Bone 134 (2020) 115303

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Bone
journal homepage: www.elsevier.com/locate/bone

Full Length Article

miR-99a in bone homeostasis: Regulating osteogenic lineage commitment T

and osteoclast differentiation
Sara Reis Mouraa,b, Joao Paulo Brasa,b,c, Jaime Freitasa,b, Hugo Osórioa,d,e,
Mario Adolfo Barbosaa,b,c, Susana Gomes Santosa,b,c, Maria Ines Almeidaa,b,c,∗
a
i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
b
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 4200-135 Porto, Portugal
c
ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
d
Ipatimup - Instituto de Patologia e Imunologia Molecular da Universidade do Porto, 4200-135 Porto, Portugal
e
FMUP – Faculdade de Medicina da Universidade do Porto, 4200-319 Porto, Portugal

ARTICLE INFO ABSTRACT

Keywords: Background: The tight coupling between osteoblasts and osteoclasts is essential to maintain bone homeostasis.
Cell differentiation Deregulation of this process leads to loss and deterioration of the bone tissue causing diseases, such as osteo-
Osteoporosis porosis. MicroRNAs are able to control bone-related mechanisms and have been explored as therapeutic tools. In
Bone remodeling this study, we investigated the potential of miR-99a-5p to modulate osteogenic differentiation, osteoclasto-
Osteoprotegerin
genesis, and the osteoblasts-osteoclasts crosstalk.
Gene therapy
Methods: To achieve this goal, human primary Mesenchymal Stem/Stromal Cells (MSC) were differentiated into
osteoblasts and adipocytes, and miR-99a-5p expression was evaluated by RT-qPCR. Knockdown and over-
expression experiments were conducted to modulate miR-99a-5p expression in MC3T3 cells. Cell proliferation
and cell death/apoptosis were evaluated by resazurin assay and flow cytometry, respectively. Proteomic analysis
was used to identify the miR-99a-5p regulatory network, and ELISA to evaluate OPG levels in the cell culture
supernatant. Conditioned media from MC3T3-transfected cells was used to culture RAW 264.7 cells and the
effect on osteoclast differentiation was assessed. Human primary monocytes were isolated to induce osteo-
clastogenesis and evaluate miR-99a-5p expression. Finally, levels of miR-99a-5p were modulated in RAW 264.7
cells to understand the impact on osteoclastogenesis.
Results: The results show that miR-99a-5p is significantly downregulated during the early stages of human
primary MSCs osteogenic differentiation and during MC3T3 osteogenic differentiation. On the other hand, miR-
99a-5p levels are increased during the initial stages of adipogenic differentiation. Inhibition of miR-99a-5p in
MC3T3 pre-osteoblastic cells promoted osteogenic differentiation, whereas its overexpression suppressed the
levels of osteogenic specific genes (Runx2 and Alpl), as well as mineralization, with no effect on proliferation or
apoptosis. Proteomic analysis of miR-99a-5p-transfected cells showed that numerous proteins known to be in-
volved in cell differentiation were altered, including osteogenic differentiation markers and extracellular matrix
proteins. While inhibition of miR-99a-5p increased the Tnfrsf11b (OPG encoding gene)/Tnfsf11 (RANKL en-
coding gene) mRNA expression ratio, in addition to increasing OPG secretion, miR-99a-5p overexpression re-
sulted in the opposite effect. The cell culture supernatant of miR-99a-5p-inhibited MC3T3 cells impaired the
osteoclastogenic potential of RAW 264.7 cells by decreasing the number of multinucleated cells and reducing the
expression of osteoclastogenic markers. Interestingly, miR-99a-5p expression is increased during osteoclasts
differentiation, both in human primary monocytes and RAW 264.7. These results show that miR-99a-5p per se is
a positive regulator of osteoclastogenic differentiation.
Conclusions: Globally, our findings show that miR-99a-5p inhibition promotes the commitment into osteogenic
differentiation, impairs osteoclastogenic differentiation, and control bone cells communication. Ultimately, it
supports miR-99a-5p as a target candidate for future miRNA-based therapies for bone diseases associated with
bone remodeling deregulation.

⁎
Corresponding author at: Rua Alfredo Allen, 208, 4200-135 Porto, Portugal.
E-mail address: ines.almeida@ineb.up.pt (M.I. Almeida).

https://doi.org/10.1016/j.bone.2020.115303
Received 1 October 2019; Received in revised form 4 February 2020; Accepted 25 February 2020
Available online 29 February 2020
8756-3282/ © 2020 Elsevier Inc. All rights reserved.
S.R. Moura, et al.
1. Introduction
Bone remodeling involves the coupling between new bone forma-
tion by osteoblasts and bone resorption by osteoclasts [1]. In healthy
conditions, this process is tightly regulated, particularly through the
RANKL/RANK/OPG signalling pathway [2,3]. However, bone re-
modeling can be disrupted by intrinsic or extrinsic factors. An im-
balance in the bone remodeling process may lead to the development of
bone disorders, such as osteoporosis, which is a systemic progressive
skeletal disease, with an increased vulnerability to bone fragility frac-
tures [4,5]. The formation and growth of bone tissue after a fracture,
whether as a consequence of a pathological disease or an injury, is a
complex process that involves distinct cell types, including the re-
cruitment of Mesenchymal Stem/stromal Cells (MSCs) to the injury site,
which then differentiate into osteoblasts [6]. During bone regenera-
tion/repair, it is crucial for MSCs to commit into the osteogenic lineage
in detriment of adipogenic differentiation [7]. In this context, the
identification and dissection of novel regulatory players in MSCs dif-
ferentiation, and in the crosstalk with osteoclasts, is essential to better
understand the molecular processes that contribute to bone homeostasis
and to bone regeneration/repair.
MicroRNAs (miRNAs) are small endogenous non-coding RNAs that
regulate gene expression post-transcriptionally, through com-
plementary binding to RNA transcripts, leading to RNA degradation or
inhibition of translation [8,9]. Recently, several studies described
miRNAs as key regulators of bone related processes [10–13]. For in-
stance, the local miRNA expression pattern is changed upon bone
fractures in healthy animal models [14,15] and in human osteoporotic
fractures [16–18]. Also, as previously demonstrated by us, miRNA
transcriptome is systemically changed in a timely manner upon a cri-
tical fracture in the rat femur [19]. We and others have identified
miRNAs as regulators of MSCs proliferation, differentiation and com-
munication with other cell types involved in bone repair, such as en-
dothelial and immune cells [11,20,21]. Recently, miRNA microarray
data analysis performed by us in the pre-osteoblast mouse cell line
MC3T3, and further confirmed in human primary MSCs, showed that
miR-29b-3p, miR-29c-3p and miR-20a-5p were overexpressed, whereas
miR-143-3p, miR-195-5p and miR-497-5p were downregulated during
osteogenic differentiation [11]. The microarray data also identified
miR-99a-5p to be downregulated in MC3T3 cells after 7 days of culture
in osteogenic-inducing conditions. Recently, Tang et al. has validated
miR-99a-5p role in the osteogenic differentiation of mouse-derived
MSCs and bone healing in a mice model [22]. Interestingly, a miR-99a
family member, miR-100, was shown to be upregulated in bone tissue
and serum samples from osteoporotic patients compared with non-os-
teoporotic patients [17]. Furthermore, the expression of this miR-99a
family member negatively correlate with bone mineral density, both in
plasma [23], and in bone tissue samples [24]. Therefore, dissecting the
mechanisms underlying
miR-99a-5p in bone cells is essential to support its usage as a
therapeutic target for future clinical approaches.
Currently, the miRbase database (v22) includes > 30,000 entries,
out of which, > 2500 correspond to human mature miRNA sequences
[23]. Hence, selecting which miRNA are candidates for future bone-
related clinical applications remains challenging. Envisioning future
miRNA-based therapies for bone repair/regeneration in the context of
fragility fractures, as a consequence of osteoporosis or other patholo-
gies, the ideal miRNA candidate should be able to promotes MSCs os-
teogenic differentiation in detriment of other lineages, while con-
comitantly decrease osteoclasts formation or activity. In this context,
we explored the role of miR-99a-5p as a potential therapeutic target
using in vitro tools. Specifically, we determined the involvement of
miR-99a-5p in MSC lineage commitment by analysing it expression
during osteogenic and adipogenic in in vitro differentiation of human
primary MSCs, determined the proteins and pathways most differently
affected by miR-99a under pro-osteogenic conditions thought high-
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Bone 134 (2020) 115303
throughput proteomics, evaluated modulation of OPG secretion, ex-
plored miR-99a-5p as a player in the crosstalk between osteoblasts and
osteoclasts, and lastly, investigated miR-99a-5p levels and its impact on
osteoclastogenesis.
2. Materials and methods
2.1. Human primary bone marrow-derived MSCs
Human primary MSCs (hMSCs) were isolated from the discarded
bone marrow of patients that underwent total hip arthroplasty or
anterior cruciate ligament injury at Centro Hospitalar de São João
(CHSJ, Porto, Portugal), after signing informed consent. These patients
did not suffer from known inflammatory diseases. Information about
the donors' sex, age, site of bone marrow collection and type of surgery
is provided in Supplemental Table 1. The protocol was approved by
CHSJ Ethics Committee for Health, and conforms to the declaration of
Helsinki. hMSCs from 6 donors were cultured in low-glucose Dulbecco's
Modified Eagle's Medium (DMEM, Corning) supplemented with 10%
(v/v) fetal bovine serum (FBS, mesenchymal stem cell-qualified, Gibco)
and 1% (v/v) penicillin/streptomycin (P/S, Invitrogen) in a humidified
atmosphere at 37 °C and 5% (v/v) CO2. hMSCs purity was confirmed by
flow cytometry through the expression of specific surface antigens
CD73, CD90 and CD105 (positive markers) and lack of expression of
HLA-DR, CD19, CD14, CD34 and CD45 (negative markers), according
to the criteria described by the International Society for Cellular
Therapy [25].
2.2. Human primary monocyte isolation
Human primary monocytes were isolated from buffy coats of 6
healthy blood donors kindly provided by CHSJ. The protocol was ap-
proved by CHSJ Ethics Committee for Health, and conforms to the
declaration of Helsinki. RosetteSep human monocyte enrichment iso-
lation kit was used (StemCell Technologies), as previously described by
us [20]. Briefly, buffy coats were centrifuged at 1200g, for 30 min, at
room temperature (RT), without active acceleration or brake. The
Peripheral Blood Mononuclear Cells (PBMCs) layer was collected, to-
gether with some red blood cells necessary for the formation of im-
munorosettes. Following the manufacturer's instructions, the Ro-
setteSep human monocyte enrichment isolation kit was added to the
cells and incubated during 20 min at RT in a horizontal shaker. Samples
were then 1:1 diluted in phosphate buffer saline 1× (PBS) supple-
mented with 2% (v/v) FBS (Biowest), before being carefully laid over
Histopaque®-1077 (Sigma-Aldrich). After centrifugation at 1200g, for
20 min, at RT, without acceleration and brake, a second gradient was
formed and the enriched monocyte layer (intermediate layer) was
collected and washed three times with PBS 1× by centrifugation at
300g during 20 min, before plating. Lastly, cells were resuspended in
Roswell Park Memorial Institute (RPMI, Gibco) with 10% (v/v) FBS
(Biowest) and 1% P/S (v/v) and counted using trypan blue dye (Sigma-
Aldrich) exclusion assay. Monocyte purity was routinely assessed by
flow cytometry analysis for positive CD14 marker (Immunotools), as
previously described [20,26]. Cells labelled with matching isotype were
used as negative control. Monocyte population contained > 80% CD14
positive cells (Supplemental Fig. 1).
2.3. Cell lines
MC3T3-E1 pre-osteoblastic cells, widely used in osteoblast biology
studies [27], and RAW 264.7 monocytic cells, commonly used in os-
teoclasts studies due to their osteoclastogenic potential [28], were
maintained in alpha-Minimum Essential Medium (α-MEM, Gibco)
supplemented with 10% heat inactivated FBS (Biowest) and 1% (v/v)
P/S (Invitrogen). All cells were expanded in a humidified atmosphere,
at 37 °C and 5% (v/v) CO2.
S.R. Moura, et al.
2.4. Osteogenic differentiation
To induce osteogenic differentiation, MC3T3 cells and hMSCs were
cultured in media supplemented with 10−7 M dexamethasone, 10−2 β-
glycerophosphate and 5 × 10−5 M ascorbic acid (all from Sigma-
Aldrich) and allowed to differentiate for 14 and 28 days, respectively.
In parallel, cells were grown without osteogenic supplements (basal
conditions), as a control. RNA was collected at days 0, 3, 7 and 14
(MC3T3) or 0, 3, 7, 14, 21 and 28 (hMSCs) of culture. Alkaline phos-
phatase (ALP) staining was performed at days 7 and 14 for MC3T3 and
hMSCs, respectively; Alizarin Red S staining was performed at days 14
and 28 for MC3T3 and hMSCs, respectively (Supplemental Methods).
2.5. Adipogenic differentiation
To induce adipogenic differentiation, hMSCs from 6 donors were
cultured for 28 days with the following supplements: 10−4 M dex-
amethasone, 10−1 mM Indomethacin, 5 × 10−4 M IBMX and 10 μg/mL
insulin (all from Sigma-Aldrich). RNA was collected at days 0, 3, 7, 14,
21 and 28 of culture. Oil Red O staining was performed to confirm
adipogenic differentiation into mature adipocytes at day 21 and day 28
(Supplemental Methods).
2.6. Osteoclastogenesis
Osteoclastogenesis in human primary monocytes (6 donors) was
induced with 30 ng/mL human macrophage colony-stimulating factor
(M-CSF, ImmunoTools) and 50 ng/mL human receptor activator of
nuclear factor kappa-Β ligand (RANKL, ImmunoTools). Cultures were
maintained during 21 days, and the media was carefully changed twice
a week. RNA was collected at days 0, 7, 14 and 21. F-actin/DAPI and
TRAP stainings were performed at days 7, 14 and 21 (Supplemental
Methods). RAW 264.7 cells were supplemented with 50 ng/mL mouse
RANKL (ImmunoTools). RNA was collected and TRAP staining assay
was performed each day, for 5 days. Multinucleated cells where con-
sidered when ≥3 nuclei [29–32].
2.7. Reverse transcription and real-time quantitative polymerase chain
reaction (RT-qPCR)
Total RNA was extracted using TRIzol® Reagent (Invitrogen), ac-
cording to the manufacturer's instructions. RNA concentration and
purity were determined using the NanoDrop Spectrophotometer ND-
1000 (ThermoFisher Scientific). RNA integrity was evaluated by gel
electrophoresis.
RNA was digested with TURBO DNA-free kit (Invitrogen), following
manufacturer's protocol, to remove potential DNA contaminants.
Complementary DNA (cDNA) was synthetized using random hexamers
(Invitrogen), dNTPs (Bioline) and SuperScript® III Reverse
Transcriptase kit (Invitrogen). qPCR reactions were performed using
cDNA, primers (Supplemental Table 2) and iQ SYBR Green Supermix
(Bio-Rad) in a CFX Real-Time PCR Detection System (Bio-Rad) with the
following conditions: 3 min at 95 °C and 40 cycles of 30 s at 95 °C, 30 s
at 58 °C and 30 s at 72 °C. GAPDH was used as a reference gene.
miRNA expression was evaluated by RT-qPCR using TaqMan miRNA
assays (Applied Biosystems). Briefly, cDNA was synthesized using total
RNA, TaqMan MicroRNA Reverse Transcription Kit (Applied
Biosystems) and gene specific stem-loop Reverse Transcription primers
(hsa-miR-99a-5p and small nuclear RNA U6, Applied Biosystems), ac-
cording to the manufacturer's protocol. qPCR was carried out in CFX
Real-Time PCR Detection System (Bio-Rad), using cDNA,
SsoAdvanced™ Universal Probes Supermix (Bio-Rad) and hsa-miR-99a-
5p or small nuclear RNA U6 TaqMan assays (Applied Biosystems),
under the following conditions: 10 min at 95 °C, 40 cycles of 15 s at
95 °C and 1 min at 60 °C. Small nuclear RNA U6 was used as a reference
gene.
3
Bone 134 (2020) 115303
Relative expression levels were calculated using the quantification
cycle (Cq) method, according to MIQE guidelines [33]. Data was ana-
lyzed using Bio-Rad CFX Manager software and all the reactions were
performed in duplicate.
2.8. miRNA mimics and inhibitor transfection
MC3T3 and RAW264.7 cells were transfected with 50 nM of miR-
99a-5p mimics (mimics), miR-99a-5p inhibitor (inhibitor), or the re-
spective controls, namely miRNA mimics negative control (NC-mimics),
or miRNA inhibitor negative control (NC-inhibitor), all from Ambion,
using Lipofectamine 2000 transfection reagent (Invitrogen), according
to manufacturer's instructions. Cells were incubated with the miRNA-
lipid complexes for 12 h and then collected for the distinct assays.
MC3T3-transfected cells were plated in osteogenic differentiation con-
ditioned media, as described in 2.4, for ALP (day 7) and for Alizarin
(day 10) staining, RNA extraction (days 3 and 7), protein extraction
(day 7), collection of supernatants (day 7). MC3T3-transfected cells
were also plated in growth condition media for evaluation of metabolic
activity (Section 2.9), apoptosis and cell death (Section 2.10). RAW
264.7-transfected cells were plated in osteoclastogenic differentiation
conditions, as described in Section 2.6, for TRAP staining (day 2) and
RNA extraction (day 2).
2.9. Resazurin reduction assay
To assess the effect of miR-99a-5p on cell metabolism/proliferation,
MC3T3 cells transfected with either miR-99a-5p mimics, miR-99a-5p
inhibitor or the controls were seeded in 96-well plates. Resazurin redox
dye (0.01 mg/mL, Sigma-Aldrich) was added to the cells (10% (v/v)),
and incubated for 2 h at 37 °C. Experiments were performed for 7 re-
plicates in 5 time-points: days 0, 1, 2, 3 and 4. The supernatant was
transferred to a dark wall 96 well plate and fluorescence levels fol-
lowing resazurin (non-fluorescent blue dye) reduction into resorufin
(fluorescent and pink) were measured at an excitation wavelength of
530 nm and an emission wavelength of 590 nm, using the spectro-
photometer microplate reader Synergy MX (Biotek Synergy).
2.10. Apoptosis assay by flow cytometry
MC3T3 cells transfected with either miR-99a-5p mimics, miR-99a-
5p inhibitor or the respective controls were collected at day 7 post-
transfection. Cells were washed 3 times with PBS 1× and labelled with
FITC Annexin V Apoptosis Detection Kit I (BD Biosciences), according
to manufacturer's instructions. Briefly, cells were re-suspended in
binding buffer and incubated with FITC-Annexin V and Propidium
Iodide (PI) for 15 min, at RT, in the dark. Then, the cells were run
within 1 h on the BD Accuri™ C6 system and the results were analyzed
with FlowJo software.
2.11. Enzyme-linked immunosorbent assay
The culture media from MC3T3 transfected cells was collected at
day 7 of osteogenic differentiation and centrifuged at 800g for 10 min,
to remove cell debris. Osteoprotegerin (OPG) concentration in the su-
pernatants was determined using the commercial Quantikine ELISA kit
(MOP00 for OPG, R&D Systems), according to manufacturer's instruc-
tions. Absorbance was measured at 450 nm with correction set to
540 nm.
2.12. Effect of osteogenic-conditioned media on osteoclastogenesis
Cell culture media derived from MC3T3 cells transfected with miR-
99a-5p mimics, miR-99a-5p inhibitor, or the respective controls, was
collected following 7 days of culture in osteogenic-inducing conditions.
Media was centrifuged at 800g, for 10 min, at 4 °C, to remove cells and
S.R. Moura, et al.
cell debris. RAW 264.7 cells were incubated with the MC3T3-derived
cell culture media at a proportion of 3:1 (3 volumes of conditioned
media and 1 volume of α-MEM with FBS) for 72 h. As a control, RAW
264.7 cells were also independently incubated with α-MEM + 10% (v/
v) FBS (negative control) or α-MEM + 10% (v/v) FBS + 50 ng/mL
RANKL (positive control). Expression of osteoclastogenic associated
genes was evaluated by RT-qPCR and the number of multinucleated
cells was quantified by microscopy.
2.13. Proteomic analysis
MC3T3 cells transfected with miR-99a-5p mimics, miR-99a-5p in-
hibitor or the respective controls were cultured in osteogenic differ-
entiation conditions. Cells were harvested at day 7 of differentiation
and washed 6 times with cold PBS 1×, before being lysed with cold
RIPA buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-
100 and 1% (v/v) NonidetP-40] in the presence of protease inhibitors
[(10 μL/mL phenylmethylsulfonyl fluoride (PMSF, 200 mM), 10 μL/mL
Leupeptin (1 ng/mL) and 10 μL/mL Aprotinin (1 mg/mL)] and phos-
phatase inhibitors [20 μL/mL NaVO3 (50 nM), 50 μL/mL Na4P2O7
(50 mg/mL) and 10 μL/mL NaF (10 mM)], for 30 min. Cell lysates were
centrifugation at 20,000g, for 10 min, at 4 °C and the supernatant-
containing protein was quantified using DC protein assay kit (Bio-Rad).
Protein identification and label-free quantitation was performed by
nanoLC-MS/MS, composed by an Ultimate 3000 liquid chromatography
system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass
spectrometer (Thermo Scientific, Bremen, Germany). Samples were
loaded onto a trapping cartridge for 3 min and further separated on an
nano-C18 column at 300 nL/min. Peptide separation gradient was the
following (A: 0.1% (v/v) FA, B: 80% (v/v) ACN 0.1% (v/v)): 5 min
(2.5% (v/v) B to 10% (v/v) B), 100 min (10% (v/v) B to 35% (v/v) B),
20 min (35% (v/v) B to 55% (v/v) B), 3 min (55% (v/v) B to 99% (v/v)
B) and 12 min (hold 99% (v/v) B). Data acquisition was controlled by
Xcalibur and Tune software (Thermo Scientific).
The mass spectrometer was operated in data-dependent positive
acquisition mode alternating between a full scan (m/z 380–1580) and
subsequent HCD MS/MS of the 10 most intense peaks from full scan.
Raw data was processed using Proteome Discoverer 2.3.0.523 software
(Thermo Scientific). Protein identification was performed with Sequest
HT search engine against the Mus musculus entries from the UniProt
database (https://www.uniprot.org/). Mass tolerance was 10 ppm for
precursor and 0.02 Da for fragment ions, respectively. Maximum al-
lowed missing cleavage sites was set 2. Cysteine carbamidomethylation
was defined as constant modification. Methionine oxidation and protein
N-terminus acetylation were defined as variable modifications. Protein
and peptide confidence were set to high. The processing node
Percolator was enabled with the following settings: maximum delta Cn
0.05; decoy database search target FDR 1%, validation of based on q-
value. Analyzed samples were normalized against to the total peptide
signal in each experiment and its quantitative evaluation was achieved
by pairwise comparisons of the detected peptides. Data was corrected
using Benjamin Hochberg method. Protein levels were compared by
using the median ratio.
Bioinformatics and data analysis was performed for proteins ex-
pressed in the two independent experiments with fold-change (FC)
differences ≥1.25 or ≤−1.25. A minimum of two unique peptides
were required for further evaluation. The differentially expressed pro-
teins were classified according to Gene Ontology (GO) annotations. The
enriched Biological Process analysis was performed using Protein
ANalysis THrough Evolutionary Relationships (PANTHER) tool.
miRWalk2.0 database was used for miRNA target prediction (http://
mirwalk.umm.uni-heidelberg.de/). For the analysis of the top 20 pro-
teins with opposite profiles between the miR-99a-mimics/NC-mimics
and in the miR-99a-5p-inhibitor/NC-inhibitor groups, only the proteins
with greater than or equal to 6 unique peptides were considered.
4
Bone 134 (2020) 115303
2.14. Western blot
Protein extracts (30 μg of protein) were prepared in reducing
loading buffer, denatured at 95 °C for 5 min, and resolved in 10% (v/v)
polyacrylamide SDS-PAGE gels at 100 V, along with the molecular
weight marker. Proteins were wet-transferred to nitrocellulose mem-
branes, blocked with a 5 g/L non-fat dry milk solution, and probed
overnight at 4 °C using the following primary antibodies: anti-
Fibromodulin (H-11, Santa Cruz Biotechnology, 1:500), anti-Lumican
(B-9, Santa Cruz Biotechnology, 1:500), and anti-GAPDH (Cell
Signalling, 1:1000). Next, the membranes were probed with an ap-
propriate secondary antibody conjugated to horseradish peroxidase
(HRP, GE Healthcare, 1:10000) for 1 h, at RT. After washing, mem-
branes were incubated with chemiluminescent substrate (ECL, GE
Healthcare), for signal detection in autoradiographic films (GE
Healthcare). Protein bands were quantified on Fiji, and relative protein
levels were calculated using GAPDH bands as normalizer.
2.15. Statistical analysis
Statistical data analysis was performed on Prism 7 (GraphPad
Software, Inc.). Gaussian distribution was tested by the Shapiro-Wilk
and Kolmogorov-Smirnov normality tests. Data was only considered to
follow a normal distribution when passed both tests (alpha = 0.05). For
non-normal distribution data, or when n < 5, non-parametric tests
were used to evaluate significant differences between samples, namely
two-tailed Wilcoxon matched pairs test (between 2 groups) or Friedman
test (> 2 groups) followed by uncorrected Dunn's multiple comparison
test. When the data passed normality tests, one-way ANOVA (> 2
groups), followed by Sidak's multiple comparison or Turkey's multiple
comparisons tests were used. For the RT-qPCR data, the fold change
differences (2−∆∆CT) were tested using the Wilcoxon signed-ranked test.
Statistical significance was considered for P < 0.05 (* P < 0.05; **
P < 0.01 and *** P < 0.001).
3. Results
3.1. miR-99a-5p is downregulated during early stages of osteogenic
differentiation of human primary MSCs and in mouse MC3T3 cell line
When hMSCs were induced to differentiate into the osteogenic
lineage, the ALP staining and the mineralized area, assessed by Alizarin
staining, were significantly increased when compared to basal condi-
tions (Fig. 1A and B). The expression profile of miR-99a-5p in 6 hMSCs
donors shows that miR-99a-5p levels were significantly downregulated
at days 3, 7 and 14 of osteogenic differentiation compared to the basal
control (P < 0.05, days 3, 7 and 14), while at days 21 and 28 miR-99a-
5p expression was restored to basal levels (Fig. 1C). This indicates a
potential effect of this miRNA in the early stages of hMSCs osteogenic
differentiation rather than in the later mineralization stages. In the
MC3T3 mouse pre-osteoblastic cell line, osteogenic differentiation also
significantly increased ALP staining, and the deposition of calcium at
days 7 and 14, respectively (Fig. 1D and E). RT-qPCR results show that
miR-99a-5p was significantly downregulated during osteogenic differ-
entiation, specifically at day 7 (P < 0.05), which is in agreement with
our previously published microarray results [11], and at day 14
(P < 0.05), compared to the basal control in each time point (Fig. 1F).
3.2. miR-99a-5p is upregulated during the early stages of adipogenic
differentiation of human primary MSCs
To determine the impact of miR-99a-5p in the hMSCs' adipogenesis,
we induced their commitment into the adipogenic lineage. Results show
an increased Red O staining of the lipid droplets in terminally differ-
entiated mature adipocytes, after 21 and 28 days of culture in adipo-
genic-inducing conditions, compared to basal controls (Fig. 2A). The
S.R. Moura, et al. Bone 134 (2020) 115303

Fig. 1. miR-99a-5p expression during osteogenic differentiation of hMSCs and MC3T3 cells. A) Representative image of ALP staining at day 14 and detection of
calcium deposits by Alizarin Red S staining (stained red) at day 28 in hMSCs under osteogenic differentiation (Dif) and basal conditions (5×, scale 500 μm). B) ALP
staining and Alizarin quantification in hMSCs after 14 and 28 days, respectively (median, N = 6, two-tailed Wilcoxon matched pairs test, *P < 0.05). C) miR-99a-5p
expression in hMSCs after 3, 7, 14, 21 and 28 days (tdif) of culture in osteogenic versus basal conditions (median, N = 6, Wilcoxon signed ranked test, * P < 0.05).
D) Representative image of ALP staining at day 7 and detection of calcium deposits by Alizarin Red S staining (stained red) at day 14 in MC3T3 cells under osteogenic
differentiation and basal conditions (5×, scale 500 μm). E) ALP staining and Alizarin Red S quantification in MC3T3 after 7 and 14 days, respectively (median,
N = 6, two-tailed Wilcoxon matched pairs test, * P < 0.05). F) miR-99a-5p expression in MC3T3 pre-osteoblasts after 3, 7 and 14 days (tdif) of culture in osteogenic
versus basal condition (median, N = 6, Wilcoxon signed ranked test, * P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

expression of the adipogenic-specific markers ADIPOQ (Adiponectin) negatively with osteogenic differentiation.
and PPARG2 (Peroxisome proliferator activated receptor gamma) was
upregulated after 21 and 28 days (P < 0.05) (Fig. 2B). Interestingly, at
3.3. miR-99a-5p regulates negatively the osteogenic differentiation but has
early time points of adipogenic differentiation, miR-99a-5p was sig-
no effect on proliferation or apoptosis of MC3T3 cells
nificantly upregulated (P < 0.05, days 3 and 7), compared with basal
conditions (Fig. 2C), while decreased at days 21 and 28 (P < 0.05).
Next, we evaluated the biological impact of miR-99a-5p through
Therefore, at early stages of hMSCs differentiation, miR-99a-5p ex-
gain- and loss-of-function studies in MC3T3 cells, which were efficiently
pression levels associate positively with adipogenic differentiation, but
transfected (Fig. 3A). miR-99a-5p overexpression decreased ALP

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Fig. 2. miR-99a-5p expression during adipogenic differentiation of hMSCs.

A) Representative image of Oil Red O staining (lipids stained red) at days 21 and 28 under adipogenic differentiation and basal conditions (10×, scale 200 μm; 5×,
scale 500 μm, respectively). B) Expression of adipogenic specific genes, ADIPOQ and PPARG2 (median, N = 6, Wilcoxon signed ranked test, * P < 0.05). C) miR-
99a-5p expression in hMSCs grown in adipogenic-inducing conditions relative to basal conditions after 3, 7, 14, 21 and 28 days (tdif) in culture (median, N = 6,
Wilcoxon signed ranked test,* P < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

staining in 42.20% (P < 0.01), whereas miR-99a-5p inhibition in-
creased ALP staining in 70.77% (P < 0.05), after 7 days in osteogenic
inducing conditions, compared to controls (Fig. 3B). Also, results show
a 60.29% decrease (P < 0.001) on the formation of calcium deposits in
miR-99a-mimics transfected cells, and a 88.64% increase (P < 0.01) in
miR-99a-inhibitor transfected cells, both after 10 days of differentiation
and compared to their respective controls (Fig. 3C). In agreement with
these results, mRNA levels of key the osteogenic markers Alpl and
Runx2 were significantly decreased (P < 0.05) in miR-99a-5p
overexpressing cells cultured under osteogenic inducing conditions at
days 3 and 7 (Fig. 3D). Conversely, Alp and Runx2 were significantly
increased at day 3 (P < 0.05) and day 7 (P < 0.05) in response to
miR-99a-5p inhibition (Fig. 3D). In order to understand if the effect of
miR-99a-5p on osteogenic differentiation could be a consequence of its
impact on cell proliferation, the metabolic activity was measured as an
indirect method to evaluate cell proliferation. Results show that neither
upregulation nor downregulation of miR-99a-5p had a significantly
effect on cell proliferation compared to controls (Fig. 3E). The Annexin

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S.R. Moura, et al. Bone 134 (2020) 115303

Fig. 3. Effect of miR-99a-5p in osteogenic differentiation, cell proliferation and apoptosis. A) miR-99a-5p expression after transfection of MC3T3 cells with miR-99a
mimics, miR-99a inhibitor or respective controls (miR-99a NC-mimics, miR-99a NC-inhibitor), and culture in osteogenic-inducing conditions (median, N = 7,
Wilcoxon signed ranked test, * P < 0.05). B) ALP and C) Alizarin staining after transfection and culture for 7 and 10 days, respectively, in osteogenic-inducing
conditions (mean ± SD, N = 7, two-tailed Wilcoxon matched pairs test, * P < 0.05, ** P < 0.01 and *** P < 0.001). D) Alpl and Runx2 expression levels at days
3 and 7 after transfection and incubation in osteogenic-inducing conditions (median, N = 7, Wilcoxon signed ranked test, * P < 0.05). E) Representative fluor-
escence profile (resorufin) of 4 independent experiments measured every 24 h for 4 days in transfected cells (N = 4, Friedman test, followed by uncorrected Dunn's
multiple comparison test; n.s.: non-significant). F) Representative image of flow cytometry analysis of AnnexinV/PI staining and quantification of viable (Annexin
V− PI−), early apoptotic (Annexin V+ PI−), or late apoptosis and dead cells (Annexin V− PI+ and Annexin V+ PI+) (median, N = 3, Friedman test, followed by
uncorrected Dunn's multiple comparison test, n.s.: non-significant).

V/PI staining was used to evaluate apoptosis at days 3 and 7. Flow
cytometry results show that overexpression or inhibition of miR-99a-5p
in MC3T3 cells did not significantly affect early apoptosis or late
apoptosis/cell death (Fig. 3F). Taken together, the results indicate that
miR-99a-5p exerted an osteogenic-suppressive effect in MC3T3 cells in
vitro, but has no effect on proliferation or apoptosis.
3.4. Modulation of miR-99a-5p levels changes cellular proteomic profile
To identify novel miR-99a-5p targets and elucidate relevant me-
chanisms and/or pathways regulated by this miRNA, cells transfected
with miR-99a-5p-mimics, miR-99a-5p-inhibitor, or the respective con-
trols, were analyzed by mass spectrometry-based proteomics.
Proteomic profiling identified and quantified a total of 3907 proteins.
Analysis revealed differences in 1736 proteins when comparing miR-
99a-5p overexpressing cells versus NC-mimics transfected cells
(FC ≥ |1.25|), and in 809 proteins when comparing miR-99a-5p in-
hibited cells versus NC-inhibitor transfected cells (FC ≥ |1.25|). Out of
those, 64 differentially expressed proteins were simultaneously down-
regulated by miR-99a-5p mimics and upregulated by miR-99a-5p in-
hibitor. When only considering the statistically different proteins
(P < .05) that were downregulated by the miR-99a-5p mimics or
upregulated by miR-99a-5p inhibitor, results show 44 significantly
different proteins (Supplemental Table 3). In silico analysis (miRWalk
2.0. database [34]) revealed that 6 of those proteins were predicted by
at least one algorithm to originate from transcripts with putative miR-
99a-5p binding sites (Supplemental Table 4). Conversely, 316 proteins
were upregulated in miR-99a-overexpressing cells and downregulated
in miR-99a-5p-knowckdown cells (Fig. 4A).
Gene ontology analysis shows that the molecular function mostly
affected by changes in miR-99a-5p expression, either by its over-
expression or inhibition, is the cellular catalytic activity (GO: 0003824,
Fig. 4B). Included in this term, the protein kinase activity (GO:
0004672) shows the highest percentage of gene hits compared to the
total number of function hits (Supplemental Table 5). Fig. 4C shows the
20 most differently expressed proteins with opposite regulation by miR-
99a-5p mimics and its inhibitor. These include extracellular matrix
(ECM) components that are members of the small interstitial leucine-
rich repeat proteoglycans family (SLRPs), namely Fibromodulin (Fmod)
and Lumican (Lum), which are increased in miR-99a-5p-inhibitor
transfected cells (FC = 1.90 and 1.25, respectively) and decreased by
miR-99a-5p-mimics transfected cells (FC = −1.44 and −1.38, re-
spectively) (Fig. 4C). This effect was validated by western blot for Fmod
(Fig. 4D), but not for Lum (Supplemental Fig. 2), which is in agreement
with the higher differential effect found for Fmod by mass spectometry
analysis (Fig. 4C). Additionally, Fmod mRNA expression was reduced by
miR-99a-5p overexpression (P < 0.05), while increased by miR-99a-
5p inhibition (P < 0.05) (Fig. 4D).
Most of the differently expressed proteins were detected after miR-
99a-5p overexpression. According to GO term analysis, 76 of those
proteins are associated with “cell differentiation” and, of those, the top
20 include the osteogenic-specific markers ALPL (FC = −2.10),
RUNX2 (FC = −2.07), and Osterix (SP7, FC = −1.53). Additionally,
changes were also found in proteins with roles both in “cell differ-
entiation” and in “extracellular matrix production”, COL8A1
(FC = −1.82) and COL4A1 (FC = −1.60) (Fig. 4E).
Globally, this high-throuput proteomic analysis in MC3T3-trans-
fected cells shows that distinct intracellular proteins related to osteo-
genic differentiation, as well as matrix proteins, are modulated by miR-
99a-5p.
3.5. miR-99a-5p regulates osteoclastogenesis through a paracrine effect
Bone homeostasis requires the crosstalk between osteoblasts and
osteoclasts, which involves secreted mediators. As miR-99a-5p may
affect levels of secreted protein, we analyzed the expression levels of
the two main mediators of osteoblast to osteoclast communication:
OPG, as a protector of excessive bone resorption; RANKL, as a positive
regulator of bone resorption [2]. qRT-PCR results show that Tnfrsf11b
mRNA (OPG encoding gene) is decreased by miR-99a-5p over-
expression (P < 0.05), while increased by miR-99a-5p inhibition
(P < 0.05, Fig. 5A). On the other hand, Tnfrsf11 (RANKL encoding
gene) expression does not change following miR-99a-5p mimics/in-
hibitor transfection (Fig. 5A). Interestingly, in the control conditions,
Tnfrsf11b is expressed at much higher levels than Tnfrsf11. Therefore,
Tnfrsf11b is the main contributor to the decreased in Tnfrsf11b/Tnfrsf1
expression ratio by the miR-99a-5p-mimics, and the increase by miR-
99a-5p-inhibitor (P < 0.05). Next, we investigated whether the OPG
protein levels in the conditioned media were altered. In agreement with
the gene expression levels, results show that secreted OPG levels are
significantly reduced in supernatants of miR-99a-5p-overexpressing
cells (P < 0.001), while increased in supernatants of miR-99a-5p-in-
hibitor transfected cells (P < 0.001) (Fig. 5B).
Next, we tested the effect of MC3T3-conditioned media on osteo-
clast differentiation, following miR-99a-5p overexpression or inhibi-
tion. RAW 264.7 cells were cultured during 2 days with media derived
from MC3T3-transfected with miR-99a-5p-mimics or with miR-99a-5p-
inhibitor, in the absence of RANKL supplementation. As a positive
control, RAW 264.7 cells were independently treated with RANKL.
Remarkably, the number of multinucleated cells showed an increase by
33.6% (P < 0.05) and a decrease by 32.8% (P < 0.001), following
culture with the conditioned media derived from miR-99a-5p-mimics or
miR-99a-5p-inhibitor transfected MC3T3 cells, respectively (Fig. 5C).
The expression levels of osteoclast fusion markers Dcstamp, Ccl2, and
osteoclastogenic-specific marker Ctsk, were increased in RAW 264.7
cells stimulated by miR-99a-5p-mimics-MC3T3 conditioned media
(Dcstamp: FC = 1.56, P < 0.05; Ccl2: FC = 1.85, P < 0.05; Ctsk:
FC = 1.57, P < 0.05), whereas the opposite effect was observed when
cells were exposed to miR-99a-5p-inhibitor-MC3T3 conditioned media
(Dcstamp: FC = 0.745, P < 0.05; Ccl2: FC = 0.862, P < 0.05; Ctsk:
FC = 0.835, P < 0.05) (Fig. 5D). These results suggest that the con-
ditioned media from miR-99a-5p transfected cells influences the dif-
ferentiation of osteoclast-like cells by affecting the expression of os-
teoclastogenic markers, as well as their ability to form multinucleated
cells.
Interestingly, intracellular levels of miR-99a-5p in RAW 264.7 cells
were significantly increased after culture with miR-99a-5p-MC3T3
conditioned media and decreased after culture with anti-miR-99a-5p-
MC3T3 conditioned media (Supplemental Fig. 3). Taken together these
results support a role for miR-99a-5p in osteoclastogenesis via a para-
crine effect.

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S.R. Moura, et al. Bone 134 (2020) 115303

Fig. 4. Proteomic analysis of miR-99a-5p downstream targets and processes. A) Number of differently expressed proteins in MC3T3 cells after transfection with miR-
99a-5p mimics, inhibitor or respective controls (FC ≥ |1.25|). Number of proteins significantly increased by miR-99a inhibition or decreased by miR-99a over-
expression (FC ≥ |1.25| and P < 0.05) with miR-99a-5p predicted binding sites by miRWalk 2.0. B) Gene ontology enrichment analysis, according to PANTHER
classification system. The Y-axis shows the number of proteins associated with determined GO term (X-axis). C) Levels (fold change) of the most antagonistic proteins
between miR-99a-5p-inibition and miR-99a-5p-overexpressing cells (≥6 unique peptides). D) Fibromodulin (FMOD) protein levels by Western blot in MC3T3 cells
after transfection with miR-99a-5p mimics, inhibitor, or respective controls, and following 7 days of culture in osteogenic-inducing conditions. GAPDH was used as a
normalyzer. Representative image (left panel) and quantification FMOD levels of 5 independent experiments using FIJI software (quantification FMOD/GAPDH
expression in 5 independent experiments (mean ± SD, N = 5, one-way ANOVA, followed by Sidak's multiple comparison test, * P < 0.05). Fmod mRNA expression
levels (right panel, median, N = 6, Wilcoxon signed ranked test, * P < 0.05). E) List of the top 20 most differential proteins associated with the GO term “cell
differentiation” and decreased by miR-99a-5p-overexpressing cells.

Fig. 5. Effect of conditioned media from miR-99a-5p mimics/inhibitor MC3T3-transfected cells on osteoclastogenesis. A) Expression of Tnfrsf11b, Tnfsf11 and
Tnfrsf11b/Tnfsf11 ratio in MC3T3-transfected cells with either miR-99a-5p mimics, inhibitor or respective controls, at day 7 of osteogenic differentiation (median,
N = 6, Wilcoxon signed ranked test, * P < 0.05, n.s.: non-significant). B) OPG protein levels in conditioned media from MC3T3-transfected cells relative to control
(mean ± SD, N = 8, one-way ANOVA, followed by Sidak's multiple comparison test, *P < 0.05). C) Number of multinucleated cells (mean ± SD, N = 6, one-way
ANOVA, followed by Sidak's multiple comparison test, * P < 0.05, ** P < 0.01 and *** P < 0.001) and E) expression of osteoclastogenic-markers Dcstamp, Ccl2
and Ctsk (median, N = 6, Wilcoxon signed ranked test, * P < 0.05) of RAW 264.7 cells grown in the conditioned media from MC3T3-transfected cells.

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S.R. Moura, et al. Bone 134 (2020) 115303

Fig. 6. Time-dependent increase of miR-99a-5p during monocyte to osteoclast differentiation. A) Osteoclastogenesis of human monocytes. F-actin and nuclei staining
at days 7, 14 and 21 of differentiation (supplemented with MCS-F and RANKL) and in control cells (MCS-F) (10×, scale 100 μm). B) Number of cells with 1, 2, 3–9
and > 10 nuclei per cell across 6 separate random fields (median, N = 6, Friedman test, followed by uncorrected Dunn's multiple comparison test, * P < 0.05 and **
P < 0.01). C) Average of the area of the human osteoclasts after 7, 14 and 21 days of differentiation (mean + SD, N = 6, one-way ANOVA, followed by Turkey's
multiple comparison test, ** P < 0.01 and *** P < 0.001). D) Expression levels of human osteoclast specific genes ACP5, CTSK and CALCR in the presence of
osteoclastogenic-inducing supplements (median, N = 6, Wilcoxon signed ranked test, * P < 0.05). E) miR-99a-5p expression levels in human monocyte-derived
osteoclasts grown in osteoclastogenic-inducing conditions after 7, 14 and 21 days, relative to day 0/non-stimulated monocytes (median, N = 6, Wilcoxon signed
ranked test, * P < 0.05). F) miR-99a-5p expression levels of RAW 264.7 cells grown in osteoclastogenic-inducing conditions after 1, 2, 3, 4 and 5 days, relative to
day 0 (median, N = 6, Wilcoxon signed ranked test, * P < 0.05).

3.6. miR-99a-5p expression increases during osteoclastogenesis of human 4. Discussion

primary monocytes and of mouse RAW 264.7 cells
The results obtained this far prompted us to investigate the levels of
miR-99a-5p during osteoclastogenesis. As such, human primary
monocytes isolated from healthy blood donors were seeded in the
presence of RANKL and M-CSF, and the number of multinucleated cells
was quantified over time. Monocytes stimulated with only M-CSF were
used as a negative control (Fig. 6A, Supplemental Fig. 4). As expected,
the number of nuclei per cell increases in a time-dependent manner
(Fig. 6B). While the number of cells with only one or two nuclei pro-
gressively decreases during differentiation into OCs at day 21 (1 nuclei:
P < 0.01; 2 nuclei: P < 0.05), the number of cells with > 10 nuclei is
significantly higher at day 21 (P < 0.01), compared to day 7 of dif-
ferentiation (Fig. 6B). Accordingly, along differentiation the cells were
able to merge and originate large OCs, increasing the cells size over
time (day 14: P < 0.001; day 21: P < 0.01) (Fig. 6C). Also, the ex-
pression levels of the osteoclastogenic markers ACP5 (TRAP encoding
gene), CTSK, and CALCR (CTR encoding gene) were significantly in-
creased over time (Fig. 6D), confirming a successful osteoclast differ-
entiation for all 6 donors. Finally, the expression of miR-99a-5p was
evaluated during osteoclastogenesis in these cells. As illustrated in
Fig. 6E, miR-99a-5p is significantly overexpressed during the differ-
entiation of human monocyte-derived OCs (day 7: FC = 4.12,
P < 0.05; day 14: FC = 6.31, P < 0.05; day 21: FC = 10.52,
P < 0.05), compared to unstimulated cells (day 0). Similar to the
human monocytes-derived OCs, miR-99a-5p is upregulated during os-
teoclastogenesis of RAW 264.7 murine monocytes, particularly at the
later time points (day 4: FC = 5.20, P < 0.05; day 5: FC = 6.35,
P < 0.01; (Fig. 6F). The expression of osteoclastogenic genes was also
evaluated during 5 days of RANKL-induced differentiation in RAW
264.7 cells (Supplemental Fig. 5).
3.7. miR-99a-5p positively regulates osteoclastogenesis
To investigate the role of miR-99a-5p in osteoclast differentiation,
gain- and loss-of-functions studies were performed in RAW 264.7 cells.
Levels of miR-99a-5p were increased upon transfection with miR-99a-
5p mimics and decreased after transfection with its inhibitor (Fig. 7A).
Two days after transfection, and in the presence of RANKL stimulation,
miR-99a-5p-overexpressing RAW 264.7 cells show a significant increase
in Dcstamp expression (P < 0.05), and a decrease with the inhibition of
miR-99a-5p (P < 0.05) (Fig. 7B). Also, Ccl2 is upregulated by miR-99a
(P < 0.05). Although not statistically significant, expression of Ctsk
follow the same tendency, with an upregulation of by miR-99a-mimics
(P = 0.125) and a reduction by miR-99a-inhibitor (P = 0.094)
(Fig. 7B). Finally, we tested the ability of these cells to fuse and form
osteoclast-like cells under RANKL stimulation. Results show that miR-
99a-5p overexpression promotes the formation of multinucleated cells
(FC = 1.54, P < 0.05), while miR-99a-5p inhibition impairs cell fu-
sion (FC = 0.776, P < 0.05) (Fig. 7C). Taken together, these results
demonstrate that miR-99a-5p has a functional effect on osteoclasto-
genesis.
Cell lineage commitment is crucial when considering the potential
use of miRNA-based engineered MSCs for bone diseases or fractures [6].
Herein, we show that, in human primary bone-marrow-derived MSCs,
miR-99a-5p expression is decreased during early stages of osteogenic
differentiation, and that miRNA inhibition promotes a pro-osteogenic
profile. The miR-99a-5p expression profile during osteogenic differ-
entiation is in agreement with our published microarray data in a
mouse pre-osteoblastic cell line [11] and with Tang et al. findings in
mice MSCs [22]. Furthermore, we show, for the first time in human
MSCs, that miR-99a-5p increases in early stages of adipogenic differ-
entiation, suggesting an opposite expression profile in comparison with
osteogenic differentiation. Interestingly, in distinct cancer cell lines,
miR-99a-5p has been shown to act as a tumour suppressor through
negative regulation of proliferation (e.g. Bladder [35], esophageal
squamous cell [36], and renal cell carcinoma [37]) and inducing
apoptosis (e.g. breast cancer [38]). However, in this study, we show
that in a non-tumoral cell line (MC3T3), miR-99a-5p does not affect
proliferation or apoptosis/cell death. Therefore, the miR-99a-5p-medi-
ated control of osteogenic differentiation is a specific effect and not an
indirect consequence of cell proliferation/apoptosis. This study shows
that levels of miR-99a-5p are timely controlled during human in vitro
osteogenic/adipogenic differentiation and during osteoclastogenesis.
However, it does not uncover the upstream mechanisms that regulate
the expression of this miRNA, which can occur at multiple levels.
Generally, transcriptional and post-transcriptional modifications can
control miRNA expression [39]. Transcriptional control can be depen-
dent or independent of the miRNA host gene, while post-transcriptional
modifications are dependent of the miRNA processing and stability.
Additionally, the expression of a given miRNA can be influenced by cell
endogenous factors, like their production of cytokines, and exogenous
stimuli that cells may be exposed to, such as chemical compounds [39].
The assessment of miRNA expression during osteogenic/adipogenic
differentiation and during osteoclastogenesis described here, was per-
formed in human primary cells, while miRNA gain- and loss-of-function
experiments were performed using mouse cell lines. Nonviral gene
delivery (transfection/electroporation) in primary cells (either MSCs or
monocytes) presents low efficiency and toxic effects, while viral
mediated delivery presents safety issues and unexpected side effects,
which is particularly relevant when envisioning a future in vivo ap-
plication [40]. This is a limitation of this study, as we cannot exclude
that the observed effects could be cell line or species specific. Still, miR-
99a-5p is evolutionary conserved between human and mouse (http://
www.mirbase.org/), and it mature sequence shows 100% homology
between these species. RAW 264.7 cells have been widely used to study
monocyte/osteoclast differentiation in vitro because these cells are easy
to culture, to genetically manipulate, and can overcome the limited
lifespan of primary cells. RAW264.7 present similarities with the pri-
mary monocytes, for instance, both require RANKL supplementation for
in vitro differentiation. Nevertheless, their usage should be carefully
considered. High-throughput proteomics analysis performed by Ng
et al. shows a distinct protein profile during osteoclast formation be-
tween RAW 264.7 and mice bone marrow macrophages [41]. The most

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S.R. Moura, et al. Bone 134 (2020) 115303

Fig. 7. miR-99a-5p promotes osteoclastogenesis of RAW 264.7 cells.

A) miR-99a-5p expression in RAW 264.7-transfected cells with miR-99a mimics, miR-99a inhibitor, and respective controls (median, N = 6, Wilcoxon signed ranked
test, * P < 0.05). B) Dcstamp, Ctsk and Ccl2 expression levels 3 days after transfection and culture in the presence of RANKL (median, N = 6, Wilcoxon signed ranked
test, * P < 0.05). C) Number of multinucleated cells (≥3 nuclei) 3 days after transfection and culture in the presence of RANKL, relative to controls (mean ± SD,
N = 6, one-way ANOVA, followed by Sidak's multiple comparison test, * P < 0.05 and ** P < 0.01).

differently affected biological processes are cell cycle, cytoskeleton re-
organization and apoptosis [41]. Additionally, RAW 264.7 cells do not
exclusively differentiate into osteoclast but also in other multinucleated
cells, including macrophage polykaryons [42]. To mitigate some of
these limitations, our study not only evaluated the number of multi-
nucleated cells but also tested the levels of key osteoclastogenic mar-
kers.
An advantage of miRNA-based therapies is that a single miRNA can
affect multiple targets and simultaneously control distinct pathways. In
fact, our proteomic analysis shows that several cell differentiation and
extracellular matrix classes of proteins are affected by miR-99a-5p le-
vels. Among these, a class II small leucine-rich proteoglycan was
identified to be regulated by this miRNA, namely Fmod. In the litera-
ture, Fmod is described as a promotor of mineralization [43] and its
ablation negatively affects bone mineral density [44]. The advantage of
using a high-throughput screening method is that we can identify many
distinct proteins and pathways associated to the expression of a specific
miRNA. This is particularly relevant when studying miRNA, since these
small ncRNAs are known to have multiple targets, contrary to silencing
RNAs (siRNA) that are exogenous sequences specifically designed to
target a single mRNA. Therefore, our approach has 1) resulted in a
global analysis on the molecular and biological processes most affected
by miR-99a-5p expression, 2) identified novel players regulated by miR-
99a-5p during osteogenic differentiation, as well as 3) detected the
impact of miR-99a-5p on known key osteogenic markers. However, this
analysis was performed in proteins isolated from cell lysates and thus,
excludes secreted proteins present in the supernatant. To partially
overcome this limitation, and considering the importance of RANK/
RANLK/OPG pathway in bone remodeling, the secreted levels of OPG
were measured. OPG, an endogenous anti-osteoclastogenic decoy for
RANKL [45,46], was increased by miR-99a-5p inhibition, while abro-
gated by miR-99a-5p mimics. As expected, OPG could only be detected
by ELISA and was not identified in the cell proteomic analysis [47].
Importantly, OPG is a central player in bone metabolism and potentially
a therapeutic candidate. In vivo studies showed that OPG is able to
reverse osteoporosis and that OPG-engineered MSCs are able to pro-
moted critical-size bone repair in an osteoporosis mice model [48,49].
Nonetheless, our analysis does not excluded the involvement of other
mediators that may be present in the supernatant of MC3T3-transfected
cells that could be able to impact the communication with osteoclast.

13
S.R. Moura, et al.
These may include different mediators like cytokines/chemokines, or
even nucleic acids, such as miRNAs encapsulated or not in extracellular
vesicles [50–52].
Therefore, the effect that the total secretome produced by miR-99a-
5p/anti-miR-99a-5p-transfected MC3T3 cells have on osteoclastogen-
esis was investigated. Surprisingly, supernatants derived from anti-miR-
99a-5p cells could not only reduce osteoclastogenesis but also decrease
the levels of miR-99a-5p expression by the osteoclasts, suggesting a role
for this miRNA in osteoclasts. Herein, we show that
miR-99a-5p is increased during osteoclastogenesis of primary
human monocytes and that miR-99a-5p promotes the differentiation of
osteoclasts. This is in line with De la Rica et al. [53] and Franceschetti
et al. [54] findings that miR-99b, a miR-99a-5p close related family
member, has a pro-osteoclastogenesis activity. However, Zhou et al.
[55] demonstrated that miR-100, other family member, inhibits os-
teoclast differentiation. Importantly, the nucleotide differences in the
mature sequences from miRNA family members are sufficient to cause
alterations in the targets binding capacity, through modulation of target
specificity or the strength of repression [56].
Globally, our results show that miR-99a-5p is a bidirectional reg-
ulator of bone cellular processes, by reciprocally acting as an inhibitor
of osteogenic differentiation and as a promoter of osteoclastogenesis.
Furthermore, it also interferes with the intercellular communication
from the bone forming to bone resorbing cells. Taken together, our
results support miR-99a-5p as a potential target for the regeneration of
bone defects and/or treatment of bone disorders, such as osteoporosis.
Acknowledgments
This project is supported by Fundação para a Ciência e a Tecnologia
(FCT) - in the framework of the project POCI-01-0145-FEDER-031402-
R2Bone, under the PORTUGAL 2020 Partnership Agreement, through
ERDF, co-funded by FEDER/FNR, and national funding (through FCT –
Fundação para a Ciência e a Tecnologia, I.P., provided by the contract-
program and according to numbers 4, 5 and 6 of art. 23 of Law No. 57/
2016 of 29 August 2016, as amended by Law No. 57/2017 of 19 July
2017). SRM and JB are supported by FCT (SFRH/BD/147229/2019,
PD/BD/135490/2018, respectively; BiotechHealth PhD program). The
mass spectrometry technique was performed at the i3S Proteomics
Scientific Platform. This work had support from the Portuguese Mass
Spectrometry Network, integrated in the National Roadmap of Research
Infrastructures of Strategic Relevance (ROTEIRO/0028/2013; LISBOA-
01-0145-FEDER-022125).
Author contribution
SM and MIA: planned all the experiments. SM: performed all the
experiments, data acquisition and wrote the main manuscript text. SM,
JB, HO: proteomics analysis; SM, JB, JF, MAB, SGS and MIA: discussed
the experiments, reviewed and approved the manuscript.
Declaration of competing interest
The authors declare no conflict of interest.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bone.2020.115303.
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