miR-99a in bone homeostasis: Regulating osteogenic lineage commitment and osteoclast differentiation

Journal Pre-proof miR-99a in bone homeostasis: Regulating osteogenic lineage commitment and osteoclast differentiation

Sara Reis Moura, Joao Paulo Bras, Jaime Freitas, Hugo Osório, Mario Adolfo Barbosa, Susana Gomes Santos, Maria Ines Almeida PII:

S8756-3282(20)30083-1

DOI:

https://doi.org/10.1016/j.bone.2020.115303

Reference:

BON 115303

To appear in:

Bone

Received date:

1 October 2019

Revised date:

4 February 2020

Accepted date:

25 February 2020

Please cite this article as: S.R. Moura, J.P. Bras, J. Freitas, et al., miR-99a in bone homeostasis: Regulating osteogenic lineage commitment and osteoclast differentiation, Bone(2018), https://doi.org/10.1016/j.bone.2020.115303

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© 2018 Published by Elsevier.

Journal Pre-proof Title: miR-99a in bone homeostasis: regulating osteogenic lineage commitment and osteoclast differentiation Authors: Sara Reis Moura a,b, Joao Paulo Bras a,b,c , Jaime Freitas

a,b

, Hugo Osório

a,d,e

, Mario

Adolfo Barbosa a,b,c, , Susana Gomes Santos a,b,c , Maria Ines Almeida a,b,# Affiliations a

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-

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b

f

135 Porto, Portugal

INEB - Instituto de Engenharia Biomédica, Universidade do Porto, 4200-135 Porto,

Portugal

ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-

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c

d

e-

313 Porto, Portugal

Ipatimup - Instituto de Patologia e Imunologia Molecular da Universidade do Porto,

e

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4200-135 Porto, Portugal

FMUP – Faculdade de Medicina da Universidade do Porto, 4200-319 Porto, Portugal

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#Corresponding author

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Address: Rua Alfredo Allen, 208, 4200-135 Porto, Portugal Email: [email protected]

Running title

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Phone: +351 220 408 800

Role of miR-99a in osteoblasts and osteoclasts

Keywords Cell differentiation, Osteoporosis, Bone remodeling, Osteoprotegerin, Gene therapy

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Journal Pre-proof

Abstract Background: The tight coupling between osteoblasts and osteoclasts is essential to maintain bone homeostasis. Deregulation of this process leads to loss and deterioration of the bone tissue causing diseases, such as osteoporosis. MicroRNAs are able to control cell differentiation of bone cells and thus, have been explored as therapeutic tools. In this study, we explored the potential of miR-99a-5p to concurrently modulate

osteogenic

differentiation, osteoclastogenesis, and the osteoblasts-

osteoclasts crosstalk. Methods: To achieve this goal, human primary Mesenchymal Stem/Stromal

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Cells were differentiated into osteoblasts and adipocytes, and miR-99a-5p expression was evaluated by RT-qPCR. Knockdown and overexpression experiments were

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conducted to modulate miR-99a-5p expression in MC3T3 cells. Cell proliferation and cell death/apoptosis were evaluated by resazurin assay and flow cytometry,

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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

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media from MC3T3-transfected cells was incubated with RAW 264.7 cells and the effect on osteoclast differentiation was assessed. Human primary monocytes were isolated to induce osteoclastogenesis and evaluate miR-99a-5p expression. Finally,

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osteoclastogenesis.

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levels of miR-99a-5p were modulated in RAW 264.7 cells to understand the impact on Results: The results show that miR-99a-5p is significantly downregulated during

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the early stages of human primary MSCs osteogenic differentiation and during MC3T3 osteogenic differentiation. On the other hand, in hMSCs, 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 involved 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 encoding gene) mRNA expression ratio, in addition to increasing OPG secretion, miR-99a-5p overexpression resulted 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.

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Journal Pre-proof Interestingly, miR-99-5p 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

simultaneously promotes the commitment into osteogenic differentiation, impairs osteoclastogenic differentiation, and control bone cells communication. Ultimately, it supports miR-99a-5p as a candidate for future novel miRNA-based therapies for bone diseases associated with bone remodeling deregulation.

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1. Introduction

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Bone remodeling involves the coupling between new bone formation by osteoblasts and bone resorption by osteoclasts [1]. In healthy conditions, this process

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is tightly regulated, particularly through the RANKL/RANK/OPG signalling pathway [2, 3]. However, bone remodelling can be disrupted by intrinsic or extrinsic factors. An

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imbalance in the bone remodelling process may lead to the development of bone disorders, such as osteoporosis, which is a systemic progressive skeletal disease, with

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an increased vulnerability to bone fragility fractures [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 recruitment of

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Mesenchymal Stem/stromal Cells (MSCs) to the injury site, which then differentiate into

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osteoblasts [6]. During bone regeneration/repair, it is crucial for MSCs to commit into the osteogenic lineage in detriment of adipogenic differentiation [7]. In this context, the

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identification and dissection of novel regulatory players in MSCs differentiation, 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 complementary 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 instance, 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 critical fracture in the rat femur [19]. We and others have identified

miRNAs

as

regulators

of

MSCs

proliferation,

differentiation

and

communication with other cell types involved in bone repair, such as endothelial and immune cells [11, 20, 21]. Recently, miRNA microarray data analysis performed by us 3

Journal Pre-proof 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-osteoporotic patients [17]. Furthermore, the expression of this miR-99a family member negatively correlate with dissecting

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bone mineral density, both in plasma [23], and in bone tissue samples [24]. Therefore, the

mechanisms

underlying

miR-99a-5p in bone cells is essential to support its usage as a therapeutic target for

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future clinical approaches.

Currently, the miRbase database (v22) includes more than 30 000 entries, out

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of which, more than 2 500 correspond to human mature miRNA sequences [23].

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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

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osteoporosis or other pathologies, the ideal miRNA candidate should be able to promotes MSCs osteogenic differentiation in detriment of other lineages, while

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concomitantly decrease osteoclasts formation or activity. In this context, we explored

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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-throughput proteomics, evaluated modulation of OPG secretion, explored 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 (hMSC) 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 4

Journal Pre-proof 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) CO 2. 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

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Society for Cellular Therapy [25]. 2.2 Human primary monocyte isolation

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Human primary monocytes were isolated from buffy coats of 6 healthy blood donors kindly provided by CHSJ. The protocol was approved by CHSJ Ethics

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Committee for Health, and conforms to the declaration of Helsinki. RosetteSep human

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monocyte enrichment isolation kit was used (StemCell Technologies), as previously described by us [20]. Briefly, buffy coats were centrifuged at 1200 g, for 30 min, at room temperature (RT), without active acceleration or brake. The Peripheral Blood

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Mononuclear Cells (PBMCs) layer was collected, together with some red blood cells necessary for the formation of immunorosettes. Following the manufacturer’s

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instructions, the RosetteSep human monocyte enrichment isolation kit was added to

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the cells and incubated during 20 min at RT in a horizontal shaker. Samples were then 1:1 diluted in phosphate buffer saline 1x (PBS) supplemented with 2 % (v/v) FBS (Biowest), before being carefully laid over Histopaque®-1077 (Sigma-Aldrich). After centrifugation at 1200 g, 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 1x by centrifugation at 300 g 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 Figure 1). 2.3 Cell lines 5

Journal Pre-proof MC3T3-E1 pre-osteoblastic cells, widely used in osteoblast biology studies [27], and RAW 264.7 monocytic cells, commonly used in osteoclasts 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. 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 5x10-5

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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 day 0, 3, 7 and 14 (MC3T3) or 0, 3, 7,

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14, 21 and 28 (hMSCs) of culture. Alkaline phosphatase (ALP) staining was performed at day 7 and 14 for MC3T3 and hMSCs, respectively; Alizarin Red S staining was Methods). 2.5 Adipogenic differentiation

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performed at day 14 and 28 for MC3T3 and hMSCs, respectively (Supplemental

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To induce adipogenic differentiation, hMSCs from 6 donors were cultured for 28 days with the following supplements: 10-4 M dexamethasone, 10-1 mM Indomethacin,

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5x10-4 M IBMX and 10 μg/mL insulin (all from Sigma-Aldrich). RNA was collected at

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day 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 day 0, 7, 14 and 21. F-actin/DAPI and TRAP stainings were performed at day 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 considered when ≥ 3 nuclei [29-32].

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Journal Pre-proof 2.7 Reverse transcription and real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA was extracted using TRIzol® Reagent (Invitrogen), according 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

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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 °C. GAPDH was used as a reference gene.

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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 miRNA expression was evaluated by RT-qPCR using TaqMan miRNA assays

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(Applied Biosystems). Briefly, cDNA was synthesized using total RNA, TaqMan

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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), according to the manufacturer’s protocol. qPCR was carried out in CFX

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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

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assays (Applied Biosystems), under the following conditions: 10 min at 95 °C, 40

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cycles of 15 s at 95 °C and 1 min at 60 °C. Small nuclear RNA U6 was used as a reference gene.

Relative expression levels were calculated using the quantification cycle (Cq) method, according to MIQE guidelines [33]. Data was analyzed 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 respective controls, namely miRNA mimics negative control (NC-mimics), or miRNA inhibitor negative control (NCinhibitor), all from Ambion, using Lipofectamine 2000 transfection reagent (Invitrogen), according to manufacturer’s instructions. Cells were incubated with the miRNA-lipid complexes for 12 hours and then collected for the distinct assays. MC3T3-transfected cells were plated in osteogenic differentiation conditioned media, as described in 2.4, for ALP (day 7) and for Alizarin (day 10) staining, RNA extraction (day 3 and 7), protein 7

Journal Pre-proof 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

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added to the cells (10 % (v/v)), and incubated for 2 h at 37 °C. Experiments were performed for 7 replicates in 5 time-points: day 0, 1, 2 ,3 and 4. The supernatant was transferred to a dark wall 96 well plate and fluorescence levels following resazurin

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(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

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nm, using the spectrophotometer 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

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the respective controls were collected at day 7 post-transfection. Cells were washed 3 times with PBS 1x and labelled with FITC Annexin V Apoptosis Detection Kit I (BD

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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

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min, at RT, in the dark. Then, the cells were run within one hour on the BD Accuri™ C6 system and the results were analysed 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 800 g for 10 min, to remove cell debris. Osteoprotegerin (OPG) concentration in the supernatants was determined using the commercial Quantikine ELISA kit (MOP00 for OPG, R&D Systems), according to manufacturer’s instructions. 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 8

Journal Pre-proof of culture in osteogenic-inducing conditions. Media was centrifuged at 800 g, for 10 min, at 4 °C, to remove cells and 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 αMEM + 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

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MC3T3 cells transfected with miR-99a-5p mimics, miR-99a-5p inhibitor or the respective controls were cultured in osteogenic differentiation conditions. Cells were harvested at day 7 of differentiation and washed 6 times with cold PBS 1x, before

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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

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μL/mL phenylmethylsulfonyl fluoride (PMSF, 200 mM), 10 μL/mL Leupeptin (1 ng/mL)

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and 10 μL/mL Aprotinin (1 mg/mL)] and phosphatase inhibitors [20 μL/mL NaVO 3 (50 nM), 50 μL/mL Na4P2O7 (50 mg/mL) and 10 μL/mL NaF (10 mM)], for 30 min. Cell lysates were centrifugation at 20 000 g, for 10 min, at 4 °C and the supernatant-

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containing protein was quantified using DC protein assay kit (Bio-Rad). Protein identification and label-free quantitation was performed by nanoLC-

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MS/MS, composed by an Ultimate 3000 liquid chromatography system coupled to a Q-

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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 allowed missing cleavage sites was set 2. Cysteine carbamidomethylation was defined as constant modification. 9

Journal Pre-proof 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 expressed in the two independent experiments with fold-change (FC) differences ≥1.25 or ≤-1.25. A

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minimum of two unique peptides were required for further evaluation. The differentially expressed proteins were classified according to Gene Ontology (GO) annotations. The enriched Biological Process analysis was performed using Protein ANalysis THrough

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Evolutionary Relationships (PANTHER) tool. miRWalk2.0 database was used for miRNA target prediction (http://mirwalk.umm.uni-heidelberg.de/). For the analysis of the

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top 20 proteins with opposite profiles between the miR-99a-mimics/NC-mimics and in

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the miR-99a-5p-inhibitor/NC-inhibitor groups, only the proteins with greater than or equal to 6 unique peptides were considered.

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2.15 Western blot Protein extracts (30 µg of protein) were prepared in reducing loading buffer,

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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

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nitrocellulose membranes, 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 Signaling, 1:1000). Next, the membranes were probed with an appropriate secondary antibody conjugated to horseradish peroxidase (HRP, GE Healthcare, 1:10 000) for 1 h, at RT. After washing, membranes 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 10

Journal Pre-proof 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 (more than 2 groups) followed by uncorrected Dunn’s multiple comparison test. When the data passed normality tests, one-way ANOVA (more than 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).

miR-99a-5p

is

downregulated

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3.1

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3. Results during

early stages

of

osteogenic

differentiation of human primary MSCs and in mouse MC3T3 cell line

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When hMSCs were induced to differentiate into the osteogenic lineage, the ALP staining and the mineralized area, assessed by Alizarin staining, were significantly

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increased when compared to basal conditions (Figure 1A and 1B). The expression

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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, day 3, 7 and 14), while at day 21 and 28 miR-99a-5p

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expression was restored to basal levels (Figure 1C). This indicates a potential effect of this miRNA in the early stages of hMSCs osteogenic differentiation rather than in the

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later mineralization stages. In the MC3T3 mouse pre-osteoblastic cell line, osteogenic

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differentiation also significantly increased ALP staining, and the deposition of calcium at days 7 and 14, respectively (Figure 1D and 1E). RT-qPCR results show that miR99a-5p was significantly downregulated during osteogenic differentiation, 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 (Figure 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 differentiated mature adipocytes, after 21 and 28 days of culture in adipogenic-inducing conditions, compared to basal controls (Figure 2A). The expression of the adipogenic-specific markers ADIPOQ (Adiponectin) and PPARG2 (Peroxisome proliferator activated receptor gamma) was upregulated 11

Journal Pre-proof after 21 and 28 days (P<0.05) (Figure 2B). Interestingly, at early time points of adipogenic differentiation, miR-99a-5p was significantly upregulated (P<0.05, day 3 and 7), compared with basal conditions (Figure 2C), while decreased at days 21 and 28 (P<0.05). Therefore, at early stages of hMSCs differentiation, miR-99a-5p expression levels associate positively with adipogenic differentiation, but negatively with osteogenic differentiation. 3.3 miR-99a-5p regulates negatively the osteogenic differentiation but has no effect on proliferation or apoptosis of MC3T3 cells Next, we evaluated the biological impact of miR-99a-5p through gain- and loss-

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of-function studies in MC3T3 cells, which were efficiently transfected (Figure 3A). miR99a-5p overexpression decreased ALP staining in 42.20 % (P<0.01), whereas miR99a-5p inhibition increased ALP staining in 70.77 % (P<0.05), after 7 days in

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osteogenic inducing conditions, compared to controls (Figure 3B). Also, results show a 60.29 % decrease (P<0.001) on the formation of calcium deposits in miR-99a-mimics

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transfected cells, and a 88.64 % increase (P<0.01) in miR-99a-inhibitor transfected

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cells, both after 10 days of differentiation and compared to their respective controls (Figure 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

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overexpressing cells cultured under osteogenic inducing conditions at day 3 and 7 (Figure 3D). Conversely, Alp and Runx2 were significantly increased at day 3 (P<0.05)

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and day 7 (P<0.05) in response to miR-99a-5p inhibition (Figure 3D). In order to

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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 (Figure 3E). The Annexin V/PI staining was used to evaluate apoptosis at day 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 (Figure 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 mechanisms and/or pathways regulated by this miRNA, cells transfected with miR-99a-5p-mimics, miR99a-5p-inhibitor, or the respective controls, were analyzed by mass spectrometry12

Journal Pre-proof 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 inhibited cells versus NC-inhibitor transfected cells (FC≥|1.25|). Out of those, 64 differentially expressed proteins were simultaneously downregulated by miR-99a-5p mimics and upregulated by miR-99a-5p inhibitor. When only considering the statistically different proteins (P<0.05) 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

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algorithm to originate from transcripts with putative miR-99a-5p binding sites (Supplemental Table 4). Conversely, 316 proteins were upregulated in miR-99aoverexpressing cells and downregulated in miR-99a-5p-knowckdown cells (Figure 4A).

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Gene ontology analysis shows that the molecular function mostly affected by changes in miR-99a-5p expression, either by its overexpression or inhibition, is the

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cellular catalytic activity (GO: 0003824, Figure 4B). Included in this term, the protein

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kinase activity (GO: 0004672) shows the highest percentage of gene hits compared to the total number of function hits (Supplemental Table 5). Figure 4C shows the 20 most differently expressed proteins with opposite regulation by miR-99a-5p mimics and its

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inhibitor. These include extracellular matrix (ECM) components that are members of the small interstitial leucine-rich repeat proteoglycans family (SLRPs), namely

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Fibromodulin (Fmod) and Lumican (Lum), which are increased in miR-99a-5p-inhibitor

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transfected cells (FC=1.90 and 1.25, respectively) and decreased by miR-99a-5pmimics transfected cells (FC=-1.44 and -1.38, respectively) (Figure 4C). This effect was validated by western blot for Fmod (Figure 4D), but not for Lum (Supplemental Figure 2), which is in agreement with the higher differential effect found for Fmod by mass spectometry analysis (Figure 4C). Additionally, Fmod mRNA expression was reduced by miR-99a-5p overxepression (P<0.05), while increased by miR-99a-5p inhibition (P<0.05) (Figure 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 differentiation” and in “extracellular matrix production”, COL8A1 (FC=-1.82) and COL4A1 (FC=-1.60) (Figure 4E).

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Journal Pre-proof Globally, this high-throuput proteomic analysis in MC3T3-transfected cells shows that distinct intracellular proteins related to osteogenic 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 analysed 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

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mRNA (OPG encoding gene) is decreased by miR-99a-5p overexpression (P<0.05), while increased by miR-99a-5p inhibition (P<0.05, Figure 5A). On the other hand, Tnfrsf11 (RANKL encoding gene) expression does not change following miR-99a-5p

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mimics/inhibitor transfection (Figure 5A). Interestingly, in the control conditions, Tnfrsf11b is expressed at much higher levels than Tnfrsf11. Therefore, Tnfrsf11b is the

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main contributor to the decreased in Tnfrsf11b/ Tnfrsf1 expression ratio by the miR-

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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

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significantly reduced in supernatants of miR-99a-5p-overexpressing cells (P<0.001), while increased in supernatants of miR-99a-5p-inhibitor transfected cells (P<0.001)

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(Figure 5B).

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Next, we tested the effect of MC3T3-conditioned media on osteoclast differentiation, following miR-99a-5p overexpression or inhibition. RAW 264.7 cells were cultured during 2 days with media derived from MC3T3-transfected with miR-99a5p-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 (Figure 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) (Figure 5D). These results suggest that the conditioned media from 14

Journal Pre-proof miR-99a-5p transfected cells influences the differentiation of osteoclast-like cells by affecting the expression of osteoclastogenic 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 Figure 3).

Taken together these results support a role for miR-99a-5p in

osteoclastogenesis via a paracrine effect.

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primary monocytes and of mouse RAW 264.7 cells

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

The results obtained this far prompted us to investigate the levels of miR-99a5p during osteoclastogenesis. As such, human primary monocytes isolated from

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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

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only M-CSF were used as a negative control (Figure 6A, Supplemental Figure 4). As

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expected, the number of nuclei per cell increases in a time-dependent manner (Figure 6B). While the number of cells with only one or two nuclei progressively decreases during differentiation into OCs at day 21 (1 nuclei: P<0.01; 2 nuclei: P<0.05), the

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number of cells with more than 10 nuclei is significantly higher at day 21 (P<0.01), compared to day 7 of differentiation (Figure 6B). Accordingly, along differentiation the

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cells were able to merge and originate large OCs, increasing the cells size over time

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(day 14: P<0.001; day 21: P<0.01) (Figure 6C). Also, the expression levels of the osteoclastogenic markers ACP5 (TRAP encoding gene), CTSK, and CALCR (CTR encoding gene) were significantly increased over time (Figure 6D), confirming a successful osteoclast differentiation for all 6 donors. Finally, the expression of miR-99a5p was evaluated during osteoclastogenesis in these cells. As illustrated in Figure 6E, miR-99a-5p is significantly overexpressed during the differentiation 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 osteoclastogenesis 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; (Figure 6F). The expression of osteoclastogenic genes was also evaluated during 5 days of RANKL-induced differentiation in RAW 264.7 cells (Supplemental Figure 5). 3.7 miR-99a-5p positively regulates osteoclastogenesis 15

Journal Pre-proof 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 (Figure 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) (Figure 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) (Figure 7B). Finally, we tested the ability of these cells to fuse and form

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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 fusion (FC=0.776, P<0.05) (Figure 7C). Taken

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together, these results demonstrate that miR-99a-5p has a functional effect on

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osteoclastogenesis.

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4. Discussion

Cell lineage commitment is crucial when considering the potential use of miRNA-based engineered MSCs for bone diseases or fractures [6]. Herein, we show

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that, in human primary bone-marrow-derived MSCs, miR-99a-5p expression is

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decreased during early stages of osteogenic differentiation, and that miRNA inhibition promotes a pro-osteogenic profile. The miR-99a-5p expression profile during

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osteogenic differentiation 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 differentiation, 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 (eg. 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-mediated 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, 16

Journal Pre-proof transcriptional and post-transcriptional modifications can control miRNA expression [39]. Transcriptional control can be dependent 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 performed 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

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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 application [40]. This is a

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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

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mouse (http://www.mirbase.org/), and it mature sequence shows 100% homology

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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.

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RAW264.7 present similarities with the primary monocytes, for instance, both require RANKL supplementation for in vitro differentiation. Nevertheless, their usage should be

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carefully considered. High-throughput proteomics analysis performed by Ng et al.

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shows a distinct protein profile during osteoclast formation between RAW 264.7 and mice bone marrow macrophages [41]. The most differently affected biological processes are cell cycle, cytoskeleton reorganization 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 multinucleated cells but also tested the levels of key osteoclastogenic markers. 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 levels. Among these, a class II small leucine-rich proteoglycan was identified to be regulated by this miRNA, namely Fmod. In the literature, 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 17

Journal Pre-proof 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

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RANK/RANLK/OPG pathway in bone remodelling, 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 abrogated by miR-99a-5p mimics. As

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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

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potentially a therapeutic candidate. In vivo studies showed that OPG is able to reverse

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osteoporosis and that OPG-engineered MSCs are able to promoted 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

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MC3T3-transfected cells that could be able to impact the communication with osteoclast. These may include different mediators like cytokines/chemokines, or even

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nucleic acids, such as miRNAs encapsulated or not in extracellular vesicles [50-52].

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Therefore, the effect that the total secretome produced by miR-99a-5p/anti-miR99a-5p-transfected MC3T3 cells have on osteoclastogenesis 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 osteoclast 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 regulator of bone cellular processes, by reciprocally acting as an inhibitor of osteogenic differentiation 18

Journal Pre-proof and as a promoter of osteoclatogenesis. 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-031402R2Bone, under the PORTUGAL 2020 Partnership Agreement, through ERDF, cofunded 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

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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). JB and MIA are supported by FCT, through BiotechHealth PhD program fellowship (PD/BD/135490/2018) and DL 57/2016/CP1360/CT0008,

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respectively. The mass spectrometry technique was performed at the i3S Proteomics Scientific Platform. This work had support from the Portuguese Mass Spectrometry

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Network, integrated in the National Roadmap of Research Infrastructures of Strategic

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Relevance (ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125). Author contribution

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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,

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JB, JF, MAB, SGS and MIA: discussed the experiments, reviewed and approved the

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manuscript. Conflict of interest

The authors declare no conflict of interest. Figures

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Figure 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 (5x, 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 (5x, scale 500 μm). E) ALP staining and Alizarin Red S quantification in MC3T3 after 7 and 14 days, respectively (median, N=6, twotailed 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). 20

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Figure 2 - miR-99a-5p expression during adipogenic differentiation of hMSCs. A) Representative image of Oil Red O staining (lipids stained red) at day 21 and 28 under adipogenic differentiation and basal conditions (10x, scale 200 µm; 5x, 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). 21

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Figure 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 NCinhibitor), and culture in osteogenic-inducing conditions (median, N=7, Wilcoxon signed 22

Journal Pre-proof 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, twotailed Wilcoxon matched pairs test, * P<0.05, ** P<0.01 and *** P<0.001). D) Alpl and Runx2 expression levels at day 3 and 7 after transfection and incubation in osteogenicinducing conditions (median, N=7, Wilcoxon signed ranked test, * P<0.05). E) Representative fluorescence profile (resorufin) of 4 independent experiments measured every 24 hours 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 PI+ and Annexin V+ PI+) (median, N=3,

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apoptosis and dead cells (Annexin V-

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Friedman test, followed by uncorrected Dunn’s multiple comparison test, n.s.: non-

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significant).

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Figure 4 - Proteomic analysis of miR-99a-5p downstream targets and processes. A) Number of differently expressed proteins in MC3T3 cells after transfection with miR99a-5p mimics, inhibitor or respective controls (FC≥|1.25|). Number of proteins significantly increased by miR-99a inhibition or decreased by miR-99a overexpression (FC≥|1.25| and P<0.05) with miR-99a-5p predicted binding sites by miRWalk 2.0. B) 24

Journal Pre-proof 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, *

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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

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the GO term “cell differentiation” and decreased by miR-99a-5p-overexpressing cells.

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Figure 5 - Effect of conditioned media from miR-99a-5p mimics/inhibitor MC3T3transfected 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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Figure 6 - Time-dependent increase of miR-99a-5p during monocyte to osteoclast differentiation. A) Osteoclastogenesis of human monocytes. F-actin and nuclei staining at day 7, 14 and 21 of differentiation (supplemented with MCS-F and RANKL) and in control cells (MCS-F) (10x, scale 100 μm). B) Number of cells with 1, 2, 3-9 and more than 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 27

Journal Pre-proof 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 osteoclastogenicinducing 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

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and 5 days, relative to day 0 (median, N=6, Wilcoxon signed ranked test, * P<0.05).

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

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A) miR-99a-5p expression in RAW 264.7-transfected cells with miR-99a mimics, miR99a inhibitor, and respective controls (median, N=6, Wilcoxon signed ranked test, *

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P<0.05). B) Dcstamp, Ctsk and Ccl2 expression levels 2 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).

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Journal Pre-proof References

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[1] X. Chen, Z. Wang, N. Duan, G. Zhu, E.M. Schwarz, C. Xie, Osteoblast-osteoclast interactions, Connect Tissue Res 59(2) (2018) 99-107. doi: 10.1080/03008207.2017.1290085 [2] B.F. Boyce, L. Xing, Functions of RANKL/RANK/OPG in bone modeling and remodeling, Arch Biochem Biophys 473(2) (2008) 139-46. doi: 10.1016/j.abb.2008.03.018 [3] A.E. Kearns, S. Khosla, P.J. Kostenuik, Receptor activator of nuclear factor kappaB ligand and osteoprotegerin regulation of bone remodeling in health and disease, Endocr Rev 29(2) (2008) 155-92. doi: 10.1210/er.2007-0014 [4] J.A. Kanis, E.V. McCloskey, H. Johansson, A. Oden, Approaches to the targeting of treatment for osteoporosis, Nat Rev Rheumatol 5(8) (2009) 425-31. doi: 10.1038/nrrheum.2009.139 [5] X. Feng, J.M. McDonald, Disorders of bone remodeling, Annu Rev Pathol 6 (2011) 121-45. doi: 10.1146/annurev-pathol-011110-130203 [6] J. Freitas, S.G. Santos, R.M. Goncalves, J.H. Teixeira, M.A. Barbosa, M.I. Almeida, Genetically Engineered-MSC Therapies for Non-unions, Delayed Unions and Critical-size Bone Defects, Int J Mol Sci 20(14) (2019). doi: 10.3390/ijms20143430 [7] Q. Chen, P. Shou, C. Zheng, M. Jiang, G. Cao, Q. Yang, J. Cao, N. Xie, T. Velletri, X. Zhang, C. Xu, L. Zhang, H. Yang, J. Hou, Y. Wang, Y. Shi, Fate decision of mesenchymal stem cells: adipocytes or osteoblasts?, Cell Death Differ 23(7) (2016) 1128-39. doi: 10.1038/cdd.2015.168 [8] A.M. Silva, S.R. Moura, J.H. Teixeira, M.A. Barbosa, S.G. Santos, M.I. Almeida, Long noncoding RNAs: a missing link in osteoporosis, Bone Res 7 (2019) 10. doi: 10.1038/s41413019-0048-9 [9] M.I. Almeida, R.M. Reis, G.A. Calin, MicroRNA history: discovery, recent applications, and next frontiers, Mutat Res 717(1-2) (2011) 1-8. doi: 10.1016/j.mrfmmm.2011.03.009 [10] G. Papaioannou, F. Mirzamohammadi, T. Kobayashi, MicroRNAs involved in bone formation, Cell Mol Life Sci 71(24) (2014) 4747-61. doi: 10.1007/s00018-014-1700-6 [11] M.I. Almeida, A.M. Silva, D.M. Vasconcelos, C.R. Almeida, H. Caires, M.T. Pinto, G.A. Calin, S.G. Santos, M.A. Barbosa, miR-195 in human primary mesenchymal stromal/stem cells regulates proliferation, osteogenesis and paracrine effect on angiogenesis, Oncotarget 7(1) (2016) 7-22. doi: 10.18632/oncotarget.6589 [12] D. Li, J. Liu, B. Guo, C. Liang, L. Dang, C. Lu, X. He, H.Y. Cheung, L. Xu, C. Lu, B. He, B. Liu, A.B. Shaikh, F. Li, L. Wang, Z. Yang, D.W. Au, S. Peng, Z. Zhang, B.T. Zhang, X. Pan, A. Qian, P. Shang, L. Xiao, B. Jiang, C.K. Wong, J. Xu, Z. Bian, Z. Liang, D.A. Guo, H. Zhu, W. Tan, A. Lu, G. Zhang, Osteoclast-derived exosomal miR-214-3p inhibits osteoblastic bone formation, Nat Commun 7 (2016) 10872. doi: 10.1038/ncomms10872 [13] J.Y. Krzeszinski, W. Wei, H. Huynh, Z. Jin, X. Wang, T.C. Chang, X.J. Xie, L. He, L.S. Mangala, G. Lopez-Berestein, A.K. Sood, J.T. Mendell, Y. Wan, miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2, Nature 512(7515) (2014) 431-5. doi: 10.1038/nature13375 [14] M. Hadjiargyrou, J. Zhi, D.E. Komatsu, Identification of the microRNA transcriptome during the early phases of mammalian fracture repair, Bone 87 (2016) 78-88. doi: 10.1016/j.bone.2016.03.011 [15] T. Waki, S.Y. Lee, T. Niikura, T. Iwakura, Y. Dogaki, E. Okumachi, K. Oe, R. Kuroda, M. Kurosaka, Profiling microRNA expression during fracture healing, BMC Musculoskelet Disord 17 (2016) 83. doi: 10.1186/s12891-016-0931-0 [16] L. De-Ugarte, G. Yoskovitz, S. Balcells, R. Guerri-Fernandez, S. Martinez-Diaz, L. Mellibovsky, R. Urreizti, X. Nogues, D. Grinberg, N. Garcia-Giralt, A. Diez-Perez, MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones, BMC Med Genomics 8 (2015) 75. doi: 10.1186/s12920-015-0149-2 [17] C. Seeliger, K. Karpinski, A.T. Haug, H. Vester, A. Schmitt, J.S. Bauer, M. van Griensven, Five freely circulating miRNAs and bone tissue miRNAs are associated with osteoporotic fractures, J Bone Miner Res 29(8) (2014) 1718-28. doi: 10.1002/jbmr.2175 30

Journal Pre-proof

Jo u

rn

al

Pr

e-

pr

oo

f

[18] P. Garmilla-Ezquerra, C. Sanudo, J. Delgado-Calle, M.I. Perez-Nunez, M. Sumillera, J.A. Riancho, Analysis of the bone microRNome in osteoporotic fractures, Calcif Tissue Int 96(1) (2015) 30-7. doi: 10.1007/s00223-014-9935-7 [19] A.M. Silva, M.I. Almeida, J.H. Teixeira, C. Ivan, J. Oliveira, D. Vasconcelos, N. Neves, C. Ribeiro-Machado, C. Cunha, M.A. Barbosa, G.A. Calin, S.G. Santos, Profiling the circulating miRnome reveals a temporal regulation of the bone injury response, Theranostics 8(14) (2018) 3902-3917. doi: 10.7150/thno.24444 [20] J.P. Bras, A.M. Silva, G.A. Calin, M.A. Barbosa, S.G. Santos, M.I. Al meida, miR-195 inhibits macrophages pro-inflammatory profile and impacts the crosstalk with smooth muscle cells, PLoS One 12(11) (2017) e0188530. doi: 10.1371/journal.pone.0188530 [21] X. Liang, L. Zhang, S. Wang, Q. Han, R.C. Zhao, Exosomes secreted by me senchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a, J Cell Sci 129(11) (2016) 2182-9. doi: 10.1242/jcs.170373 [22] Y. Tang, L. Zhang, T. Tu, Y. Li, D. Murray, Q. Tu, J.J. Chen, MicroRNA -99a is a novel regulator of KDM6B-mediated osteogenic differentiation of BMSCs, J Cell Mol Med 22(4) (2018) 2162-2176. doi: 10.1111/jcmm.13490 [23] W. Ding, S. Ding, J. Li, Z. Peng, P. Hu, T. Zhang, L. Pan, Aberrant Expression of miR-100 in Plasma of Patients with Osteoporosis and its Potential Diagnostic Value, Clin Lab 65(9) (2019). doi: 10.7754/Clin.Lab.2019.190327 [24] S. Kelch, E.R. Balmayor, C. Seeliger, H. Vester, J.S. Kirschke, M. van Griensven, miRNAs in bone tissue correlate to bone mineral density and circulating miRNAs are gender i ndependent in osteoporotic patients, Sci Rep 7(1) (2017) 15861. doi: 10.1038/s41598-017-16113-x [25] M. Dominici, K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop, E. Horwitz, Minimal criteria for defini ng multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, Cytotherapy 8(4) (2006) 315-7. doi: 10.1080/14653240600855905 [26] M.I. Oliveira, S.G. Santos, M.J. Oliveira, A.L. Torres, M.A. Barbosa, Chitosan drives antiinflammatory macrophage polarisation and pro-inflammatory dendritic cell stimulation, Eur Cell Mater 24 (2012) 136-52; discussion 152-3. doi: 10.22203/ecm.v024a10 [27] H. Sudo, H.A. Kodama, Y. Amagai, S. Yamamoto, S. Kasai, In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria, J Cell Biol 96(1) (1983) 191-8. doi: 10.1083/jcb.96.1.191 [28] P. Collin-Osdoby, P. Osdoby, RANKL-mediated osteoclast formation from murine RAW 264.7 cells, Methods Mol Biol 816 (2012) 187-202. doi: 10.1007/978-1-61779-415-5_13 [29] A.H. Lourenco, A.L. Torres, D.P. Vasconcelos, C. Ribeiro-Machado, J.N. Barbosa, M.A. Barbosa, C.C. Barrias, C.C. Ribeiro, Osteogenic, anti-osteoclastogenic and immunomodulatory properties of a strontium-releasing hybrid scaffold for bone repair, Mater Sci Eng C Mater Biol Appl 99 (2019) 1289-1303. doi: 10.1016/j.msec.2019.02.053 [30] S. Yan, L. Miao, Y. Lu, L. Wang, Sirtuin 1 inhibits TNF-alpha-mediated osteoclastogenesis of bone marrow-derived macrophages through both ROS generation and TRPV1 activation, Mol Cell Biochem 455(1-2) (2019) 135-145. doi: 10.1007/s11010-018-3477-7 [31] A.L. Torres, S.G. Santos, M.I. Oliveira, M.A. Barbosa, Fibrinogen promotes resorption of chitosan by human osteoclasts, Acta Biomater 9(5) (2013) 6553-62. doi: 10.1016/j.actbio.2013.01.015 [32] C.R. Gardner, Morphological analysis of osteoclastogenesis induced by RANKL in mouse bone marrow cell cultures, Cell Biol Int 31(7) (2007) 672-82. doi: 10.1016/j.cellbi.2006.12.008 [33] S.A. Bustin, V. Benes, J.A. Garson, J. Hellemans, J. Huggett, M. Kubista, R. Mueller, T. Nolan, M.W. Pfaffl, G.L. Shipley, J. Vandesompele, C.T. Wittwer, The MIQE guidelines: minimum information for publication of quantitative real -time PCR experiments, Clin Chem 55(4) (2009) 611-22. doi: 10.1373/clinchem.2008.112797

31

Journal Pre-proof

Jo u

rn

al

Pr

e-

pr

oo

f

[34] H. Dweep, C. Sticht, P. Pandey, N. Gretz, miRWalk--database: prediction of possible miRNA binding sites by "walking" the genes of three genomes, J Biomed Inform 44(5) (2011) 839-47. doi: 10.1016/j.jbi.2011.05.002 [35] T.F. Tsai, J.F. Lin, K.Y. Chou, Y.C. Lin, H.E. Chen, T.I. Hwang, miR-99a-5p acts as tumor suppressor via targeting to mTOR and enhances RAD001-induced apoptosis in human urinary bladder urothelial carcinoma cells, Onco Targets Ther 11 (2018) 239-252. doi: 10.2147/OTT.S114276 [36] L.L. Mei, Y.T. Qiu, M.B. Huang, W.J. Wang, J. Bai, Z.Z. Shi, MiR-99a suppresses proliferation, migration and invasion of esophageal squamous cell carcinoma cells through inhibiting the IGF1R signaling pathway, Cancer Biomark 20(4) (2017) 527-537. doi: 10.3233/CBM-170345 [37] L. Cui, H. Zhou, H. Zhao, Y. Zhou, R. Xu, X. Xu, L. Zheng, Z. Xue, W. Xia, B. Zhang, T. Ding, Y. Cao, Z. Tian, Q. Shi, X. He, MicroRNA-99a induces G1-phase cell cycle arrest and suppresses tumorigenicity in renal cell carcinoma, BMC Cancer 12 (2012) 546. doi: 10.1186/1471-2407-12546 [38] H. Qin, W. Liu, MicroRNA-99a-5p suppresses breast cancer progression and cell -cycle pathway through downregulating CDC25A, J Cell Physiol 234(4) (2019) 3526-3537. doi: 10.1002/jcp.26906 [39] L.F. Gulyaeva, N.E. Kushlinskiy, Regulatory mechanisms of microRNA expression, J Transl Med 14(1) (2016) 143. doi: 10.1186/s12967-016-0893-x [40] A. Hamann , A. Nguyen, A.K. Pannier, Nucleic acid delivery to mesenchymal stem cells: a review of nonviral methods and applications, J Biol Eng 13 (2019) 7. doi: 10.1186/s13036-019-0140-0 [41] A.Y. Ng, C. Tu, S. Shen, D. Xu, M.J. Oursler, J. Qu, S. Yang, Comparative Characterization of Osteoclasts Derived From Murine Bone Marrow Macrophages and RAW 264.7 Cells Using Quantitative Proteomics, JBMR Plus 2(6) (2018) 328-340. doi: 10.1002/jbm4.10058 [42] J. Nguyen, A. Nohe, Factors that Affect the Osteoclastogenesis of RAW264.7 Cells, J Biochem Anal Stud 2(1) (2017). doi: 10.16966/2576-5833.109 [43] M. Goldberg, D. Septier, A. Oldberg, M.F. Young, L.G. Ameye, Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation, J Histochem Cytochem 54(5) (2006) 525-37. doi: 10.1369/jhc.5A6650.2005 [44] C.J. Li, Y. Xiao, M. Yang, T. Su, X. Sun, Q. Guo, Y. Huang, X.H. Luo, Long noncoding RNA Bmncr regulates mesenchymal stem cell fate during skeletal aging, J Clin Invest 128(12) (2018) 5251-5266. doi: 10.1172/JCI99044 [45] X. Ren, Q. Zhou, D. Foulad, A.S. Tiffany, M.J. Dewey, D. Bischoff, T.A. Miller, R.R. Reid, T.C. He, D.T. Yamaguchi, B.A.C. Harley, J.C. Lee, Osteoprotegerin reduces osteoclast resorption activity without affecting osteogenesis on nanoparticulate mineralized collagen scaffolds, Sci Adv 5(6) (2019) eaaw4991. doi: 10.1126/sciadv.aaw4991 [46] W.S. Simonet, D.L. Lacey, C.R. Dunstan, M. Kelley, M.S. Chang, R. Luthy, H.Q. Nguyen, S. Wooden, L. Bennett, T. Boone, G. Shimamoto, M. DeRose, R. Elliott, A. Colombero, H.L. Tan, G. Trail, J. Sullivan, E. Davy, N. Bucay, L. Renshaw-Gegg, T.M. Hughes, D. Hill, W. Pattison, P. Campbell, S. Sander, G. Van, J. Tarpley, P. Derby, R. Lee, W.J. Boyle, Osteoprotegerin: a novel secreted protein involved in the regulation of bone density, Cell 89(2) (1997) 309-19. doi: 10.1016/s0092-8674(00)80209-3 [47] H. Brandstrom, T. Bjorkman, O. Ljunggren, Regulation of osteoprotegerin secretion from primary cultures of human bone marrow stromal cells, Bioche m Biophys Res Commun 280(3) (2001) 831-5. doi: 10.1006/bbrc.2000.4223 [48] H. Min, S. Morony, I. Sarosi, C.R. Dunstan, C. Capparelli, S. Scully, G. Van, S. Kaufman, P.J. Kostenuik, D.L. Lacey, W.J. Boyle, W.S. Simonet, Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis, J Exp Med 192(4) (2000) 463-74. doi: 10.1084/jem.192.4.463 32

Journal Pre-proof

Jo u

rn

al

Pr

e-

pr

oo

f

[49] X. Liu, C. Bao, H.H.K. Xu, J. Pan, J. Hu, P. Wang, E. Luo, Osteoprotegerin gene-modified BMSCs with hydroxyapatite scaffold for treating critical -sized mandibular defects in ovariectomized osteoporotic rats, Acta Biomater 42 (2016) 378-388. doi: 10.1016/j.actbio.2016.06.019 [50] A. Pinheiro, A.M. Silva, J.H. Teixeira, R.M. Goncalves, M.I. Almeida, M.A. Barbosa, S.G. Santos, Extracellular vesicles: intelligent delivery strategies for therapeutic applications, J Control Release 289 (2018) 56-69. doi: 10.1016/j.jconrel.2018.09.019 [51] A.M. Silva, J.H. Teixeira, M.I. Almeida, R.M. Goncalves, M.A. Barbosa, S.G. Santos, Extracellular Vesicles: Immunomodulatory messengers in the context of tissue repair/regeneration, Eur J Pharm Sci 98 (2017) 86-95. doi: 10.1016/j.ejps.2016.09.017 [52] A. Turchinovich, L. Weiz, A. Langheinz, B. Burwinkel, Characterization of extracellular circulating microRNA, Nucleic Acids Res 39(16) (2011) 7223-33. doi: 10.1093/nar/gkr254 [53] L. de la Rica, A. Garcia-Gomez, N.R. Comet, J. Rodriguez-Ubreva, L. Ciudad, R. VentoTormo, C. Company, D. Alvarez-Errico, M. Garcia, C. Gomez-Vaquero, E. Ballestar, NF-kappaBdirect activation of microRNAs with repressive effects on monocyte -specific genes is critical for osteoclast differentiation, Genome Biol 16 (2015) 2. doi: 10.1186/s13059-014-0561-5 [54] T. Franceschetti, N.S. Dole, C.B. Kessler, S.K. Lee, A.M. Delany, Pathway analysis of microRNA expression profile during murine osteoclastogenesis, PLoS One 9(9) (2014) e107262. doi: 10.1371/journal.pone.0107262 [55] L. Zhou, H.Y. Song, L.L. Gao, L.Y. Yang, S. Mu, Q. Fu, MicroRNA1005p inhibits osteoclastogenesis and bone resorption by regulating fibroblast growth factor 21, Int J Mol Med 43(2) (2019) 727-738. doi: 10.3892/ijmm.2018.4017 [56] J.M. Wolter, H.H. Le, A. Linse, V.A. Godlove, T.D. Nguyen, K. Kotagama, A. Lynch, A. Rawls, M. Mangone, Evolutionary patterns of metazoan microRNAs reveal targeting principles in the let-7 and miR-10 families, Genome Res 27(1) (2017) 53-63. doi: 10.1101/gr.209361.116

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Highlights  miR-99a-5p shows an opposite profile during human MSC osteogenic/adipogenic differentiation  miR-99a-5p inhibits the osteogenic differentiation of pre-osteoblasts, by modulating key markers of cell differentiation and extracellular matrix.  miR-99a-5p affects the osteoclastogenesis of murine monocytes via a paracrine effect  miR-99a-5p increases during osteoclastogenesis of murine and human primary monocytes  miR-99a-5p positively regulates the osteoclastogenesis of murine monocytes

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