Abstract
Background
Exosomes derived from dental pulp stem cells (DPSCs) can be used as
biomimetic tools to induce odontogenic differentiation of stem cells,
but the regulatory mechanisms and functions of exosome-encapsulated
microRNAs are still unknown. The present study aimed to clarify the
role of microRNAs contained in the exosomes derived from human DPSCs
and their potential signaling cascade in odontogenic differentiation.
Methods
Exosomes were isolated from human DPSCs cultured undergrowth and
odontogenic differentiation conditions, named UN-Exo and OD-Exo,
respectively. The microRNA sequencing was performed to explore the
microRNA profile contained in UN-Exo and OD-Exo. Pathway analysis was
taken to detect enriched pathways associated with the predicted target
genes of microRNAs. The regulatory roles of a highly expressed microRNA
in OD-Exo were investigated through its inhibition or overexpression
(miRNA inhibitors and miRNA mimics). Automated western blot was used to
identify the function of exosomal microRNA and the roles of TGFβ1/smads
pathway in odontogenic differentiation of DPSCs. A luciferase reporter
gene assay was used to verify the direct target gene of exosomal
miR-27a-5p.
Results
Endocytosis of OD-Exo triggered odontogenic differentiation of DPSCs by
upregulating DSP, DMP-1, ALP, and RUNX2 proteins. MicroRNA sequencing
showed that 28 microRNAs significantly changed in OD-Exo, of which 7
increased and 21 decreased. Pathway analysis showed genes targeted by
differentially expressed microRNAs were involved in multiple signal
transductions, including TGFβ pathway. 16 genes targeted by 15
differentially expressed microRNAs were involved in TGFβ signaling.
Consistently, automated western blot found that OD-Exo activated TGFβ1
pathway by upregulating TGFβ1, TGFR1, p-Smad2/3, and Smad4 in DPSCs.
Accordingly, once the TGFβ1 signaling pathway was inhibited by
SB525334, protein levels of p-Smad2/3, DSP, and DMP-1 were
significantly decreased in DPSCs treated with OD-Exo. MiR-27a-5p was
expressed 11 times higher in OD-Exo, while miR-27a-5p promoted
odontogenic differentiation of DPSCs and significantly upregulated
TGFβ1, TGFR1, p-Smad2/3, and Smad4 by downregulating the inhibitory
molecule LTBP1.
Conclusions
The microRNA expression profiles of exosomes derived from DPSCs were
identified. OD-Exo isolated under odontogenic conditions were better
inducers of DPSC differentiation. Exosomal microRNAs promoted
odontogenic differentiation via TGFβ1/smads signaling pathway by
downregulating LTBP1.
Electronic supplementary material
The online version of this article (10.1186/s13287-019-1278-x) contains
supplementary material, which is available to authorized users.
Keywords: Exosomes, TGFβ pathway, MicroRNAs, Odontogenesis, DPSCs
Introduction
Clinically relevant dental pulp tissue regeneration can serve as a
treatment to replace existing root canal therapy used to treat necrotic
permanent teeth [[35]1]. Regenerative endodontic treatment is attempted
by using a variety of mesenchymal stem cells (MSCs), growth factors,
and biomaterials [[36]2, [37]3]. Exosomes are reported as an ideal
biomaterial in regenerative endodontic treatment, with the properties
of immunomodulation [[38]4, [39]5], promoting angiogenesis [[40]6] and
inducing differentiation of stem cells [[41]7, [42]8].
Exosomes are nano-sized vesicles ranging from 30 to 150 nm in diameter,
which are secreted by many cell types to mediate intercellular
communication [[43]9]. The cargo of exosomes is shown to contain both
ubiquitous and cell type-specific biological molecules such as protein,
mRNA, and microRNA [[44]9]. It has been revealed that exosomal
microRNAs are transferred between cells and can regulate the
post-transcriptional gene expression in recipient cells [[45]10].
microRNAs can negatively regulate gene expression at the
post-transcriptional level by binding to their target mRNAs through
base pairing to the 3′-untranslated region (UTR), causing translational
repression of the mRNA [[46]11]. Exosomal microRNAs have been shown to
be critical components in stem cell differentiation. For example,
exosomal miR-320c enhanced chondrogenic differentiation of bone marrow
MSCs by upregulating SOX9 and downregulating MMP13 expression [[47]12].
Similarly, exosomal miR-let-7 has been shown to initiate the osteogenic
differentiation of MSCs [[48]13].
Exosomes derived from dental pulp stem cells (DPSCs) can be used as
biomimetic tools to induce odontogenic differentiation of stem cells
during dental pulp regeneration [[49]14]; however, the function and
regulatory mechanism of microRNAs encapsulated in exosomes derived from
DPSCs is still unknown. To date, no study has been conducted on the
microRNA expression profiles of exosomes derived from human DPSCs. The
present study aimed to clarify the role of microRNAs, encapsulated in
the exosomes from human DPSCs, and their potential signaling cascade in
odontogenic differentiation.
Materials and methods
Isolation and culture of human DPSCs
All experimental protocols were approved by the Ethics Committee of Sun
Yat-sen University. Human DPSCs were harvested from healthy pulp
tissues isolated from caries-free teeth of patients (5 females, age
24~35 years; 5 males, age 22~36 years) undergoing extraction of fully
erupted third molars. Healthy pulp tissues were digested for isolation
of DPSCs as described previously [[50]15]. Cells were cultured at
37 °C, in a 5% CO[2] incubator, using а-MEM supplemented with 10% FBS
(GIBCO, USA) as a growth medium, 10 mg/ml streptomycin, and 10 U/ml of
penicillin (Sigma, USA). Experiments were performed with DPSCs from
passages 3 to 7.
Investigation of DPSC surface markers
100 μl DPSCs at a concentration of 1 × 10^6 cells/ml were stained by
5 μl of each of the following human antibodies: CD45-PE, CD73-PE,
CD90-APC, and CD166-PE (BD, USA). The samples were incubated at 37 °C
for 30 min, centrifuged, washed twice with PBS, and examined by flow
cytometry (BD, USA).
Determination of DPSC differentiation capacity
We determined the multi-potential differentiation of DPSCs into
osteoblasts, adipocytes, and chondrocytes in vitro. To explore the
potential of differentiation into osteoblasts, DPSCs were induced for
14 days in osteogenic medium supplemented with 100 nmol dexamethasone,
10 mmol β-glycerophosphate, and 0.2 mmol l-ascorbic acid (Sigma, USA),
osteogenic differentiation was measured by Alizarin Red S staining. To
verify the adipogenic differentiation potential, DPSCs were induced for
27 days in adipogenic medium supplemented with 0.5 μM
isobutyl-methylxanthine, 50 μM indomethacin, 0.5 μM dexamethasone, and
5 μg/mL insulin (Sigma, USA), adipogenic differentiation was determined
by Oil Red O staining. For chondrogenic differentiation, the cell
pellets were prepared for a three-dimensional culture system.
Approximately 4 × 10^5 cells were placed in a 15-ml polypropylene tube
and centrifuged at 500 g for 5 min. The cells were cultured in human
mesenchymal stem cell chondrogenic differentiation medium (Cyagen, USA)
for 28 days, in the presence of 10 ng/mL recombinant human TGF-b3.
Alcian blue staining was utilized to examine the cartilage nodules.
Isolation and identification of exosomes
Exosomes were isolated from the culture medium of DPSCs cultured in the
presence of either growth (UN-Exo) or odontogenic differentiation media
(OD-Exo) for a period of 10 days. Exosomes were isolated as per
previously published protocols [[51]16, [52]17]. Briefly, 2 days prior
to isolation, the cell cultures were washed in serum-free PBS and
cultured for 48 h in serum-free а-MEM. When odontogenic media were
used, the serum-free а-MEM was supplemented with the odontogenic media
cocktail of 100 nmol dexamethasone, 10 mmol β-glycerophosphate, and
0.2 mmol l-ascorbic acid (Sigma, USA). The exosomes from the culture
medium were isolated using the Exo-spin (Cell Guidance, UK) exosome
isolation reagent as per the manufacturer’s protocol. The exosome
protein concentration was quantified with a BCA Protein Assay Kit
(Bocai, Shanghai, China). The exosomal markers CD9 and CD63 (Affinity
Biosciences, USA) in the UN-Exo and OD-Exo were measured by automated
western blot analysis.
Transmission electron microscopy (TEM)
TEM was used to identify the presence of UN-Exo and OD-Exo. 10 μl
exosomes suspension was placed on to formvar/carbon-coated nickel TEM
grids and incubated for 30 min. The grids were then washed, dried, and
imaged using an H-7650 transmission electron microscope (HITACHI,
Japan) to identify the morphology of exosomes.
Fluorescent labeling of exosomes
UN-Exo and OD-Exo were collected from supernatants of DPSC and isolated
by ultracentrifugation and sucrose cushion centrifugation. Briefly,
cell culture supernatant (48 h, serum-free medium) was cleared
(2 × 10 min, 500×g; 1 × 20 min, 2000×g; 1 × 30 min, 10,000×g),
centrifuged (90 min, 100,000×g), washed (PBS, 90 min, 100,000×g), and
further purified by sucrose-gradient centrifugation. 1 μl PKH26
(Sigma-Aldrich, St Louis, MO) was added to 250 μl diluent C, which was
then immediately mixed with the exosomes by pipetting. After 5 min of
incubation at room temperature, the staining was stopped by the
addition of an equal volume of exosome-free FBS. The exosomes were
harvested and washed twice with PBS by centrifugation (100,000 g for
1 h) and resuspended in 100 μl exosome-free culture medium. The
PKH26-labeled exosomes were added to the DPSCs and incubated for 24 h.
Then, the cells were washed 3 times with PBS, fixed with 4%
paraformaldehyde for 10 min, and stained with 4,
6-diamidino-2-phenylindole for 5 min. Confocal laser scanning
microscopy (Zeiss, Oberkochen, Germany) was used to visualize the
endocytosis of UN-Exo and OD-Exo by DPSCs.
Exosome-mediated odontogenic differentiation of DPSCs
DPSCs were seeded into 6-well plates at an initial density of 1 × 10^5
cells/well and incubated for 48 h with exosomes isolated from DPSCs
cultured for 10 days using growth media (UN-Exo, 30 μg/ml) as well as
odontogenic differentiation media (OD-Exo, 30 μg/ml), the growth media
supplemented with 10% exosome-free FBS was used as control medium.
Odontogenic differentiation was measured by automated western blot to
explore the protein expression of ALP, RUNX2, odontoblast-specific
marker DSP, and DMP-1.
Automated western blot analysis
The automated western blot was performed using Simple Wes (Protein
Simple, USA) following the manufacturer’s protocol. Briefly, 1.5 μg of
protein from the cell lysates or exosomes was added to the standard
fluorescent mastermix, then was loaded into corresponding wells of the
prefilled Wes assay plate, along with antibody diluent (Protein Simple,
USA), anti-DSP (Santa Cruz, USA), anti-DMP1, anti-RUNX2, anti-ALP,
anti-CD9, anti-CD63, anti-TGFβ1, anti-TGFR1, anti-Smad2/3,
anti-p-Smad2/3, anti-Smad4, anti-β-Tublin (Affinity, USA), anti-LTBP1
(Affinity, USA), anti-rabbit secondary antibody (Protein Simple, USA),
and Streptavidin-HRP, followed by luminal peroxide mix. The imaging and
analysis were done with compass software (Protein Simple, USA).
microRNA sequencing
MicroRNAs of 18–30 nucleotides (nt) were obtained from 100 μg of total
RNA isolated from exosomes using 15% denaturing polyacrylamide gel
electrophoresis (PAGE). After PCR amplification, the products were
purified and submitted for sequencing via an Illumina Hi-Seq 2000
platform. Library preparation and microRNA sequencing were performed by
RiboBio Ltd. (Guangzhou, China). Differentially expressed microRNAs
with 2 fold change in expression (p < 0.05) were analyzed. Predicted
microRNA target genes were detected using four publicly available
bioinformatics tools (TargetScan, miRTarBase, miRDB, and miRWalk
databases). Gene Ontology (GO, [53]http://www.geneontology.org)
analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG,
[54]http://www.genome.jp/kegg/pathway.html) Pathway analysis were
performed to detect molecular functions, biological processes, and
pathways associated with the predicted microRNA target genes.
GO analysis, all the targeted genes of miRNAs were input into the Gene
Ontology database, and each term number of genes was calculated.
Compared with the whole genome background, the hypergeometric test was
used to find out the GO terms that were significantly enriched in the
targeted genes of miRNAs. The formula is as follows:
[MATH:
p=1−∑i<
/mi>=0m−1MiN−Mn−iNn
:MATH]
, where N is the number of all genes with GO annotation, n is the
number of targeted genes of miRNAs in N, M is the number of all genes
that are annotated to certain GO terms, m is the number of targeted
genes of miRNAs in M. The calculated p value goes through Bonferroni
correction, taking corrected p value < 0.05 as a threshold. GO terms
fulfilling this condition are defined as significantly enriched GO
terms in targeted genes of miRNAs. This analysis is able to recognize
the main biological functions that targeted genes of miRNA exercise.
KEGG, the major public pathway-related database, is used to perform
pathway enrichment analysis of targeted genes of miRNAs. This analysis
identifies significantly enriched metabolic pathways or signal
transduction pathways in targeted genes of miRNAs comparing with the
whole genome background. The calculating formula is the same as that in
the GO analysis. Here, N is the number of all genes that with KEGG
annotation, n is the number of targeted genes of miRNAs in N, M is the
number of all genes annotated to specific pathways, and m is the number
of targeted genes of miRNAs in M.
Real-time PCR
Total RNA was extracted from cells by RNA extraction kit (Qiagen,
China). Then, 2 μg of total RNA was reversely transcribed into cDNA
using a reverse transcription polymerase chain reaction (RT-PCR) system
(Promega, USA). The qRT-PCR was performed by SYBR-Green PCR kit
(Qiagen, China) according to the manufacturer’s instructions on a
LightCycler 480 (Roche, USA). The data are representative of three
independent experiments, and the relative microRNA expression was
determined using the comparative Ct (ΔΔCt) method. The sequences of the
primers are shown in Additional file [55]1: Table S1.
Determination and inhibition of TGFβ1/smads signaling pathway
For evaluation of the involvement of TGFβ1/smads signaling pathway,
DPSCs were seeded into 6-well plates at an initial density of 1 × 10^5
cells/well and were incubated for 48 h with 30 μg/ml UN-Exo and
30 μg/ml OD-Exo, the growth media supplemented with 10% exosomes-free
FBS was used as the control medium. TGFβ1/smads signaling pathway was
inhibited by 10 μM inhibitor SB525334 (Selleck, USA) for 48 h.
Transfection of miR-27a-5p mimics and inhibitor
DPSCs were treated with OD-Exo in 6-well culture plates and transfected
with the miR-27a-5p mimics and inhibitor using Lipofectamine 2000
(Invitrogen, USA) according to the manufacturer’s instructions. DPSCs
were harvested after 48 h.
Dual-luciferase reporter assay
A luciferase reporter gene assay was used to verify whether LTBP1 was
the direct target gene of miR-27a-5p. Luciferase reporter constructs
encoding the wild-type 3′-UTRs of LTBP1 (LTBP1-WT) or mutant 3′-UTRs of
AXL (LTBP1-MUT) were synthesized. The 3′-UTR luciferase vector (150 ng)
was co-transfected into cells with either miR-27a-5p mimic or
miR-27a-5p mimic-control using Lipofectamine 2000 (Invitrogen). After
incubation for 48 h, the cells were collected and lysed, and their
luciferase activities were detected by the Dual-Luciferase Reporter
Assay Kit (Beyotime Biotechnology, Shanghai, China) according to the
manufacturer’s protocol.
Statistical analysis
Each experiment was repeated three times. All values were expressed as
the mean ± SD and were evaluated by the independent samples t test
using SPSS 17.0 (SPSS Inc., USA). p < 0.05 was considered statistically
significant.
Results
Characterization of DPSCs
The results showed that DPSCs had the potential of differentiation into
osteoblasts, adipocytes, and chondrocytes (Fig. [56]1a), indicating the
multi-lineage differentiation potential of DPSCs. DPSCs expressed high
levels of the mesenchymal stem cell marker CD73 (Fig. [57]1b), CD90
(Fig. [58]1c), and CD166 (Fig. [59]1d), but expressed low levels of the
hematopoietic cell marker CD45(Fig. [60]1e).
Fig. 1.
[61]Fig. 1
[62]Open in a new tab
Characterization of DPSCs. a DPSCs had the potential of differentiation
into osteoblasts, adipocytes, and chondrocytes. b–e DPSCs expressed
high levels of the mesenchymal stem cell marker CD73, CD90, and CD166,
but expressed low levels of the hematopoietic cell marker CD45
Endocytosis of UN-Exo and OD-Exo by DPSCs
To characterize the presence of exosomes in the isolates, the bilayer
membrane and “saucerlike” appearance of representative exosomes were
examined by TEM, which verified the presence of UN-Exo and OD-Exo
ranging from 30 to 150 nm in diameter (Fig. [63]2a). Automated western
blot analysis revealed that exosomal markers CD9 and CD63 were
expressed in the UN-Exo and OD-Exo (Fig. [64]2b). To confirm whether
UN-Exo and OD-Exo could be taken up by DPSCs, the isolated UN-Exo and
OD-Exo were labeled with PKH26, and DPSC cultures were incubated with
the labeled exosomes at 37 °C. After 24 h, PKH26-labeled UN-Exo and
OD-Exo were taken up by DPSCs into the cytoplasm (Fig. [65]2c).
Fig. 2.
[66]Fig. 2
[67]Open in a new tab
Endocytosis of UN-Exo and OD-Exo by DPSCs. a The morphology of UN-Exo
and OD-Exo was determined by transmission electron microscopy. b
Automated western blot analysis revealed that exosomal markers CD9 and
CD63 were expressed in the UN-Exo and OD-Exo. c Endocytosis of exosomes
by DPSCs was visualized by fluorescent labeling with PKH26
Endocytosis of OD-Exo by DPSCs triggered odontogenic differentiation
We evaluated whether the endocytosis of exosomes triggered odontogenic
differentiation of DPSCs by affecting the expression of regulatory
proteins. When DPSCs were treated with OD-Exo for 48 h, the protein
expressions of DSP, DMP-1, ALP, and RUNX2 significantly increased,
compared to the control group and UN-Exo treated group (p < 0.05).
However, DPSCs treated with UN-Exo only expressed a higher level of DSP
than the control group (p < 0.05), but no significant differences in
DMP-1, ALP, and RUNX2 (Fig. [68]3). These results showed that OD-Exo
isolated under odontogenic conditions are better inducers of DPSCs
differentiation.
Fig. 3.
[69]Fig. 3
[70]Open in a new tab
Endocytosis of OD-Exo by DPSCs triggered odontogenic differentiation.
Endocytosis of OD-Exo isolated under odontogenic conditions triggered
odontogenic differentiation of DPSCs by upregulating protein
expressions of DSP, DMP-1, ALP, and RUNX2, when compared to control
group (without exosomes) and UN-Exo group (p < 0.05). And DPSCs treated
with UN-Exo only expressed higher protein level of DSP than the control
group (p < 0.05), but no significant differences in DMP-1, ALP, and
RUNX2
MicroRNA profiles of UN-Exo and OD-Exo
As exosomes transfer microRNAs between cells, post-transcriptional gene
expression in recipient cells can be regulated by microRNAs contained
in exosomes [[71]18]. Thus, we hypothesized that this process may be
exploited by DPSCs to promote odontogenic differentiation. To explore
this possibility, we analyzed the microRNA profiles of UN-Exo and
OD-Exo via Ion Torrent/MiSeq sequencing. The results showed microRNA
levels in OD-Exo significantly changed when compared with that in
UN-Exo. There were 28 microRNAs significantly changed in OD-Exo
isolated under odontogenic conditions, of which 7 microRNAs increased
(miR-5100, miR-27a-5p, miR-652-3p, miR-1260a, miR-1260b, let-7f-1-3p,
and miR-370-3p) and 21 microRNAs decreased (miR-193a-5p, miR-4792,
miR-505-3p, miR-629-5p, miR-140-3p, miR-185-5p, miR-146b-5p,
miR-339-5p, miR-1246, miR-107, miR-320d, miR-451a, miR-215-5p,
miR-126-3p, miR-3687, miR-31-5p, miR-210-3p, miR-1-3p, miR-10a-5p,
miR-10b-5p, and miR-619-5p) (Fig. [72]4a, Table [73]1). The qRT-PCR
analysis showed that miR-5100 and miR-1260a levels in OD-Exo increased,
while miR-210-3p and miR-10b-5p decreased, which were consistent with
the microRNA sequencing (Fig. [74]4e).
Fig. 4.
[75]Fig. 4
[76]Open in a new tab
microRNA profiles of UN-Exo and OD-Exo via microRNA sequencing. a
MicroRNA levels in OD-Exo significantly changed when compared with that
in UN-Exo, 28 microRNAs significantly changed, of which 7 increased and
21 decreased. b Four bioinformatics tools (TargetScan, miRTarBase,
miRDB, and miRWalk) were used to analyze genes targeted by
differentially expressed microRNAs, for example, hsa-miR-27a-5p. c All
genes targeted by differentially expressed microRNAs were shown in the
mRNA-microRNA network. d mRNA-microRNA network showed 16 genes in TGFβ
signaling were targeted by 15 differentially expressed microRNAs. e
Consistent with the microRNA sequencing, hsa-miR-5100 and hsa-miR-1260a
were increased; hsa-miR-210-3p and hsa-miR-10b-5p were decreased by
qPCR analysis
Table 1.
There were 28 microRNAs significantly changed in OD-Exo isolated under
odontogenic conditions, of which 7 increased and 21 decreased
miRNA_ID Up/down Fold change Significance
hsa-miR-5100 Up 29.91 **
hsa-miR-27a-5p Up 11.42 **
hsa-miR-652-3p Up 10.62 *
hsa-miR-1260a Up 4.65 **
hsa-miR-1260b Up 4.00 **
hsa-let-7f-1-3p Up 2.61 **
hsa-miR-370-3p Up 2.54 **
hsa-miR-193a-5p Down 2.09 *
hsa-miR-4792 Down 2.13 *
hsa-miR-505-3p Down 2.27 *
hsa-miR-629-5p Down 2.35 *
hsa-miR-140-3p Down 2.72 **
hsa-miR-185-5p Down 2.79 *
hsa-miR-146b-5p Down 2.94 **
hsa-miR-339-5p Down 3.08 *
hsa-miR-1246 Down 3.56 *
hsa-miR-107 Down 3.65 *
hsa-miR-320d Down 3.79 *
hsa-miR-451a Down 4.20 *
hsa-miR-215-5p Down 4.39 **
hsa-miR-126-3p Down 4.44 *
hsa-miR-3687 Down 4.64 **
hsa-miR-31-5p Down 4.93 *
hsa-miR-210-3p Down 5.23 **
hsa-miR-1-3p Down 9.15 *
hsa-miR-10a-5p Down 11.39 **
hsa-miR-10b-5p Down 14.91 **
hsa-miR-619-5p Down 17.70 **
[77]Open in a new tab
*p < 0.05, **p < 0.01
Pathway and GO analysis of genes targeted by differentially expressed
microRNAs
Four publicly available bioinformatics tools (TargetScan, miRTarBase,
miRDB, and miRWalk) were used to analyze genes targeted by
differentially expressed microRNAs, for example, miR-27a-5p
(Fig. [78]4b). All genes targeted by differentially expressed microRNAs
were shown in Fig. [79]4c and Additional file [80]2: Table S2. Pathway
analysis showed targeted genes involved in multiple signal
transductions, including TGFβ signaling pathway (Fig. [81]5a). 16 genes
in TGFβ signaling targeted by 15 differentially expressed microRNAs
were shown in Fig. [82]4d. GO analysis of targeted genes showed that
the most significant biological processes consisted of DNA binding,
catalytic activity, cellular metabolic processes, and regulation of
cellular processes (Fig. [83]5b).
Fig. 5.
[84]Fig. 5
[85]Open in a new tab
Pathway and GO analysis of genes targeted by differentially expressed
microRNAs. a Pathway analysis showed that targeted genes involved in
multiple signal transductions, including TGFβ signaling pathway. b GO
analysis of targeted genes showed that the most significant biological
processes consisted of DNA binding, catalytic activity, cellular
metabolic process, and regulation of the cellular process
Exosomal microRNAs promoted odontogenic differentiation via TGFβ1/smads
signaling pathway by downregulating LTBP1
To confirm the results of pathway analysis as to whether TGFβ signaling
was activated by exosomal microRNAs encapsulated in OD-Exo, we
performed the automated western blot to evaluate the key proteins in
TGFβ signaling. Consistent with pathway analysis, automated western
blot found that OD-Exo activated TGFβ1 pathway by upregulating TGFβ1,
TGFR1, p-Smad2/3, and Smad4 in DPSCs, compared to control group and
UN-Exo-treated group (Fig. [86]6a). Accordingly, once we inhibited the
TGFβ1 signaling pathways by SB525334, protein levels of p-Smad2/3, DSP,
and DMP-1 were significantly reduced in DPSCs during odontogenic
differentiation induced by OD-Exo for 48 h (Fig. [87]6b).
Fig. 6.
[88]Fig. 6
[89]Open in a new tab
OD-Exo promoted odontogenic differentiation via TGFβ1/smads signaling
pathway. a Consistent with pathway analysis, automated western blot
analysis found significantly upregulated protein expressions of TGFβ1,
TGFR1, p-Smad2/3, and Smad4 in DPSCs treated with OD-Exo, when compared
to the control group and UN-Exo-treated group. b In accordance, once we
inhibited the TGFβ1 signaling pathways by SB525334, protein levels of
p-Smad2/3, DSP, and DMP-1 were significantly reduced in DPSCs treated
with OD-Exo
MiR-27a-5p was expressed 11 times higher in OD-Exo isolated under
odontogenic conditions (Table [90]1) and was predicted to be involved
in TGFβ pathway (Fig. [91]4d). Automated western blot showed that
miR-27a-5p mimics promoted odontogenic differentiation of DPSCs by
upregulating the protein expressions of DSP, DMP-1, ALP, and RUNX2
(Fig. [92]7a), and activated TGFβ1/smads signaling pathway by
increasing TGFβ1, TGFR1, p-Smad2/3, and Smad4 proteins (Fig. [93]7b).
Fig. 7.
[94]Fig. 7
[95]Open in a new tab
Exosomal miR-27a-5p regulated odontogenic differentiation via
TGFβ1/smads signaling pathway by downregulating LTBP1. a miR-27a-5p
mimics promoted odontogenic differentiation of DPSCs by upregulating
the protein expressions of DSP, DMP-1, ALP, and RUNX2. b miR-27a-5p
mimics activated TGFβ1/smads signaling pathway by increasing TGFβ1,
TGFR1, p-Smad2/3, and Smad4 proteins. c The western blot showed LTBP1
was downregulated bymiR-27a-5p mimics. d The predicted miRNA binding
sites in the 3′-UTR of LTBP1. e Luciferase reporter assay found that
miR-27a-5p could significantly reduce LTBP1-WT luciferase activity and
that miR-27a-5p had no effect on the luciferase activity of LTBP1-MUT
group
As is known, exosomal microRNAs can negatively regulate gene expression
by binding to their target mRNAs through base pairing to the 3′-UTR,
there may exist some other inhibitory molecules between the miR-27a-5p
and TGFβ1 signaling. Latent TGF-β-binding protein 1 (LTBP1), one of the
inhibitory molecules of TGFβ1 signaling, which form latent complexes
with TGFβ by covalently binding the TGFβ propeptide (LAP) via disulfide
bonds, plays a role in maintaining TGFβ latency by anchoring TGF-β to
the extracellular matrix [[96]19]. It was found that the mutation of
LTBP-1 in mice resulted in excess active TGF-β, which caused increased
signaling through its receptor and accumulation of nuclear p-Smad2/3
[[97]20]. Using four publicly available bioinformatics tools, LTBP1 was
found to be targeted by miR-27a-5p (Fig. [98]4b, d). We used western
blot and double luciferase assay to test whether LTBP1 could be
directly downregulated by miR-27a-5p. Western blot showed that LTBP1
was downregulated by miR-27a-5p mimics (Fig. [99]7c). Figure [100]7d
showed the predicted miRNA binding sites in the 3′-UTR of LTBP1. The
luciferase reporter assay was used to determine whether miR-27a-5p
could target the 3′-UTR of LTBP1 directly. The 3′-UTR fragment
(LTBP1-WT) of LTBP1 containing a miR-27a-5p binding site and mutant
fragments (LTBP1-MUT) was cloned into luciferase reporter vectors.
miR-27a-5p was found to significantly reduce LTBP1-WT luciferase
activity while it had no effect on the luciferase activity of LTBP1-MUT
group (Fig. [101]7e). Hence, the exosomal microRNAs promoted
odontogenic differentiation via TGFβ1/smads signaling pathway by
downregulating the inhibitory molecule LTBP1 (Fig. [102]8).
Fig. 8.
[103]Fig. 8
[104]Open in a new tab
Summary of the function of OD-Exo in the odontogenic differentiation of
DPSCs through TGFβ1/smads signaling pathway via the transfer of
mircoRNAs
Discussion
Regenerative endodontic treatment which has been defined as
biologically based procedures designed to replace damaged structures,
including dentin and root structures, as well as cells of the
pulp–dentin complex, has great potential in treating endodontic disease
[[105]21]. Stem cells can be implanted into the root canal via a
suitable medium, induced to proliferate, migrate, and differentiate
into different cell types by biomaterials and growth factors to
regenerate damaged tissue [[106]22]. DPSCs is a suitable source for
cells in regenerative endodontic treatment, and its differentiation
into odontogenic lineage induced by biomaterials and growth factors is
essential in dental pulp regeneration [[107]1]. As reported, exosomes
can be used as biomimetic tools to induce odontoblast-specific
differentiation of DPSCs in regenerative endodontic treatment
[[108]14]. The present study determined that endocytosis of exosomes
triggered odontogenic differentiation of DPSCs by evaluating the
expression of ALP, RUNX2, odontoblast-specific marker DMP-1, and DSP
proteins. Moreover, this study showed that exosomes isolated from DPSCs
undergoing odontogenic differentiation (OD-Exo) were better inducers of
DPSC differentiation than exosomes undergrowth medium (UN-Exo).
Consistent with previous studies, cell type-specific exosomes can
induce lineage-specific differentiation of stem cells. For example,
DPSC-derived exosomes can direct the differentiation of the bone marrow
MSCs towards an odontogenic lineage [[109]14]. Similarly,
osteoblast-derived exosomes containing instructive factors promoted the
osteogenic differentiation of MSCs, while the adipocyte-derived
exosomes triggered adipogenic differentiation of MSCs [[110]7].
Exosomes, by carrying cell-type specific biological molecules such as
protein, mRNA, and microRNA, serve as a mode of intercellular
communications during tissue formation and repair [[111]7]. Exosomal
microRNAs can negatively regulate gene expression by binding to their
target mRNAs through base pairing to the 3′-UTR, causing translational
repression of the mRNA in recipient cells [[112]23]. Exosomal microRNAs
play important roles in stem cell differentiation [[113]24]; however,
the function of microRNAs encapsulated in exosomes derived from DPSCs
is still unrevealed. In the present study, we performed microRNA
sequencing to clarify microRNA expression profiles of exosomes derived
from DPSCs. The results showed microRNA levels in OD-Exo were
significantly changed when compared with that in UN-Exo. There were 28
microRNAs significantly changed in OD-Exo isolated under odontogenic
conditions, of which 7 microRNAs increased (miR-5100, miR-27a-5p,
miR-652-3p, miR-1260a, miR-1260b, let-7f-1-3p, and miR-370-3p) and 21
microRNAs decreased (miR-193a-5p, miR-4792, miR-505-3p, miR-629-5p,
miR-140-3p, miR-185-5p, miR-146b-5p, miR-339-5p, miR-1246, miR-107,
miR-320d, miR-451a, miR-215-5p, miR-126-3p, miR-3687, miR-31-5p,
miR-210-3p, miR-1-3p, miR-10a-5p, miR-10b-5p, and miR-619-5p). Among
these, 2 upregulated microRNAs (miR-5100 [[114]25], miR-652-3p
[[115]26]), and 5 downregulated microRNAs (miR-185-5p [[116]27],
miR-107 [[117]28], miR-215-5p [[118]29], miR-31-5p [[119]30], and
miR-10b-5p [[120]31]) have been found to play important roles in stem
cell differentiation. miR-5100 has been verified to be upregulated
during osteoblast differentiation and promoted osteogenic
differentiation of MSCs [[121]25]. miR-652-3p has been found
significantly upregulated during neuronal differentiation and might
play a pivotal role in the promotion of neuronal differentiation
[[122]26]. On the other hand, suppression of miR-185-5p enhanced
ameloblast differentiation of LS8 cells and osteogenesis of MC3T3-E1
cells by regulating Dlx2 expression [[123]27]. miR-107 was found to
inhibit myoblast differentiation [[124]28] and adipocyte
differentiation [[125]32]. miR-215-5p inhibited adipocyte
differentiation of 3 T3-L1 cells through post-transcriptional
regulation of fibronectin type III domain containing 3B (FNDC3B) and
catenin-beta interacting protein 1 (CTNNBIP1) during early adipogenesis
[[126]29]. miR-31-5p inhibition enhanced the adipogenic differentiation
in hADSCs, since miR-31-5p directly bound to the 3′-UTR of C/EBP-α to
inhibit its expression [[127]30]. Downregulation of miR-10b-5p promoted
the differentiation of 3 T3-L1 cells and adipogenesis by upregulating
the Apol6 expression [[128]31]. In the present study, we transfected
DPSCs with miR-27a-5p which was expressed 11 times higher in OD-Exo,
and the results show miR-27a-5p mimics promoted odontogenic
differentiation of DPSCs by upregulating the protein expressions of
DSP, DMP-1, ALP, and RUNX2. Consequently, we can draw the conclusion
that exosomes isolated from DPSCs undergoing odontogenic
differentiation (OD-Exo) promoted the odontogenic differentiation via
the transfer of mircoRNAs.
In our study, genes targeted by differentially expressed microRNAs were
predicted by four publicly available bioinformatics tools (TargetScan,
miRTarBase, miRDB, and miRWalk). Pathway analysis showed genes targeted
by differentially expressed microRNAs were involved in multiple signal
transductions, including TGFβ signaling pathway. TGFβ signaling has
been shown to play important roles in odontogenic differentiation and
tooth development [[129]33, [130]34]. TGFβ signaling pathway was
activated by exosomes via upregulating p-Smad2 and promoting nuclear
localization of Smad4 [[131]35]. Exosomes regulated the TGFβ pathway by
increasing expression of Smad-3 in liver cells [[132]36]. It has been
revealed that exosomes regulated the TGFβ pathway by transferring
exosomal microRNAs. Exosomal miR-302b influenced the TGFβ pathway by
suppressing the expression of TGFβR2 [[133]37]. Exosomal miR-let7c
attenuated kidney injury by significantly downregulated TGFβ1 and TGFR1
in the kidneys [[134]38]. Exosomal miR-132 promoted lymphangiogenic
response by directly targeting Smad-7 and regulating TGF-β/Smad
signaling [[135]39]. In the present study, there were 16 genes targeted
by 15 differentially expressed microRNAs were involved in TGFβ
signaling. To confirm whether TGFβ signaling was activated by exosomal
microRNAs, we evaluated the key proteins in TGFβ signaling. Consistent
with pathway analysis, the protein expressions of TGFβ1, TGFR1,
p-Smad2/3, and Smad4 were significantly increased when DPSCs were
treated with OD-Exo for 48 h. In accordance, we inhibited the TGFβ1
signaling pathways by SB525334 and found protein levels of p-Smad2/3,
DSP, and DMP-1 were significantly reduced in DPSCs during odontogenic
differentiation induced by OD-Exo. We transfected DPSCs with miR-27a-5p
mimics which was predicted to be involved in TGFβ pathway, and the
results show miR-27a-5p mimics activated TGFβ1/smads signaling pathway
by increasing TGFβ1, TGFR1, p-Smad2/3, and Smad4 proteins. Thus, the
exosomal microRNAs promoted odontogenic differentiation via TGFβ1/smads
signaling pathway.
Since exosomal microRNAs negatively regulate gene expression by binding
to the 3′-UTRs of their target mRNAs, there may exist some inhibitory
molecules between the miR-27a-5p and TGFβ1 signaling. As reported,
latent TGF-β-binding protein 1 (LTBP1) was one of the inhibitory
molecules of TGFβ1 signaling. The LTBP1 was first identified as forming
latent complexes with TGFβ by covalently binding the TGFβ propeptide
(LAP) via disulfide bonds and was thought to primarily play a role in
maintaining TGFβ latency by anchoring TGF-β to the extracellular matrix
[[136]19]. Mutation of LTBP-1 in mice resulted in activating TGF-β
signaling through its receptor and accumulation of nuclear p-Smad2/3
[[137]20]. We performed western blot and double luciferase assay to
test whether LTBP1 could be directly downregulated by miR-27a-5p.
Interestingly, the western blot showed LTBP1 protein levels were
reduced by miR-27a-5p mimics. Moreover, we found that miR-27a-5p could
significantly reduce WT-LTBP1 luciferase activity and that miR-27a-5p
had no effect on the luciferase activity of MUT-LTBP1 group. Luciferase
reporter gene assay verified LTBP1 was the direct target gene of
miR-27a-5p. Taken together, we draw a conclusion that the exosomal
microRNAs promoted odontogenic differentiation via TGFβ1/smads
signaling pathway by downregulating the inhibitory molecule LTBP1.
Conclusions
This study identified the microRNA expression profiles of exosomes
derived from DPSCs. Our results showed that OD-Exo isolated under
odontogenic conditions were better inducers of DPSC differentiation.
Exosomal microRNAs promoted odontogenic differentiation via TGFβ1/smads
signaling pathway by downregulating the inhibitory molecule LTBP1.
Additional files
[138]Additional file 1:^ (15.7KB, docx)
Table S1. Primer pairs used in the qRT-PCR. (DOCX 15 kb)
[139]Additional file 2:^ (41.6KB, xlsx)
Table S2. All genes targeted by differentially expressed microRNAs were
analyze by four publicly available bioinformatics tools (TargetScan,
miRTarBase, miRDB, and miRWalk). (XLSX 41 kb)
Acknowledgements