleA Novel OsteomiRs Expression Signature forOsteoblast Differentiation of Human AmnioticMembrane-Derived Mesenchymal Stem Cells

Mariana Avendantildeo-Feacutelix1 Lizeth Fuentes-Mera 2 Rosal-o Ramos-Payan 1

Maribel Aguilar-Medina 1 Vanessa Peacuterez-Silos 2 Nidia Moncada-Saucedo2

Laurence A Marchat 3 Juan Antonio Gonzaacutelez-Barrios4 Erika Ruiz-Garc-a 5

Horacio Astudillo-de la Vega6 Joseacute L Cruz-Colin7 and Ceacutesar Loacutepez-Camarillo 8

1Facultad de Ciencias Quımico Biologicas Universidad Autonoma de Sinaloa Culiacan Sinaloa Mexico2Departamento de Bioquımica y Medicina Molecular Facultad de Medicina Universidad Autonoma de Nuevo LeonMonterrey NL Mexico

3Programa en Biomedicina Molecular y Red de Biotecnologıa Escuela Nacional de Medicina y HomeopatıaInstituto Politecnico Nacional Ciudad de Mexico Mexico

4Laboratorio de Medicina Genomica Hospital Regional 1 de Octubre ISSSTE Ciudad de Mexico Mexico5Laboratorio de Medicina Traslacional Instituto Nacional de Cancerologıa Ciudad de Mexico Mexico6Laboratorio de Investigacion Traslacional en Cancer y Terapia Celular Hospital de Oncologıa Centro Medico Nacional Siglo XXICiudad de Mexico Mexico

7Subdireccion de Investigacion Basica Instituto Nacional de Medicina Genomica Ciudad de Mexico Mexico8Posgrado en Ciencias Genomicas Universidad Autonoma de la Ciudad de Mexico Ciudad de Mexico Mexico

Correspondence should be addressed to Cesar Lopez-Camarillo genomicasyahoocommx

Received 30 September 2018 Revised 11 December 2018 Accepted 4 March 2019 Published 24 March 2019

Academic Editor Diego Franco

Copyright copy 2019 MarianaAvendano-Felix et alThis is an open access article distributed under theCreativeCommons AttributionLicensewhichpermits unrestricteduse distribution and reproduction in anymedium provided the original work is properly cited

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are a potential source of cells for therapeuticapplications in bone regeneration Recent evidence reveals a role for microRNAs (miRNAs) in the fine-tuning regulation ofosteogenesis (osteomiRs) suggesting that they can be potential targets for skeleton diseases treatment However the functions ofosteomiRs during differentiation of hAM-MSCs to osteogenic lineage are poorly understood In this investigation we discovereda novel miRNAs expression signature corresponding to the matrix maturation (preosteoblast) and mineralization (matureosteoblast) stages of dexamethasone-induced osteoblastic differentiation of hAM-MSCs Comprehensive miRNAs profiling usingTaqMan Low Density Arrays showed that 18 miRNAs were significantly downregulated whereas 3 were upregulated in thematrix maturation stage (7 days after osteogenic induction) in comparison to undifferentiated cells used as control Likewise 47miRNAs were suppressed and 25 were overexpressed at mineralization stage (14 days after osteogenic induction) in comparison toosteoprogenitors cells Five out 93 miRNAs (miR-19b-3p miR-335-3pmiR-197-3p miR-34b-39 andmiR-576-3p) were regulated atboth 7 and 14 days suggesting a role in coordinated guidance of osteoblastic differentiation Exhaustive bioinformatic predictionsshowed that the set ofmodulatedmiRNAsmay targetmultiple genes involved in regulatory networks driving osteogenesis includingkey members of BMP TGF-120573 and WNT120573-catenin signaling pathways Of these miRNAs we selected miR-204 a noncodingsmall RNA that was expressed at matrix maturation phase and downregulated at maturation stage for further functional studiesInterestingly gain-of-function analysis showed that restoration of miR-204 using RNAmimics at the onset of mineralization stagedramatically inhibited deposition of calcium and osteogenic maturation of hAM-MSCs Moreover in silico analysis detected aconserved miR-204 binding site at the 31015840UTR of TGF-120573R2 receptor gene Using luciferase assays we confirmed that TGF-120573R2 isa downstream effector of miR-204 In conclusion we have identified a miRNAs signature for osteoblast differentiation of hAM-MSCs The results from this study suggested that these miRNAs may act as potential inhibitors or activators of osteogenesis Ourfindings also points towards the idea that miR-204TGF-120573R2 axis has a regulatory role in differentiation of hAM-MSCs committedto osteoblastic lineage

HindawiBioMed Research InternationalVolume 2019 Article ID 8987268 13 pageshttpsdoiorg10115520198987268

2 BioMed Research International

1 Introduction

Osteoporosis disease and bone fractures are among themajorpublic health concerns worldwide as they produce a declinein patient mobility and a significant increase in medicalcare costs [1] A recent review on this subject summarizethe evidence indicating that both skeletal development andbone regeneration depend on the proper functional dif-ferentiation of mesenchymal stem cells to osteoblasts [2]Human mesenchymal stem cells are pluripotent stem cellsthat undergo a multistage differentiation process in whichthey proliferate and differentiate into osteocytes adipocytesand chondrocytes [3] From the past decade human amnioticmembrane-derived mesenchymal stem cells (hAM-MSCs)have been used for in vivo construction of tissue-engineeredcartilage bone and other soft tissues and they have becomea promising tool for the treatment of bone diseases [4]Osteoblastic differentiation from hMSCs is a highly special-ized process involving the coordinated activation ofWNT120573-catenin FGF and BMPsTGF-120573 signaling pathways [5 6]which activate runt-related transcription factor 2 (RUNX2)and Osterix (OSXSp7) transcription factors among othersthat in turn activate a complex transcriptional program driv-ing the differentiation of osteoprogenitors towards functionalmature osteocytes [7] Therefore elucidating the molecularmechanisms that regulate the osteoblastic differentiation isessential to help understand the molecular mechanisms ofosteogenesis but it also may be a guide for the developmentof novel therapies for the treatment of bone lesions andosteoporosis

MicroRNAs (miRNAs) are evolutionary conservedsingle-stranded small RNA molecules of 21-25 nucleotidesin length that function as negative regulators of geneexpression [8] MiRNAs function as guide molecules inposttranscriptional gene silencing acting by complementarybinding with the 31015840 untranslated region (UTR) of specifictranscripts leading to target mRNA degradation andortranslational repression in P-bodies [9] Recent evidencessuggests an important role for miRNAs in the fine-tuningregulation of genes involved in osteogenesis indicating thatthese so-called osteomiRs can be promising therapeutictargets for skeleton diseases [10ndash12] However the functionsof miRNAs during differentiation of hAM-MSCs toosteogenic lineage are still poorly understood Despitethis interest comprehensive miRNAs profiling studiesassociated with differentiation of hAM-MSCs to osteoblastsare scarce thus the relevance of small RNAs in osteogenesisis not yet known In this investigation we reported a novelmiRNAs expression signature in two stages of hAM-MSCsdifferentiation to osteoblasts and functionally characterizedthe role of miR-204 in this cellular process Implications onthe molecular mechanisms regulating the differentiation ofhAM-MSCs and the potential therapeutic applications arediscussed

2 Materials and Methods

21 Isolation of Stem Cells from the Human AmnioticMembranes Caesarean-delivered term placentas (n=4) were

collected from healthy donor mothers Procedures wereapproved by the Ethics Committee of the University HospitalldquoDr Jose Eleuterio Gonzalezrdquo and each donor gave herconsent Pieces of amniotic membrane (10 x10 cm) weresubjected to two enzymatic digestions by adding (i) 0125trypsin05 mM EDTA solution at 37∘C for 30min and (ii)100 UmL collagenase type II and 3mM calcium chloridein the Dulbeccorsquos Modified Eaglersquos Medium (DMEM) for2 h at 37∘C followed by washing with PBS The resultingcell suspension was filtered and cells were seeded in 25-cm2 flasks in DMEM containing 10 fetal bovine serum(FBS) and 100 UmL penicillin 100mgmL streptomycinand 025mgmL amphotericin B The resulting hAM-MSCswere cultured in a humidified 5 CO

2atmosphere at 37∘C

until 90 confluence All experiments were carried out usinghAM-MSCs below 10 passages

22 Fluorescent Activated Cell Sorting After establishing thehAM-MSCs population a CD44+CD73+CD105minus (CD105minus)subpopulation was enriched by Fluorescent Activated CellSorting (FACS Aria BD) hAM-MSCs were stained withhuman PE-conjugated CD73 APC-conjugated CD105 andPercP-Cy55-conjugated CD44 (Miltenyi Biotec) and pos-itive cells were collected After centrifugation at 400 g for5min they were plated in fresh culture medium for expan-sion and further molecular characterization

23 OsteogenicDifferentiation of hAM-MSCs Theosteogenicdifferentiation was induced as previously described [13]Briefly isolates of hAM-MSCs (2x103 cellscm2) were grownin osteogenic medium (DMEM supplemented with 10FBS 10mM 120573-glycerophosphate 025mM ascorbic acidand 10minus8M dexamethasone) Cultures were maintained withmedium changes every 2-3 days Cells were stained withAlizarin Red S dye reactive (pH 43) to evaluate calcium-rich deposits and monitor the differentiation process asdescribed [14] Cells were imaged and birefringence stainingwas quantified using the FluorChem 9900 system

24 Western Blot Assays For immunoblotting hAM-MSCswere rinsed with cooled PBS and lysed at room temper-ature for 10min in 1mL of RIPA buffer (20mM Tris-HCl pH 75 150mM NaCl 1mM EGTA 1 NP-40 1sodium deoxycholate 25mM sodium pyrophosphate 1mMb-glycerophosphate 1mMNa

3VO4 and 1mgmL leupeptin)

containing the Complete Protease Inhibitor (05mM phenyl-methylsulfonyl fluoride 10mgmL leupeptin 10mgmLaprotinin 5mgmL pepstatin 10mgmL soybean trypsininhibitor and 05mM dithiothreitol ROCHE MolecularBiochemicals) Equal amounts of protein (30120583g) were run on10 SDS-PAGE gels and transferred to a PVDF membrane(Millipore Corporation) The membrane was blocked for60min at room temperature with TBST-1 (137mM NaCl20mMTris and 01Tween-20 (pH76)) containing 5BSA(Sigma-Aldrich) and then incubated overnight at 4∘C withrabbit anti-human COL1A2 (11000) antibodies The mem-brane was washed in TBST-1 and incubated with horseradishperoxidase-conjugated goat anti-rabbit IgG antibody (13000

BioMed Research International 3

Zymed) Antibody-antigen complexes were revealed usingthe ChemiLucent system (Chemicon) and imaged Densito-metry analysis was performed using the myImage Analysissoftware

25 MicroRNAs Profiling Using TaqMan Low Density ArraysTotal RNA (70ng) was isolated from undifferentiatedhAM-MSCs (time 0) and cells collected after 7 and 14days of osteogenic induction using the TRIzol reagent(Invitrogen) MiRNAs expression profiles were obtainedusing the stem-loop qRT-PCR-based TaqMan Low Den-sity Arrays (TLDAs) covering 677 human mature miR-NAs (httpswwwthermofishercomordercatalogproduct4398965) Briefly expression of miRNAs was performed byreverse transcription and quantitative real-time polymerasechain reaction using the Megaplex TaqMan Low DensityArrays v20 system 4398965 (Applied Biosystems FosterCity CA) as described by the manufacturer In order todetect low abundant miRNAs a preamplification step wasincluded For this 125120583l TaqMan PreAmp Master Mix wasincubated with 25120583l Megaplex PreAmp Primers and waterup to 225120583l Then 25120583l of RT reaction was added to thePreAmp mix and sequences were amplified following thenext program 95∘C for 10min (enzyme activation) 55∘C for2min (annealing) 72∘C for 2min (extension) 95∘C for15 s(denaturation) 69∘C for 4min (annealingextension) and99∘C for 10min (enzyme inactivation) Then preamplifiedproducts were loaded into the TLDAs for PCR reactions andamplification signal detection was performed in the 7900FAST real-time thermal cycler (ABI) Tests were normalizedusing RNU44 as control

26 Reverse Transcription and Real-Time Polymerase ChainReaction Quantitative real-time RT-PCR (qRT-PCR) anal-ysis for miRNAs expression was performed using the Taq-Man MicroRNA Assay kits (ThermoFisher) Total RNA(100ng) was reverse transcribed using a looped RT specificprimer dNTPs (100mM) reverse transcriptase MultiScribe(50 U120583l) 10X buffer RNase inhibitor (20 U120583l) and 416 120583lRNase-free water Retrotranscription reaction (115) wasmixed with master mix TaqMan (Universal PCRMaster MixNo AmpErase UNG 2X) and the corresponding specificTaqMan PCR probe PCR reaction was performed in aGeneAmpSystem9700 (Applied Biosystems) as follows 95∘Cfor 10min and 40 cycles at 95∘C for 15 s and 60∘C for 1minTests were normalized using RNU44 as endogenous control

27 Prediction of Gene Targets and Gene Ontology AnalysisTarget genes of miRNAs were predicted using TargetScanPicTar5miRanda andDIANAmicroT softwareOnly targetspredicted by two algorithms were included in further analy-ses Cellular pathways and processes potentially affected bylet-7c-3p were predicted using DAVID 67 software

28 Transfection of miR-204 Mimics Seven days after induc-tion of osteogenic differentiation hAM-MSCs were trans-fected with miR-204 mimics (40 nM) and scramble (40 nM)sequence (AM17110ThermoFisher) as negative control using

siPORT amine transfection agent (Ambion) Briefly miR-204mimics was diluted in 25120583l of Opti-MEM to 40 nM concen-trations and then added to wells containing cultured cellsin 450120583l of DMEM After 48 h incubation total RNA wasextracted using TRIzol and efficacy of miR-204 restorationwas evaluated by qRT-PCR using specific stem-looped RToligonucleotide and TaqMan probe as implemented in theTaqMan MicroRNA assays protocol

29 Effects of miR-204Mimics on Osteogenic Differentiation ofhAM-MSCc Briefly 11 days after of osteoblastic differentia-tion the cells were transfected with 30 nMofmiR-204mimicsusing siPORT amine transfection agent (Ambion) and 48 hafter transfection the calcium-rich deposits were evaluatedwith Alizarin Red S dye reactive (pH 43) which binds tointracellular calcium ions Cells were incubated with dyefor 15min at room temperature and washed tree times withPBS 1x and then cells were imaged and birefringent stainingwas quantified using FluorChem 9900 for comparisons ofpixel intensity between control nontransfected and miR-204mimics transfected hAM-MSCs

210 Luciferase Gene Reporter Assays A DNA fragment ofthe 31015840UTR of TGF-120573R2 gene containing the predicted miR-204 binding site and as well as a mutated version of the seedregion were cloned into the p-miR-report vector (Ambion)downstream of the luciferase gene Constructs were verifiedthrough automatic sequencing Then recombinant pmiR-LUC- TGF120573R2 wild type and pmiR-LUC-TGF-120573R2 mutantplasmids were transfected into hAM-MSCs cells using lipo-fectamine 2000 (Invitrogen) At 24 h after transfection cellswere cotransfected with miR-204 mimics and scramble(40 nM) and incubated for 24 h Finally firefly and Renillareniformis luciferase activities were measured by the Dual-Glo luciferase Assay (Promega) using a Fluoroskan AscentMicroplate Fluorometer and firefly luciferase activity wasnormalized with Renilla reniformis luciferase data

211 Statistical Analyses Experiments were performed threetimes by triplicate and results were represented asmean plusmn SDOne-way analysis of variance (ANOVA) was used to comparethe differences between means A plt005 was considered asstatistically significant

3 Results

31 Osteoblastic Differentiation of hAM-MCS In Vitro Previ-ously we have isolated and characterized the biological eventsunderlying the osteoblastic differentiation of the hAM-MSCssubpopulation studied here and established the temporalityof proliferation (undifferentiated cells time 0) matrix mat-uration (preosteoblast 7 days) and mineralization (matureosteoblast 14 days) stages [13 14] To assess the ability ofhAM-MSCs isolates to differentiate into osteogenic lineagethe calcium deposition and expression of the mineralizationstage marker collagen type I-alpha (COL1A2) were mea-sured The hAM-MSCs were induced to differentiation inosteogenic media and maintained for three weeks at 37∘C


7 140

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Figure 1 Osteogenic differentiation of AM-hMSCs (a) Alizarin Red staining showing mineral deposition of calcium in undifferentiated AM-hMSCs (time 0) and after 7 and 14 days of differentiation to osteoblastic lineage (b) Quantification of the calcium deposition shown in (a)(c) Western blot assays using whole protein extract and antibodies raised against COL1A2 protein after 0 7 and 14 days of induction Datawere normalized using control GAPDH expression (d) Densitometry analysis of the immunodetected bands in (c) Data represent the meanplusmn SD of three independent experiments (lowastlowastplt005 lowast lowast lowastplt0001)

Cultures were stained with Alizarin Red at 0 7 and 14days after induction and imaged Results showed a gradualand significant time-dependent increase of calcium-richdeposits in preosteoblasts and osteoblasts in comparisonto undifferentiated control cells (time 0) (Figures 1(a) and1(b)) Congruently immunoblot assays revealed that theCOL1A2 protein expression was significantly increased at 7and 14 days after induction relative to undifferentiated controlcells (Figures 1(c) and 1(d)) These data confirmed that theisolates of hAM-MSCs used in this study efficiently initiatedthe process of differentiation to osteoblasts as previouslydescribed [13 14]

32MicroRNAsExpression Signatures of hAM-MSCsDifferen-tiation to Osteoblastic Lineage To evaluate the potential con-tribution ofmiRNAs in osteoblastmaturation of hAM-MSCswe comprehensively analyzed 667 miRNAs using stem-loop qRT-PCR in TaqMan Low Density Arrays (TLDAs) aspreviously described [15] Undifferentiated hAM-MSCs wereinduced to osteoblastic lineage in differentiation media andglobal miRNAs profiles were assessed at matrix maturation(7 days after osteogenic induction) and mineralization (14

day after osteogenic induction) stages After comparative 2-ΔΔCt analyses a total of 93 miRNAs significantly modulated(| log 2(TN)| gt 10 plt005) were identified Of these 18miRNAs showed a significant downregulation whereas threewere upregulated at matrix maturation stage in comparisonto undifferentiated cells (Table 1) Likewise 47 miRNAs weresignificantly suppressed and 25 were overexpressed at miner-alization stage (Table 2)This panel of up- and downregulatedmiRNAs was able to separate the preosteoblast and matureosteoblast groups relative to undifferentiated cells as depictedin the 2-way unsupervised hierarchical clustering shownin Figures 2(a) and 2(b) A volcano plot representation ofmiRNAs deregulated at 7 and 14 days is shown for easy visu-alization in Figures 2(c) and 2(d) respectively Interestinglyfive out 93 miRNAs were modulated at both times miR-19b-3p was upregulated whereas miR-335-3p miR-197-3p miR-34b-3p and miR-576-3p were downregulated suggesting animportant role in the coordinated guidance of the osteoblasticdifferentiation process Then we proceeded to validate thechanges in expression of four selected miRNAs using specificprobes in qRT-PCR assays Representative data at 14 daysafter induction showed that levels of miR-21 miR-125b


Table 1 MicroRNAs expression profile of hAMSC after 7 days of differentiation to osteoblasts

Down-regulated Fold change (log2) p-value Chromosomal locationmiR-425-5p -890 0003 3p2131miR-223-5p -253 0023 Xq12miR-150-5p -252 0003 19q1333miR-342-5p -173 0043 14q322Hsa-miR-197-3p -152 0039 1p133miR-99b-5p -151 0003 19q1341miR-219a-1-3p -150 0002 6p2132miR-193a-3p -148 0009 17q112miR-422a -148 0009 15q2231miR-576-3p -147 0014 4q25miR-423-5p -146 0003 17q112miR-362-3p -144 0003 Xp1123miR-140-3p -144 0001 16q221miR-34b-3p -104 0015 7q322miR-29b-2-5p -104 0007 1q322miR-335-3p -101 0010 5q333miR-148b-5p -099 0001 7p152mir-770-5p -098 0026 14q322

Up-regulated Fold change(log2) p-value Chromosomal location

miR-424-3p 105 0015 Xq263miR-769-5p 106 0009 19q1332miR-19b-3p 151 0010 13q313miRNAs previously related to osteogenesis are denoted in bold

miR-204 and miR-123 detected by qRT-PCR were similar tothose found in TLDAs platform (Figure 3)

33 Genes and Signaling Pathways Potentially Impacted byRegulated OsteomiRs To gain a comprehensive understand-ing about the impact of miRNAs abundance changes duringthe osteogenic differentiation of hAM-MSCs we performedliterature searches for reports on the set of regulatedmiRNAsResults indicated that a number of miRNAs have well-knownfunctions in osteogenesis For instance five osteomiRs (miR-223-5p miR-193a-3p miR-29b-2-5p miR-335-3p and miR-148b-5p) with a significant repression at 7 days of differen-tiation have been related to osteogenesis (Table 1) Notablyprevious studies showed that restoration of miR-223-5pimpaired the osteogenic differentiation of ST2 mesenchymalstem cells by targeting FGFR2 [16] Likewise nine of overex-pressed miRNAs (miR-119a-5p miR-21-5pmiR-145-5p miR-503-5pmiR-125b-5pmiR-181a-5pmiR-155-5p let-7f-5p andmiR-22-3p) at 14 days have been reported in osteogenesis(Table 2) For example miR-119a-5p was found upregulatedduring osteogenic differentiation of hMSCs and nanoparticledelivery of miR-119a-5p resulted in enhanced maturation ofhMSCs in vitro [17] Importantly most of miRNAs detectedhere as modulated have not been previously involved inosteoblastic differentiation suggesting that they may repre-sent novel miRNAs with potential therapeutic applications

To understand the biological impact of miRNAs varia-tions during early and late stages of osteogenesis we com-putationally predicted their gene targets using a combina-tion of TargetScan PicTar5 miRanda and DIANA microTprediction tools Exhaustive bioinformatic analysis yieldeda large list of putative gene targets In order to associatemiRNAs function to specific cellular pathways and processesthe complete list of putative gene targets was filtered usingthe Gene Ontology (GO) terms function as implemented inDAVID databases In Tables 3 and 4 we present an overviewof the main signaling pathways impacted by the completedataset of miRNAs regulated at both 7 and 14 days afterosteogenic differentiation For instance predictions showedthat 17 miRNAs modulated at 7 days may be targeting atleast 42 different genes involved in the osteogenesis-relatedTGF-120573 signaling pathway In addition 20 miRNAs werepredicted to target 65 genes involved in signaling pathwaysregulating pluripotency of stem cells (Table 3) These dataconfirmed that an important number of modulated miRNAsare related to known cellular pathways associated with hAM-MSCs osteogenic differentiation

34 Overexpressed OsteomiRs Target Multiple Genes Involvedin TGF-120573 and WNT120573-Catenin Pathways In order to bet-ter characterize the impact of modulated miRNAs on the


Table 2 MicroRNAs expression profile of hAM-MSCs after 14 days of differentiation to osteoblasts

Down-regulated Fold change (log2) p-value Chromosomal locationmiR-302b-3p -550 0008 4q25miR-223-3p -490 0008 Xq12miR-126-3p -478 0007 9q343miR-890 -453 0020 Xq273miR-548a-3p -452 0017 6p223miR-217 -449 0009 2p161miR-302a-3p -448 0009 4q25miR-517a-3p -448 0009 19q1342miR-369-3p -446 0008 14q3231miR-521 -441 0006 19q1342miR-335-3p -395 0008 7q322miR-888-5p -395 0002 Xq273miR-1-3p -392 0002 20q1333miR-511-5p -382 0004 10p1233miR-518e-3p -379 0005 19q1342miR-141-3p -358 0004 12p1331miR-545-3p -347 0009 Xq132miR-146b-3p -344 0008 10q2432miR-616-3p -333 0017 12q133miR-133b -328 0033 6p122miR-142-5p -327 0034 17q22miR-216b-5p -323 0036 2p161miR-491-3p -319 0038 9p213miR-371a-3p -310 0046 19q1342miR-142-3p -291 0020 17q22miR-127-5p -290 0022 14q322miR-204-5p -289 0028 9q2112miR-381-3p -289 0021 14q3231miR-570-3p -287 0020 3q29miR-202-3p -269 0043 10q263miR-197-3p -254 0033 1p133miR-32-5p -250 0009 9q313miR-138-5p -249 0005 3p2132miR-372-3p -247 0011 19q1342miR-190a-5p -245 0007 15q222miR-34b-3p -188 0008 11q231miR-636 -156 0014 17q251miR-539-5p -151 0001 14q3231miR-186-5p -150 0003 1p311miR-576-3p -150 0002 4q25miR-542-3p -150 0011 Xq263miR-374a-5p -149 0034 Xq132miR-449b-5p -149 0012 5q112miR-296-3p -148 0004 20q1332miR-370-3p -146 0032 14q3231miR-409-3p -095 002 14q3231miR-505-3p -090 0017 Xq271


Table 2 Continued

Up-regulated Fold change (log2) p-value Chromosomal locationmiR-136-3p 105 0013 14q322let-7i-3p 105 0018 12q141miR-191-3p 106 0009 3p2131miR-337-5p 146 0001 14q322miR-27b-3p 146 0009 9q2232miR-21-5p 148 0010 17q231miR-145-5p 149 0001 5q32miR-503-5p 149 0008 Xq263miR-19b-3p 149 0027 13q313miR-345-5p 150 0006 14q322miR-125b-5p 151 0001 21q211miR-744-5p 151 0003 17p12miR-20b-5p 153 0005 Xq262miR-143-3p 154 0002 5q32miR-181a-5p 191 0037 9q333miR-181c-5p 193 0044 19p1312miR-181a-3p 239 0023 9q333miR-155-5p 250 0017 21q213miR-27a-3p 251 0001 19p1312miR-199a-5p 288 0034 19p132miR-148b-3p 473 0029 12q1313miR-431-5p 646 0014 14q322let-7f-5p 650 0019 9q2232let-7a-5p 789 0024 9q2232miR-22-3p 851 0003 17p133miRNAs previously related to osteogenesis are denoted in bold

Table 3 Cellular signaling pathways regulated by miRNAs regulated at 7 days of differentiation to osteoblasts

KEGG pathway p-value Number of target genes Number of miRNAsTGF120573 pathway 00002 42 17PI3K-Akt pathway 00005 152 20Hippo pathway 00006 68 20FoxO pathway 00013 68 20Ubiquitin mediated proteolysis 00015 67 18Ras pathway 00020 105 21ErbB pathway 00024 46 19Rap1 pathway 00024 98 21Phosphatidylinositol signaling system 00028 43 16AMPK pathway 00062 60 18mTOR pathway 00078 33 18Signaling pathways regulating pluripotency of stem cells 00099 65 20Focal adhesion 00136 92 20MAPK pathway 00259 111 21Calcium pathway 00322 78 17

osteogenesis process we next focused on the set of upreg-ulated miRNAs and found that they may attenuate a largelist of key genes involved in TGF-120573 and WNT120573-cateninpathways which are critical for osteogenic differentiationand bone formation Several of these overexpressed miRNAs

may enhance or impair the osteoblasts differentiation bytargeting their cognate activator or repressor genes involvedin TGF-120573 signaling such as RUNX2 B-catenin ATF4and OSX transcription factors which act as activators ofosteogenic differentiation as well as FIAT p21 SMURF and


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log2(Fold Change)

(d)

Figure 2 Expression profiling of miRNAs modulated during osteoblastic differentiation of hAM-hMSCs (a) Unsupervised hierarchicalclustering analysis displaying the differential expression of miRNAs at 14 days and (b) 7 days of osteoblastic differentiation relative to time0 (undifferentiated hAM-MSCs) The heatmap (Spearman correlation Euclidean distance) represents a cluster analysis of the logarithm oftransformed ΔΔCt values of the differentially expressed microRNAs Color key upregulatedmiRNAs (red) downregulatedmiRNAs (green)(c and d) Volcano plot representations of miRNAs modulated at 7 and 14 days The y-axis represents the mean expression value of log10 (P-value) and the x-axis displays the log2-fold change value Upregulated and downregulated miRNAs are shown in red and green respectivelyBlack dots indicate genes with no significant change in expression

MSX2 which function as repressors (Figure 4) For instancemiR-19b-3p may stimulate differentiation by targeting FIATwhich is a repressor of the osteogenesis-activator ATF4(Figure 4) Likewise at both early and late times of osteogenicdifferentiationmiR-19b-3pmay also enhance osteogenesis bytargeting AXIN2 which is a repressor of 120573-catenin A more

complex regulation may be occurring for some genes Atlate stages of osteogenic differentiation let-7f-5pmiR-145-5pandmiR-148b-3pwere predicted to cooperatively target p21 arepressor of RUNX2 which could stimulate osteoblasts min-eralization In contrast miR-150-5p could inhibit osteoblasticdifferentiation by targeting 120573-catenin in undifferentiated


TLDA

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miR

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miR

-204

-5p

Figure 3Quantitative RT-PCR assays Validation of expression levels of fourmiRNAsmodulated after 14 days of differentiation to osteoblasticlineage using qRT-PCR TaqMan assays (black bars) in comparison with data obtained from TLDA (grey bars) Data were expressed as meanplusmn SD

Table 4 Cellular signaling pathways regulated by miRNAs modulated at 14 days of differentiation to osteoblasts

KEGG pathway p-value Number of target genes Number of miRNAsECM-receptor interaction 803e-11 63 56Hippo signaling pathway 262e-07 115 64Focal adhesion 311e-07 159 64ErbB signaling pathway 345e-07 72 60TGF-beta signaling pathway 550e-07 63 59PI3K-Akt signaling pathway 854e-07 247 64Ras signaling pathway 229e-05 160 63Wnt signaling pathway 770e-05 105 63MAPK signaling pathway 00002 179 63Rap1 signaling pathway 00002 152 62Signaling pathways regulating pluripotency of stem cells 00004 103 65Protein processing in endoplasmic reticulum 00021 116 59FoxO signaling pathway 00021 96 62Adherens junction 00064 58 55Ubiquitin mediated proteolysis 00123 98 60mTOR signaling pathway 00128 46 58Gap junction 00139 64 60cGMP-PKG signaling pathway 00192 113 63cAMP signaling pathway 00211 134 63p53 signaling pathway 00228 50 55Notch signaling pathway 00430 36 35AMPK signaling pathway 00467 87 61

cells Similarly at late stage of maturation the dual targetingof Osteryx (OST) by miR-449b-5p and miR-339b-5p couldresult in osteogenesis impairment (Figure 4)

In addition we found that a number of target genes couldbe recognized by more than one miRNA indicating an exten-sive target gene redundancy and at the same time revealingcomplex and intricate miRNAmRNA regulation networksAs an example in Figure 5 we depicted a miRNAmRNAinteractions network for miRNAs upregulated at 14 days andtheir predicted targets with known inhibitory functions inTGF-120573 and WNT120573-catenin pathways

35 MiR-204 Inhibits the Osteoblast Differentiation by Tar-geting TGF-120573R2 To initiate the study of the molecularmechanisms underlying the regulation of hAM-MSCs differ-entiation we focused on miR-204 a noncoding small RNAoverexpressed at the matrix maturation stage and downreg-ulated at the maturation stage Bioinformatics predictionsshowed that miR-204 may target multiple key genes involvedin osteogenesis promotion including ACVR1C ACVR2BTGBR2 BMPR1A BMPR2 SMAD2 and SMAD5 amongother thus it could have a relevant role in osteoblast dif-ferentiation To evaluate the role of miR-204 in osteogenic


AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4

Osteoblastic differentiation

Proliferation Maturation Mineralization

Undifferentiated hAM-MSCs (0 day) Pre-osteoblasts (7 day) Osteoblasts (14 day)

SMURF1

miR-19b-3p

miR-769-5p miR-21-5p

miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin

Inhibition of differentiationNegative regulation by miRNAs

Stimulation of differentiation

Negative regulation by validated miRNAs

TGF- pathway

Wnt-catenin pathway

Potential pathways and transcription factors affected by upregulated miRNAs

Transcription factor promoters

Transcription factor inhibitors

Let-7f-5p

SMAD1 SMAD5

miR-148b-3p

miR-770-5p miR-29b-5p

miR-770-5p SMAD1 SMAD5

miR-150-5p

miR-223-5pmiR-223-5p

miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p

miR-449b-5pmiR-197-3p

FIAT MSX2KLF4SMURF2 SMURF2

miR-339b-5p



Figure 4 Model for regulation of osteogenesis by miRNAs impacting TGF-120573 and WNT120573-catenin pathways The schema shows a subsetof modulated miRNAs at 7 (maturation) and 14 (mineralization) days after the dexamethasone-induced differentiation of AM-hMSCs toosteoblasts relative to control undifferentiated cells (time 0) Selected miRNAs and their predicted targets involved in WNT120573-catenin andTGF120573 signaling pathways are depicted Proteins that stimulate and repress the osteogenic differentiation process are denoted with green andred lines respectively Molecular functions of gene targets and corresponding pathways are denoted in colors

differentiation of hAM-MSCs we first restored its expressionusing RNA mimics at the onset of mineralization stage(Figure 6(a)) Data showed that miR-204 inhibited deposi-tion of calcium and osteogenic maturation of hAM-MSCs(Figure 6(b)) Then we searched for potential downstreamtargets of miR-204 using different miRNA target predictiontools In silico analysis detected a conserved miR-204 bind-ing site at the 31015840UTR of TGF-120573R2 receptor gene Thus itwas reliable to propose that restoration of miR-204 levelsmay negatively regulate osteoblast differentiation throughdirect targeting of TGF-120573 signaling To corroborate thishypothesis we performed luciferase reporter gene assaysA DNA fragment of the 31015840UTR containing the TGF-120573R2binding site for miR204 was cloned downstream of theluciferase gene into the pmiR report vector (Figure 6(d)) Inaddition point mutations in the predicted miR-204 binding

site of the 31015840UTR were included in the analysis RecombinantpmiR-LUC-TGF-120573R2 plasmid was transfected during dif-ferentiation of hAM-MSCs and luciferase activity was ana-lyzed after 24 h of transfection Data showed that ectopicexpression of miR-204 and cotransfection of pmiR-LUC-TGF-120573R2 construct resulted in a significantly reduction ofthe relative luciferase activity in comparison with controls(Figure 6(d)) When mutated sequences of the 31015840UTR TGF-120573R2were assayed no significant changes in luciferase activitywere found indicating that miR-204 binding was specific

4 Discussion

Human MSCs were initially isolated from bone marrow[18] and they have been later identified in several fetaltissues such as the liver bone marrow and pancreas in


hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs

1-14 miRNAs +15 miRNAs

Figure 5 Representative miRNAmRNA interaction network for osteogenic differentiation of hAM-MSCs at mineralization stage The pairsmiRNAsmRNA in the interaction network were generated based on the expression levels of upregulatedmiRNAs at mineralization stage (14days) and their predicted gene targets with known inhibitory functions in the TGF-120573 pathway The solid color lines connecting moleculesrepresent miRNA-mRNA interactionThe interaction network showed deregulated subnetworks that are clearly separate depending on thenumber of targets for each miRNA for instance genes potentially modulated by 1-3 upregulated miRNAs (in orange) and others by 4-6miRNAs (in green)

the endothelial umbilical vein and in the preterm blood ofthe fetus [19] The International Society of Cellular Therapy(ISCT) has described MSCs as cells that express the surfacemarkers CD73 CD105 CD90 and CD44 in the absence of

hematopoietic stem cell markers and differentiate into mes-enchymal lineages such as osteoblasts adipocytes and chon-droblasts in vitro [4] Several miRNAs modulate osteogenicdifferentiation thus they have been dubbed as osteomiRs

12 BioMed Research InternationalRe

lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)

Firefly luciferase gene

pmiR-Report

hsa-miR-204 UCCGUAUCCUACUGUUUCCCUU

UCACUACUAUACAUAAAGGGAA

UCACUACAUACAUAUACGAGAATGF-R2 mutated

TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204

plt0001lowastlowastlowast

lowastlowastlowast

3

(d)

Figure 6 MiR-204 inhibits the osteogenic differentiation of AM-hMSCs by targeting TGF-120573R2 (a) Relative expression of miR-204 aftertransfection of RNAmimics inAM-hMSCs at 13 days after dexamethasone-inducedosteoblastic differentiation (b)Quantification of AlizarinRed staining for calcium deposition (bottom images) in nontransfected AM-hMSCs (control) or transfected cells with miR-204 mimics (c)Schematic representation of p-miR report construct containing the 31015840UTR of TGF-120573R2 gene cloned downstream of the firefly luciferase geneSeed sequence is indicated in blue shaded box Point mutations in the miR-204 binding site of 31015840UTR of TGF-120573R2 gene are denoted in bold(d) Luciferase assays in AM-hMSCs cotransfected with miR-204 mimics and the constructs described in (c) Nontransfected and scrambletransfected cells were used as controls Data represent the mean plusmn SD of three independent experiments (lowast lowast lowastplt0001 lowastlowastplt005)

For instance miR-125b regulates osteoblastic differentiationof human MSCs through targeting of BMPR1b [20] whereasmiR-223 suppresses osteoblast differentiation by inhibitingDHRS3 [21] However miRNAs functions during differen-tiation of hAM-MSCs to osteogenic lineage have not beencompletely explored

In order to contribute to the understanding of miRNAsroles in osteogenic differentiation of hAM-MSCs here weperformed a miRNAs profiling in MSCs obtained fromhuman placenta Our findings indicated a novel miR-NAs expression signature corresponding to preosteoblastand mature osteoblast stages of dexamethasone-inducedosteoblastic differentiation of hAM-MSCs Bioinformaticpredictions showed that these miRNAs may target multiplegenes involved in regulatory networks driving osteogenesisincluding members of BMP TGF-120573 and WNT120573-cateninsignaling pathways Importantly our results confirmed that anumber ofmiRNAs have well-known functions in osteogene-sis For instance at 7 days of differentiation we found a signif-icant repression ofmiR-29b relative to osteo-progenitors cells(Table 1) Likewise ectopic expression of miR-29b resultedin low levels of osteogenesis inhibitors CDK6 and HDAC4and stimulated the osteogenic differentiation of unrestrictedsomatic stem cells from human cord blood [22] At 14 dayswe identified that miR-119a-5p and miR-22-3p previouslyreported in osteogenesis are overexpressed in hAM-MSCs(Table 2) Similarly it was described that upregulation ofmiR-22 promotes the osteogenic differentiation of humanadipose tissue-derived mesenchymal stem cells by repressingthe osteogenesis repressorHDAC6 [23]These data suggestedthat miRNAs detected here as regulated could be regulatingthe differentiation of hAM-MSCs importantly most of miR-NAs detected here as modulated have not been previouslyconnected to osteoblastic differentiation suggesting that they

may represent novel miRNAs with potential therapeuticapplications

In addition we found that the set of upregulated miRNAsmay regulate a large list of key genes involved in TGF-120573 and WNT120573-catenin pathways which are critical forosteogenic differentiation and bone formation This panel ofderegulatedmiRNAs represents a guide for further functionalcharacterization of the osteogenesis process To go beyondthe miRNAs profiling we initiated the functional analysis ofone deregulated miRNAs related to osteogenesis Our dataindicated that restoration of miR-204 a noncoding smallRNA that was expressed at matrix maturation stage anddownregulated in maturation stage impaired differentiationat least by targeting TGF-120573R2 which is regulator of RUNX2gene the master regulator of stem cells differentiation

5 Conclusions

In summary here we reported a novelmiRNAs signature dur-ing differentiation of hAM-MSCs to osteogenic lineagewhichmay target multiple genes involved in regulatory networksdriving osteogenesis includingmembers of BMP TGF-120573 andWNT120573-catenin signaling pathways Our findings suggestedan unexpected regulatory role for the miR-204TGF-120573R2 axisin differentiation of hAM-MSCs committed to osteoblasticlineageThese data may serve as a guide for further functionalanalysis focused in the implementation of osteomiRs as noveltherapeutic tools in bone diseases

Data Availability

All data used to support the findings of this study are includedwithin the article The raw data obtained from the miRNAs


profiling in TLDAs platform used to support the findings ofthis study are available from the corresponding author uponrequest

Conflicts of Interest

The authors declare no conflicts of interest

Acknowledgments

We thanks the Universidad Autonoma de la Ciudad deMexico Universidad Autonoma de Sinaloa and UniversidadAutonoma de Nuevo Leon for infrastructure and technicalsupportWewould like to thank Consejo Nacional de Cienciay Tecnologıa CONACYT Mexico for partially funding thisstudy (Grant 222335)

References

[1] U A Al-Sari J Tobias and E Clark ldquoHealth-related quality oflife in older people with osteoporotic vertebral fractures a sys-tematic review and meta-analysisrdquo Osteoporosis Internationalvol 27 no 10 pp 2891ndash2900 2016

[2] F P Barry and J M Murphy ldquoMesenchymal stem cells clinicalapplications and biological characterizationrdquoThe InternationalJournal of Biochemistry amp Cell Biology vol 36 no 4 pp 568ndash584 2004

[3] I Ullah R B Subbarao and G J Rho ldquoHuman mesenchymalstem cells-current trends and future prospectiverdquo BioscienceReports vol 35 no 2 Article ID e00191 2015

[4] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006

[5] M Wu G Chen and Y-P Li ldquoTgf-beta and bmp signaling inosteoblast skeletal development and bone formation home-ostasis and diseaserdquo Bone Research vol 4 16009 pages 2016

[6] Z LDeng K A SharffN Tang et al ldquoRegulation of osteogenicdifferentiation during skeletal developmentrdquo Front Biosci vol13 pp 2001ndash2021 2008

[7] P J Marie ldquoTranscription factors controlling osteoblastogene-sisrdquo Archives of Biochemistry and Biophysics vol 473 no 2 pp98ndash105 2008

[8] D P Bartel ldquoMicroRNAs genomics biogenesis mechanismand functionrdquo Cell vol 116 no 2 pp 281ndash297 2004

[9] B N Davis-Dusenbery and A Hata ldquoMechanisms of control ofmicroRNA biogenesisrdquoThe Journal of Biochemistry vol 148 no4 pp 381ndash392 2010

[10] C Huang J Geng and S Jiang ldquoMicroRNAs in regulation ofosteogenic differentiation of mesenchymal stem cellsrdquo Cell andTissue Research vol 368 no 2 pp 229ndash238 2017

[11] M T Valenti L D Carbonare and M Mottes ldquoRole ofmicroRNAs in progenitor cell commitment and osteogenicdifferentiation in health and disease (Review)rdquo InternationalJournal ofMolecularMedicine vol 41 no 5 pp 2441ndash2449 2018

[12] L Gennari S Bianciardi and DMerlotti ldquoMicroRNAs in bonediseasesrdquoOsteoporosis International vol 28 no 4 pp 1191ndash12132017

[13] M Leyva-Leyva L Barrera C Lopez-Camarillo et al ldquoChar-acterization of mesenchymal stem cell subpopulations from

human amniotic membrane with dissimilar osteoblastic poten-tialrdquo Stem Cells and Development vol 22 no 8 pp 1275ndash12872013

[14] M Leyva-Leyva A Lopez-Diaz L Barrera et al ldquoDifferentialexpression of adhesion-related proteins and MAPK pathwayslead to suitable osteoblast differentiation of human mesenchy-mal stem cells subpopulationsrdquo Stem Cells and Developmentvol 24 no 21 pp 2577ndash2590 2015

[15] A Flores-Perez L A Marchat S Rodrıguez-Cuevas et alldquoDual targeting of ANGPT1 and TGFBR2 genes by miR-204controls angiogenesis in breast cancerrdquo Scientific Reports vol6 no 1 p 34504 2016

[16] X Guan Y Gao J Zhou et al ldquoMiR-223 regulates adipogenicand osteogenic differentiation of mesenchymal stem cellsthrough a CEBPsmiR-223FGFR2 regulatory feedback looprdquoStem Cells vol 33 no 5 pp 1589ndash1600 2015

[17] X Chen S Gu B-F Chen et al ldquoNanoparticle delivery ofstablemiR-199a-5p agomir improves the osteogenesis of humanmesenchymal stem cells via the HIF1a pathwayrdquo Biomaterialsvol 53 pp 239ndash250 2015

[18] A J Friedenstein K V Petrakova A I Kurolesova and G PFrolova ldquoHeterotopic of bone marrow Analysis of precursorcells for osteogenic and hematopoietic tissuesrdquoTransplantationvol 6 no 2 pp 230ndash247 1968

[19] C Campagnoli I A G Roberts S Kumar P R Bennett IBellantuono and N M Fisk ldquoIdentification of mesenchymalstemprogenitor cells in human first-trimester fetal blood liverand bone marrowrdquo Blood vol 98 no 8 pp 2396ndash2402 2001

[20] H Wang Z Xie T Hou et al ldquoMiR-125b regulates theosteogenic differentiation of humanmesenchymal stem cells bytargeting BMPR1brdquo Cellular Physiology and Biochemistry vol41 no 2 pp 530ndash542 2017

[21] S Zhang Y Liu Z Zheng et al ldquoMicroRNA-223 suppressesosteoblast differentiation by inhibiting DHRS3rdquo Cellular Physi-ology and Biochemistry vol 47 no 2 pp 667ndash679 2018

[22] H-I Trompeter J Dreesen E Hermann et al ldquoMicroRNAsmiR-26a miR-26b and miR-29b accelerate osteogenic differ-entiation of unrestricted somatic stem cells from human cordbloodrdquo BMC Genomics vol 14 no 1 2013

[23] S Huang S Wang C Bian et al ldquoUpregulation of miR-22promotes osteogenic differentiation and inhibits adipogenicdifferentiation of human adipose tissue-derived mesenchymalstem cells by repressing HDAC6 protein expressionrdquo Stem Cellsand Development vol 21 no 13 pp 2531ndash2540 2012

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


1 Introduction

Osteoporosis disease and bone fractures are among themajorpublic health concerns worldwide as they produce a declinein patient mobility and a significant increase in medicalcare costs [1] A recent review on this subject summarizethe evidence indicating that both skeletal development andbone regeneration depend on the proper functional dif-ferentiation of mesenchymal stem cells to osteoblasts [2]Human mesenchymal stem cells are pluripotent stem cellsthat undergo a multistage differentiation process in whichthey proliferate and differentiate into osteocytes adipocytesand chondrocytes [3] From the past decade human amnioticmembrane-derived mesenchymal stem cells (hAM-MSCs)have been used for in vivo construction of tissue-engineeredcartilage bone and other soft tissues and they have becomea promising tool for the treatment of bone diseases [4]Osteoblastic differentiation from hMSCs is a highly special-ized process involving the coordinated activation ofWNT120573-catenin FGF and BMPsTGF-120573 signaling pathways [5 6]which activate runt-related transcription factor 2 (RUNX2)and Osterix (OSXSp7) transcription factors among othersthat in turn activate a complex transcriptional program driv-ing the differentiation of osteoprogenitors towards functionalmature osteocytes [7] Therefore elucidating the molecularmechanisms that regulate the osteoblastic differentiation isessential to help understand the molecular mechanisms ofosteogenesis but it also may be a guide for the developmentof novel therapies for the treatment of bone lesions andosteoporosis

MicroRNAs (miRNAs) are evolutionary conservedsingle-stranded small RNA molecules of 21-25 nucleotidesin length that function as negative regulators of geneexpression [8] MiRNAs function as guide molecules inposttranscriptional gene silencing acting by complementarybinding with the 31015840 untranslated region (UTR) of specifictranscripts leading to target mRNA degradation andortranslational repression in P-bodies [9] Recent evidencessuggests an important role for miRNAs in the fine-tuningregulation of genes involved in osteogenesis indicating thatthese so-called osteomiRs can be promising therapeutictargets for skeleton diseases [10ndash12] However the functionsof miRNAs during differentiation of hAM-MSCs toosteogenic lineage are still poorly understood Despitethis interest comprehensive miRNAs profiling studiesassociated with differentiation of hAM-MSCs to osteoblastsare scarce thus the relevance of small RNAs in osteogenesisis not yet known In this investigation we reported a novelmiRNAs expression signature in two stages of hAM-MSCsdifferentiation to osteoblasts and functionally characterizedthe role of miR-204 in this cellular process Implications onthe molecular mechanisms regulating the differentiation ofhAM-MSCs and the potential therapeutic applications arediscussed

2 Materials and Methods

21 Isolation of Stem Cells from the Human AmnioticMembranes Caesarean-delivered term placentas (n=4) were

collected from healthy donor mothers Procedures wereapproved by the Ethics Committee of the University HospitalldquoDr Jose Eleuterio Gonzalezrdquo and each donor gave herconsent Pieces of amniotic membrane (10 x10 cm) weresubjected to two enzymatic digestions by adding (i) 0125trypsin05 mM EDTA solution at 37∘C for 30min and (ii)100 UmL collagenase type II and 3mM calcium chloridein the Dulbeccorsquos Modified Eaglersquos Medium (DMEM) for2 h at 37∘C followed by washing with PBS The resultingcell suspension was filtered and cells were seeded in 25-cm2 flasks in DMEM containing 10 fetal bovine serum(FBS) and 100 UmL penicillin 100mgmL streptomycinand 025mgmL amphotericin B The resulting hAM-MSCswere cultured in a humidified 5 CO

2atmosphere at 37∘C

until 90 confluence All experiments were carried out usinghAM-MSCs below 10 passages

22 Fluorescent Activated Cell Sorting After establishing thehAM-MSCs population a CD44+CD73+CD105minus (CD105minus)subpopulation was enriched by Fluorescent Activated CellSorting (FACS Aria BD) hAM-MSCs were stained withhuman PE-conjugated CD73 APC-conjugated CD105 andPercP-Cy55-conjugated CD44 (Miltenyi Biotec) and pos-itive cells were collected After centrifugation at 400 g for5min they were plated in fresh culture medium for expan-sion and further molecular characterization

23 OsteogenicDifferentiation of hAM-MSCs Theosteogenicdifferentiation was induced as previously described [13]Briefly isolates of hAM-MSCs (2x103 cellscm2) were grownin osteogenic medium (DMEM supplemented with 10FBS 10mM 120573-glycerophosphate 025mM ascorbic acidand 10minus8M dexamethasone) Cultures were maintained withmedium changes every 2-3 days Cells were stained withAlizarin Red S dye reactive (pH 43) to evaluate calcium-rich deposits and monitor the differentiation process asdescribed [14] Cells were imaged and birefringence stainingwas quantified using the FluorChem 9900 system

24 Western Blot Assays For immunoblotting hAM-MSCswere rinsed with cooled PBS and lysed at room temper-ature for 10min in 1mL of RIPA buffer (20mM Tris-HCl pH 75 150mM NaCl 1mM EGTA 1 NP-40 1sodium deoxycholate 25mM sodium pyrophosphate 1mMb-glycerophosphate 1mMNa

3VO4 and 1mgmL leupeptin)

containing the Complete Protease Inhibitor (05mM phenyl-methylsulfonyl fluoride 10mgmL leupeptin 10mgmLaprotinin 5mgmL pepstatin 10mgmL soybean trypsininhibitor and 05mM dithiothreitol ROCHE MolecularBiochemicals) Equal amounts of protein (30120583g) were run on10 SDS-PAGE gels and transferred to a PVDF membrane(Millipore Corporation) The membrane was blocked for60min at room temperature with TBST-1 (137mM NaCl20mMTris and 01Tween-20 (pH76)) containing 5BSA(Sigma-Aldrich) and then incubated overnight at 4∘C withrabbit anti-human COL1A2 (11000) antibodies The mem-brane was washed in TBST-1 and incubated with horseradishperoxidase-conjugated goat anti-rabbit IgG antibody (13000











3 Results



7 140

Days post-induction

400 mm 400 mm 400 mm

(a)

10

20

30

40

Rela

tive c

alci

umde

posit

ion

0 7 14

Days post-induction

lowastlowast

lowastlowast

(b)

COL1A2

GAPDH

0 7 14

Days post-induction

(c)

10

20

30

40

0 7 14

Relat

ive e

xpre

ssio

n CO

L1A

2G

AD

PH

Days post-induction

lowastlowastlowast

lowastlowast

(d)


















Table 2 Continued







108726

23

5436311

7

00

-30 00 30

0da

ys

14da

ys

56608585

28304293

00

(a)

00

-30 00 30

0da

ys

7day

s

12136769

60683

846

22293066

11146533

00

(b)

Day 7th323130292827262524232221201918171615141312111009080706050403020100

-log1

0(P

-Val

ue)


log2(Fold Change)

(c)

Day 14th32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

09

08

07

06

05

04

03

02

01

00

-log1

0(P

-Val

ue)

40

39

38

37

36

35

34

33


log2(Fold Change)

(d)





TLDA

TaqMan Assay

minus4

minus2

0

2

4

Fold

chan

ge(R

Q) L

og2

miR

-21

miR

-125b

miR

-223

-3p

miR

-204

-5p








AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4




SMURF1

miR-19b-3p


miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin




TGF- pathway

Wnt-catenin pathway




Let-7f-5p

SMAD1 SMAD5

miR-148b-3p



miR-150-5p


miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p



miR-339b-5p






4 Discussion



hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





























3 Results



7 140

Days post-induction

400 mm 400 mm 400 mm

(a)

10

20

30

40

Rela

tive c

alci

umde

posit

ion

0 7 14

Days post-induction

lowastlowast

lowastlowast

(b)

COL1A2

GAPDH

0 7 14

Days post-induction

(c)

10

20

30

40

0 7 14

Relat

ive e

xpre

ssio

n CO

L1A

2G

AD

PH

Days post-induction

lowastlowastlowast

lowastlowast

(d)


















Table 2 Continued







108726

23

5436311

7

00

-30 00 30

0da

ys

14da

ys

56608585

28304293

00

(a)

00

-30 00 30

0da

ys

7day

s

12136769

60683

846

22293066

11146533

00

(b)

Day 7th323130292827262524232221201918171615141312111009080706050403020100

-log1

0(P

-Val

ue)


log2(Fold Change)

(c)

Day 14th32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

09

08

07

06

05

04

03

02

01

00

-log1

0(P

-Val

ue)

40

39

38

37

36

35

34

33


log2(Fold Change)

(d)





TLDA

TaqMan Assay

minus4

minus2

0

2

4

Fold

chan

ge(R

Q) L

og2

miR

-21

miR

-125b

miR

-223

-3p

miR

-204

-5p








AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4




SMURF1

miR-19b-3p


miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin




TGF- pathway

Wnt-catenin pathway




Let-7f-5p

SMAD1 SMAD5

miR-148b-3p



miR-150-5p


miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p



miR-339b-5p






4 Discussion



hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















7 140

Days post-induction

400 mm 400 mm 400 mm

(a)

10

20

30

40

Rela

tive c

alci

umde

posit

ion

0 7 14

Days post-induction

lowastlowast

lowastlowast

(b)

COL1A2

GAPDH

0 7 14

Days post-induction

(c)

10

20

30

40

0 7 14

Relat

ive e

xpre

ssio

n CO

L1A

2G

AD

PH

Days post-induction

lowastlowastlowast

lowastlowast

(d)


















Table 2 Continued







108726

23

5436311

7

00

-30 00 30

0da

ys

14da

ys

56608585

28304293

00

(a)

00

-30 00 30

0da

ys

7day

s

12136769

60683

846

22293066

11146533

00

(b)

Day 7th323130292827262524232221201918171615141312111009080706050403020100

-log1

0(P

-Val

ue)


log2(Fold Change)

(c)

Day 14th32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

09

08

07

06

05

04

03

02

01

00

-log1

0(P

-Val

ue)

40

39

38

37

36

35

34

33


log2(Fold Change)

(d)





TLDA

TaqMan Assay

minus4

minus2

0

2

4

Fold

chan

ge(R

Q) L

og2

miR

-21

miR

-125b

miR

-223

-3p

miR

-204

-5p








AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4




SMURF1

miR-19b-3p


miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin




TGF- pathway

Wnt-catenin pathway




Let-7f-5p

SMAD1 SMAD5

miR-148b-3p



miR-150-5p


miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p



miR-339b-5p






4 Discussion



hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
































Table 2 Continued







108726

23

5436311

7

00

-30 00 30

0da

ys

14da

ys

56608585

28304293

00

(a)

00

-30 00 30

0da

ys

7day

s

12136769

60683

846

22293066

11146533

00

(b)

Day 7th323130292827262524232221201918171615141312111009080706050403020100

-log1

0(P

-Val

ue)


log2(Fold Change)

(c)

Day 14th32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

09

08

07

06

05

04

03

02

01

00

-log1

0(P

-Val

ue)

40

39

38

37

36

35

34

33


log2(Fold Change)

(d)





TLDA

TaqMan Assay

minus4

minus2

0

2

4

Fold

chan

ge(R

Q) L

og2

miR

-21

miR

-125b

miR

-223

-3p

miR

-204

-5p








AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4




SMURF1

miR-19b-3p


miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin




TGF- pathway

Wnt-catenin pathway




Let-7f-5p

SMAD1 SMAD5

miR-148b-3p



miR-150-5p


miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p



miR-339b-5p






4 Discussion



hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















108726

23

5436311

7

00

-30 00 30

0da

ys

14da

ys

56608585

28304293

00

(a)

00

-30 00 30

0da

ys

7day

s

12136769

60683

846

22293066

11146533

00

(b)

Day 7th323130292827262524232221201918171615141312111009080706050403020100

-log1

0(P

-Val

ue)


log2(Fold Change)

(c)

Day 14th32

31

30

29

28

27

26

25

24

23

22

21

20

19

18

17

16

15

14

13

12

11

10

09

08

07

06

05

04

03

02

01

00

-log1

0(P

-Val

ue)

40

39

38

37

36

35

34

33


log2(Fold Change)

(d)





TLDA

TaqMan Assay

minus4

minus2

0

2

4

Fold

chan

ge(R

Q) L

og2

miR

-21

miR

-125b

miR

-223

-3p

miR

-204

-5p








AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4




SMURF1

miR-19b-3p


miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin




TGF- pathway

Wnt-catenin pathway




Let-7f-5p

SMAD1 SMAD5

miR-148b-3p



miR-150-5p


miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p



miR-339b-5p






4 Discussion



hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















TLDA

TaqMan Assay

minus4

minus2

0

2

4

Fold

chan

ge(R

Q) L

og2

miR

-21

miR

-125b

miR

-223

-3p

miR

-204

-5p








AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4




SMURF1

miR-19b-3p


miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin




TGF- pathway

Wnt-catenin pathway




Let-7f-5p

SMAD1 SMAD5

miR-148b-3p



miR-150-5p


miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p



miR-339b-5p






4 Discussion



hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















AXIN2

miR-19b-3p

SMAD15

BMP2

miR-148b-5p

SMAD2 SMAD2

miR-424-3p

miR-20b-5p

miR-125b-5p

miR-19b-3p

OSXATF4




SMURF1

miR-19b-3p


miR-145-5p

RUNX2

miR-22-3-p

miR-431-5p

SMAD15

SMURF1 p21

RUNX2

miR-19b-3p

AXIN2

-Catenin




TGF- pathway

Wnt-catenin pathway




Let-7f-5p

SMAD1 SMAD5

miR-148b-3p



miR-150-5p


miR-186-5p

miR-204-5p

-Catenin

-Catenin

miR-302b-3p



miR-339b-5p






4 Discussion



hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















hsa-miR-145-5p

hsa-miR-337-5p

hsa-miR-27a-3p

hsa-miR-125b-5p

hsa-miR-143-3p

hsa-miR-744-5p

hsa-miR-22-3p

hsa-miR-20b-5p

hsa-miR-345-5p

hsa-miR-503-5p

hsa-miR-191-3p

hsa-miR-27b-3p

hsa-miR-21-5p

hsa-miR-136-3p

hsa-miR-431-5p

hsa-miR-155-5p

hsa-let-7f-5p

hsa-miR-181a-5p

hsa-let-7a-5p

hsa-miR-19b-3p

hsa-miR-148b-3p

hsa-miR-181a-3p

hsa-miR-181c-5p

hsa-miR-199a-5p

ACVR2A

BAMBI

CRIM1

CTNNBIP1

DUSP2

HDAC4

MAPK1

RBL1

SKP1

SMAD7

SMURF1

SMURF2

TGFB3

1-3 miRNAs

4-6 miRNAs

7-10 miRNAs






lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















lativ

e exp

ress

ion

miR

-204

RN

U44

0

3

6

9

12

Control miR-204

plt005lowastlowast

(a)

Calcium deposition

Control miR-204

plt0001

Aliz

arin

red

stai

ning

()

100

25

75

50

0

lowastlowastlowast

(b)


pmiR-Report




TGF-R2

UTR TGF-R2

5

5

3

3

(c)

ScrambleControl

Relat

ive l

ucife

rase

ac

tivity

0020406081012

UTR-TGF-R2

TGF-R2 mutated

miR-204


lowastlowastlowast

3

(d)






5 Conclusions


Data Availability






Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























Acknowledgments


References





























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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