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1432 uMSC Exosomes Suppress Scar Formation

Figure 4. Identification of uMSC-Exo-specific microRNAs by high-throughput sequencing. (A): Exosomal miRNA abundance analysis by high-
throughput small RNA sequencing. The top 10 abundant miRNAs in uMSC-derived exosomes are color labeled. (B): Pie chart showing HEK293-
derivedexosomalmiRNAabundanceusinghigh-throughputsmallRNAsequencing.Thetop10abundantmiRNAsin(A)arelabeled.(C):ThemiRNA
abundanceanalysisinuMSCsusingGSE46989fromtheGEODataSet.Thecolor-labeledmiRNAswerethetop10abundantmiRNAsinuMSC-derived
exosomes. (D): miRNA abundance analysis in HEK293T cells using GSE56862 from the GEO DataSet. The 10 highly expressed miRNAs in uMSC-
derived exosomes are color labeled. (E): Quantitative reverse transcription-polymerase chain reaction analysis of top 10 uMSC-Exo-abundant
miRNAsinuMSC-Exo-treatedfibroblasts(upper).UEFS-treatedfibroblastsservedascontrol.Expressionlevelofpre-miRNAsintheindicatedgroups
(right).Dataarepresentedasmean6 SD;pp,p, .01;ppp,p ,.001. (F): GeneOntologyanalysisofthe TargetScan-predictedmRNAtargetsforthe
10mostabundantlyexpressedmiRNAsinuMSC-derivedexosomes.Thereddash-highlightedterm,TGF-breceptorpathway,highlycorrelatedwith
SMA expression and myofibroblasts formation. (G): The miRNA-mRNA interacting network showing the predicted targets for the top 10 abundant
miRNAsinuMSC-derivedexosomes.Targetgenespredictedtobetargetedbymorethan2ofthe10miRNAsareshown.Theyellowdotsrepresents
target mRNA, and the red arrow, miRNA. Abbreviations: Exo, exosome; hsa, Homo sapiens; miR, microRNA; miRNA, microRNA; pre-miR, before
microRNA; SMA, smooth muscle actin; TGF-b, transforming growth factor-b; UEFS, umbilical cord-derived mesenchymal stem cell exosome-free
supernatant; uMSC, umbilical cord-derived mesenchymal stem cell; uMSC-Exo, umbilical cord-derived mesenchymal stem cell-derived exosomes.

The results showed that the expression of miR-21, miR-23a, miR- to genes involved in the TGF-b/SMAD2 pathway, such as TGF-b2,
100, miR-125b, and miR-145 in the uMSC-Exo group was signifi- TGF-bR2,and SMAD2 (Fig. 4G; supplemental data file 2). Be-
cantly increased compared with that in the HEK293T-Exos group; cause this pathway is a well-known regulator of myofibroblast
pre-miRNAs were not affected (Fig. 4E), confirming the ability of formation [5, 23], we believe that these specific miRNAs could
exosomes to transport its contained mature miRNAs into target inhibit fibroblastic differentiation to myofibroblasts by suppressing
cells. TGF-b/SMAD2 pathway activities.
To further reveal the possible roles of these miRNAs, we pre-
dicted their target genes and their functions using TargetScan
(available at http://www.targetscan.org/) and Gene Ontology uMSC-Exo-Specific MicroRNAs Target the TGF-b/SMAD2
(GO) analysis. The analysis showed that the TGF-b/SMAD2 path- Pathway to Suppress Myofibroblast Formation
way was highly enriched in the GO analysis (Fig. 4F), and the To validate the functions of these exosomal microRNAs, we syn-
several most abundant microRNAs, such as miR-21, miR-23a, thesized agomirs to achieve stable overexpression. To investigate
miR-125b, and miR-145, were all found to be directly targeted the functions of specific microRNAs in uMSC-Exos, we first

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