Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2

Journal Pre-proof Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2

Jun-Xiao Yang, Peng Xie, Yu-Sheng Li, Ting Wen, Xu-Cheng Yang PII:

S0898-6568(19)30300-6

DOI:

https://doi.org/10.1016/j.cellsig.2019.109504

Reference:

CLS 109504

To appear in:

Cellular Signalling

Received date:

6 September 2019

Revised date:

10 December 2019

Accepted date:

15 December 2019

Please cite this article as: J.-X. Yang, P. Xie, Y.-S. Li, et al., Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2, Cellular Signalling(2019), https://doi.org/10.1016/j.cellsig.2019.109504

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof

Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2 Running title: The effect of exosomes-containing miR-23a-5p on osteogenic differentiation

Jun-Xiao Yang1 , Peng Xie 2 , Yu-Sheng Li1 , Ting Wen1 , Xu-Cheng Yang1,* 1

Department of orthopedics, Xiangya Hospital, Central South University, Changsha 410008, Hunan

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Province, P. R China

Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, P. R China

*

Corresponding author: Dr. Xu-Cheng Yang, Department of orthopedics, Xiangya Hospital,

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2

Central South University, No. 87, Xiangya Road, Kaifu District, Changsha 410008, Hunan

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Province, P. R China

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Tel: +86-13975156835

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Email: [email protected]

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Abstract Background: Some microRNAs (miRNAs) are involved in osteogenic differentiation. In recent years, increasing evidences have revealed that exosomes contain specific miRNAs. However, the effect and mechanism of miR-23a-5p-containing exosomes in osteoblast remain largely unclear. Methods: We extracted exosomes from RANKL-induced RAW 264.7 cells, and identified

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exosomes via transmission electron microscopy, western blot and flow cytometry analysis. In

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addition, exosome secretion was inhibited by GW4869 and Rab27a siRNAs. miR-23a-5p

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expression was analyzed by qRT-PCR, and the related protein levels were examined by western

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blot assay. Furthermore, the number and distribution of osteoclasts were detected by TRAP staining, and early osteogenesis was evaluated by ALP staining. Combination of YAP1 and Runx2 was

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luciferase reporter assay.

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verified by Co-IP assay, and the regulation of miR-23a-5p and Runx2 was measured by dual

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Results: We successfully extracted exosomes from RANKL-induced RAW 264.7 cells, and successfully verified exosomes morphology. We also indicated that miR-23a-5p was highly expressed in exosomes from RANKL- induced RAW 264.7 cells, and osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity, while its inhibition weakened osteoclasts. In mechanism, we demonstrated that Runx2 was a target gene of miR-23a-5p, YAP interacted with Runx2, and YAP or Runx2 inhibited MT1DP exp ression. In addition, we proved that knockdown of MT1DP facilitated osteogenic differentiation by regulating FoxA1 and Runx2. Conclusions: We demonstrated that osteoclast-derived miR-23a-5p-containing exosomes could efficiently suppress osteogenic differentiation by inhibiting Runx2 and promoting YAP1- mediated

Journal Pre-proof MT1DP. Therefore, we suggested miR-23a-5p in exosomes might provide a novel mechanism for osteoblast function.

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Keywords: osteoclast; miR-23a-5p; exosomes; Runx2; osteogenic differentiation

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1.Introduction Bone is a dynamic living tissue that maintains its mineralization balance and structural integrity through continuous remodeling[1, 2]. In the process of bone remodeling, the coordination of osteoblasts and osteoclasts can maintain the dynamic balance of bone remodeling, in which osteoblasts (bone formation function) and osteoclasts (bone resorption function) play a key role in the process of bone remodeling[3, 4]. The mutual regulation between osteoblasts and osteoclasts is

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the basis of bone formation and bone resorption balance during bone remodeling[5]. Research have determined that receptor activator of the nuclear factor kβ (RANK)/RANK ligand (RANKL)

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systems play a critical role in osteoclast formation, which has become important advances in the

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study of bone physiology[6, 7]. RANKL is expressed by osteoblasts, bone marrow stromal cells and

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activated T lymphocytes, which can be combined with RANK on the surface of osteoclast precursor cells or mature osteoclasts to promote osteoclast differentiation and bone resorption activity[8, 9].

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However, blocking of the RANK/RANKL pathway can lead to bone destructive diseases such as

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osteoporosis, rheumatoid arthritis and cancer bone metastasis.

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Exosomes are vesicles derived from the endocytic membrane system, with a diameter of 30-100 nm[10, 11]. Exosomes exist in almost all body fluids, and the most common methods of exosomes separation include ultracentrifugation, density gradient centrifugation, ultrafiltration, precipitation polymerization, and magnetic activated cell sorting [12]. Many studies also have revealed that exosomes are important mediators of intercellular substance exchange and signal transduction [13, 14]. Exosomes can directly activate target cells to exert their biological functions through receptor- mediated activation, or the transfer of various bioactive molecules, such as cell membrane receptors, proteins, mRNA and miRNAs, etc. [15]. Almost all cells can secrete exosomes, which

are

involved

in

various

physiological

and

pathological processes,

such

as

immunosuppression, blood coagulation, antigen presentation, waste discharge, cell adhesion, inflammation, and tumor progression. Previous research also has disclosed that osteoclasts could

Journal Pre-proof secrete microRNA-enriched exosomes, and osteoclast-derived miR-214-containing exosomes could affect osteogenesis by inducing differentiation o f osteoblast progenitor cells[16]. In addition, the study also indicated that miR-23a-5p was highly expressed in RANKL- induced RAW 264.7 exosomes[17]. However, contribution and mechanism of osteoclast-derived miR-23a-5p-containing exosomes on osteoblast differentiation remain unknown. Therefore, it is worthy of further study whether

osteoclast-derived

miR-23a-5p-containing

exosomes

could

regulate

osteogenic

differentiation.

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In the present study, we successfully separated exosomes from RANKL- induced RAW 264.7 cells, and verified exosomes morphology. We investigated the expression of miR-23a-5p in the

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extracted exosomes, and the role of osteoclast-derived miR-23a-5p-containing exosomes on

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osteoblast activity. In addition, we inhibited osteoclast-derived miR-23a-5p-containing exosomes

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by N-SMase inhibitor GW4869 and Rab27a siRNAs, and proved whether exosomes inhibition could attenuate the miR-23a-5p-mediated inhibitory effect of osteoclasts on osteoblasts. Moreover,

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we explore the osteoblastic differentiation induced by miR-23a-5p via the downstream regulatory

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mechanism (Runx2-MT1DP-Runx2 feedback pathway, that is, Runx2 inhibiting MT1DP, which in turn inhibiting Runx2). Therefore, miR-23a-5p-containing exosomes have significant effect in

remodeling.

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regulating the functions of osteoblasts and osteoclasts and maintaining the balance of bone

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2.Materials and Methods 2.1 Cell lines RAW 264.7, hFOB 1.19 and MC3T3-E1 cells were purchased from Cell Bank of Type Culture Collection, Chinese Academy of Science (Shanghai, China). Human osteoclasts (OC) were obtained from FuHeng (China, cat. no. FH-H107). RAW 264.7 cells were maintained in DMEM (Invitrogen); hFOB 1.19 cells were grown in DMEM/F12 (Invitrogen); MC3T3- E1 cells were cultured in

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α-MEM (Invitrogen); OC were cultured in MEM-α medium (GIBCO, cat. no. 12571-063) and osteoclast inducer. All media were supplemented with 10% fetal bovine serum (FBS, cat. no.

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10437), and 1% penicillin/streptomycin (Life Technologies, cat. no. 15140-122). And cells were

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the treated RAW 264.7 cells for 48 hrs.

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maintained at 37℃ under 5% CO 2 and 95% air. In addition, hFOB1.19 cells were co-cultured with

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2.2 Cell transfection

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miR-23a-5p mimics, miR-23a-5p inhibitors and miR-23a-5p NC were all obtained from

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GenePharma(Shanghai, China). Following sequences were used to construct Rab27a siRNA （Rab27a siRNA: 5’-CGGUUGUUGUGAAGACUAA-3）in this study. Osteoclasts were seeded in 6-well plates and transfected with Rab27a siRNA, NC, miR-23a-5p mimics, miR-23a-5p inhibitors and miR-23a-5p NC for 48 hrs using Lipofectamine 3000 (Thermo Fisher) according to the instructions.

2.3 shRNA oligonucleotide transfection YAP1 shRNA, Runx2 shRNA, MT1DP shRNA, FoxA1 shRNA were purchased from Sigma. The sequences were as follows: YAP1 shRNA, 5’-CCG GCG ACC AAT AGC TCA GAT CCT TCT

Journal Pre-proof CGA GAA GGA TCT GAG CTA TTG GTC GTT TTT G-3’; Runx2 shRNA 5’-CCG GGC AGA ATG GAT GAG TCT GTT TCT CGA GAA ACA GAC TCA TCC ATT CTG CTT TTT G-3’; MT1DP shRNA 5’-CCG GGC AAA GAG TAC AAA TGC ACC TCT CGA GAG GTG CAT TTG TAC TCT TTG CTT TTT TG-3’; FoxA1 shRNA 5’-CCG GGA ACA CCT ACA TGA CCA TGA ACT CGA GTT CAT GGT CAT GTA GGT GTT CTT TTT-3’. Briefly, the shRNA DNA fragments were synthesized and cloned into U6 promoter plasmid (pU6). And then the productions

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were subcloned into a pSilencer vector. Similarly, shRNA and NC were used to transfect cells using

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Lipofectamine 3000 (Thermo Fisher) according to the instructions.

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2.4 Exosome extraction

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According to previous study, exosomes were extracted through centrifugations[18]. RAW 264.7 cells induced by RANKL were maintained in exosome- free medium with FBS. The culture medium

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was used to separate exosomes by ultracentrifugation at 120,000 g for 90 min, and then the

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supernatant was then centrifugated (300 g for 10 min at 4°C). After centrifugation, the supernatant

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was filtered by using a 0.22-μm membrane. Finally, the filtering medium was used to obtain exosomes by ultracentrifugation at 120,000 g for 70 min at 4°C. The extracted exosomes were resuspended with PBS and stored at − 80 °C.

2.5 Electron microscopy The purified exosomes were resuspended with PBS and fixed with 2% paraformaldehyde. And then the mixture was dropped onto EM grids. After drying, exosomes were stained with 1% uranyl acetate, and the grids were measured with HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).

Journal Pre-proof 2.6 Flow cytometry analysis As previously noted[19], exosomes (5 μg) were incubated with 4-μm-diameter aldehyde/sulfate latex beads (10 μl) for 16 min. And then exosomes were suspended with 1 ml PBS, after 2 hrs, exosomes were centrifugated. After washing, beads were treated with PE- labeled CD63 antibody for 1 h. Finally, the results were analyzed by FACS Calibur flow cytometer (Becton Dickinson).

2.7 Tartrate-resistant acid phosphatase (TRAP) staining

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Cells were washed with PBS, and were treated with 50 μl formaldehyde solution. And then cells were treated with TRAP solution (cat. #sc-386A; Santa Cruz Biotechnology) for 30 min. Finally,

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2.8 Alkaline phosphatase (ALP) staining

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the number and distribution of osteoclasts were observed.

According to the experimental steps of Vector Blue Substrate Kit (SK-5300, Vector Laboratories),

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cells were treated with the substrate working solution for 25 min in the dark, and washed by

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min.

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deionized water for 2 min. And the slides were incubated with Mayer’s hematoxylin solution for 10

2.9 Dual-luciferase reporter assay

As in previous study[20], the wide type (wt) or mutant (mut) Runx2 were amplified and sub-cloned into the pmirGLO vector.. Cells (1 × 104 cells/well) were seeded into 24-well plates and co-transfected with wt-Runx2 or mut-Runx2 and miR-23a-5p mimics or its negative control using a lipofectamine 3000 (Invitrogen). After 24 hrs, cells were collected and the luciferase activity of cells was dectected by using a Dual-Luciferase Assay System (Promega).

2.10 RNA extraction and quantitative real-time PCR (RT-qPCR) assay

Journal Pre-proof Total RNA was extracted by using Trizol (Invitrogen), and cDNA was obtained by using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, San Diego, CA, USA) according to the manufacturer’s instructions). The expression levels of genes were analyzed by using SYBRs GREEN PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The data obtained were assessed

on

an

ABI7500

Real-time

PCR

system

(Applied

Biosystems).

Relative expression levels were analyzed using the 2-△△Ct method by normalizing to GAPDH (mRNA) or U6 (miRNA) [21]. The primer sequences were as follows: miR-23a-5p forward,

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5’-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACG GAA

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AT-3’, and reverse, 5’-GGC ATC ACA TTG CCA GGG-3’; YAP forward, 5’-AGC TGC CCG

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ACT CCT TCT TC-3’ and reverse: 5’-GAG GAA TGA GCT CGA ACA TGC-3’; FoxA1 forward,

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5'-TAA TCA TTG CCA TCG TGT GCT T-3' and reverse: 5'-ATA ATG AAA CCC GTC TGG CTA-3'; MT1DP forward 5’-GCC ACT GGT AAA GGA TGC CT3’ and reverse: 5'-ATT GGT

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CTG CTC CTG TCT GC-3’; Runx2 forward, 5'-GAC CTC TAT GCC AAC ACA GT-3' and reverse, 5'-AGT ACT TGC GCT CAG GAG GA-3'; ALP, forward: 5′-GTG AAC CGC AAC TGG

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TAC TC-3′ and reverse: 5′-GAG CTG CGT AGC GAT GTC C-3′; GAPDH, forward: 5’-AAG

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TTG TGFATT AGT CA-3’and reverse: 5’-AGA ATA GTC CTA TAA TCA-3’.

2.11 Western blot assay

Total proteins were extracted by using RIPA lysis buffer (P0013B, Beyotime) and nuclear extracts were obtained by using a Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime). The protein concentration was examined by using an enhanced BCA protein assay kit (P0010, Beyotime). 30 μg proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto polyvinylidene difluoride membranes (Millipore). After blocking with 5% non- fat milk, the bands were incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, #7074). Finally, the results were visualized by using a BeyoECL Plus kit (P0018,

Journal Pre-proof Beyotime). The primary antibodies included TFIIB (Abcam, ab154049), LaminA/C (Abcam, ab8980), Hsp70 (Abcam, ab47455), TSG101 (Abcam, ab83), Rab27a (Abcam, ab214930), Runx2 (Abcam, ab76956), YAP1 (Abcam, a ab56701) and FoxA1 (Abcam, ab55178), β-actin (Abcam, ab227387) and GAPDH (Abcam, ab37168).

2.12 Co-immunoprecipitation (Co-IP) assay We adopted Pierce® Co-IP Kit (Thermo, cat. 26149) to perform Co-IP assay. Proteins were

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obtained and subjected to immunoprecipitation with primary antibodies at 4°C for 4 hrs. Then 50 μl

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protein A/G Sepharose beads were used to treat the mixture. After incubation for 2 hrs at 4°C, the

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bound proteins were eluted by using SDS-PAGE buffer. Finally, the proteins were measured by

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western blot assay. The antibodies in this study included anti-Runx2 (Santa Cruz Biotechnology; cat no. sc-390715) and anti-YAP1 (Abcam; cat no. ab226817), normal rabbit IgG (Santa Cruz

2.13 Statistical Analysis

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Biotechnology; cat no. sc-2027) or mouse IgG (Santa Cruz Biotechnology; cat no. sc-2025).

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All data were expressed as mean ± SD. The difference between groups was calculated by One-way

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analysis of variance through Graphpad (Ver. Prism 7, GraphPad Prism Software, La Jolla, CA, USA). P value less than 0.05 was considered significant.

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3.Results 3.1 Identification of exosomes from osteoclasts To investigate the properties of the vesicles released during osteoclast formation, RANKL was used to induce the growth of osteoclast progenitor cells (RAW 264.7) for 2 days. Firstly, we observed the morphology of exosomes by using transmission electron microscopy, and the results indicated that

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vesicles were about 50-100 nm in diameter (Figure 1A). Therefore, we have identified the presence

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of exosomes. In addition, western blot results showed that the related proteins (Hsp70 and TSG101)

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existed in exosomes and the proteins (TFIIB and LaminA/C) not existed in exosomes, and the

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results showed that TFIIB, LaminA/C, Hsp70 and TSG101 were expressed in RAW 264.7 cell lysate, while only Hsp70 and TSG101 were expressed in exosomes (Figure 1B). Meanwhile, the

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results from Flow cytometry revealed that CD63 was highly express ed, suggesting that there were

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exosomes (Figure 1C).

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Previous research has identified the differently expressed miRNAs in RANKL- induced RAW 264.7

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exosomes compared with RAW 264.7 exosomes, and the results found miR-23a-5p was highly expressed in RANKL- induced RAW 264.7 exosomes compared with RAW 264.7 exosomes[22]. In our study, we further verified miR-23a-5p expression, and the results showed that miR-23a-5p was significantly up-regulated in RANKL group compared with control group (Figure 1D). To explore the role of miR-23a-5p on osteoblast function, we adopted RANKL and macrophage colony-stimulating factor (M-CSF) to stimulate RAW 264.7 cells, and the results from western blot assay indicated that TRAP was up-regulated in M-CSF+RANKL group compared with control group (Figure 1E), and TRAP staining results showed that M-CSF and RANKL could induce osteoclastogenesis (1F). In addition, we proved that miR-23a-5p was also up-regulated in M-CSF+RANKL group compared with control group (Figure 1G).

Journal Pre-proof 3.2 Osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity To further explore whether mouse preosteoblast (MC3T3- E1) cells could internalize exosomes from osteoclast cells induced by RANKL. Confocal microscopy results showed that exosomes could be in combination with MC3T3-E1 cells (Figure 2A). In addition, to further analyze the effect of miR-23a-5p on exosome function in human osteoblast hFOB1.19 cells, RANKL- induced human osteoclasts (OC) cells were transfected with miR-23a-5p mimic, miR-23a-5p inhibitor, respectively. We found that miR-23a-5p mimic increased miR-23a-5p expression in exosomes while miR-23a-5p

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inhibitor decreased miR-23a-5p expression in exosomes (Figure 2B). In addition, OC cells were co-cultured with miR-23a-5p mimic or inhibitor-treated exosomes, and miR-23a-5p and MT1DP

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were also up-regulated in miR-23a-5p mimic (Figure 2C). Furthermore, we proved that exosomes

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could decrease Runx2 and ALP expressions, and miR-23a-5p mimics also down-regulated Runx2

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and ALP expressions, while miR-23a-5p inhibitor up-regulated them (Figure 2D). And early osteogenesis was reduced in miR-23a-5p mimic compared with NC group, while was increased in

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miR-23a-5p inhibitor compared with NC group (Figure 2E).

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osteoclasts on osteoblasts

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3.3 Inhibition of exosomes release weakened the miR-23a-5p-mediated inhibitory effect of

To further demonstrate the influence of osteoclast-derived miR-23a-5p-containing exosomes on the role of osteoclasts, N-SMase inhibitor GW4869 and Rab27a siRNAs were used to inhibit exosomes release. After treatment with GW4869/Rab27a siRNA in OC cells, the results proved that miR-23a-5p, MT1DP were down-regulated, while ALP and Runx2 expressions were significantly up-regulated in GW4869 treatment group comparing with the untreated group (Figure 3A-D). In addition, the results from ALP staining also showed similar changes ( Figure 3E). Meanwhile, Rab27a siRNAs also could down-regulate the levels of miR-23a-5p and MT1DP, and up-regulate the levels of ALP and Runx2, suggesting that miR-23a-5p transfer from osteoclasts to osteoblasts was also receded (Figure 3F-H). Rab27a siRNAs also could reduce the level of ALP (Figure 3K).

Journal Pre-proof In summary, inhibition of exosomes release could weaken the miR-23a-5p-mediated inhibitory effect of osteoclasts on osteoblasts.

3.4 YAP interacted with Runx2, and inhibited MT1DP expression Next, we then explored the mechanism of osteoclast-derived miR-23a-5p-containing exosomes. Firstly, we revealed that knockdown of YAP1 up-regulated MT1DP expression (Figure 4A);

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knockdown of Runx2 also up-regulated MT1DP expression (Figure 4B). In addition, we proved that knockdown of YAP1 down-regulated Runx2 expression (Figure 4C); knockdown of Runx2

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also down-regulated YAP1 expression (Figure 4D). And the results from Co-IP showed that YAP1

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can be combined with Runx2, suggesting that YAP1 could interact with Runx2 (Figure 4E).

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Furthermore, we found that there was a binding site between miR-23a-5p and Runx2 by using bioinformatics analysis including TargetScan (Figure 4G), and the results from dual- luciferase

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reporter gene assay indicated that a decrease in luciferase intensity between wild-type Runx2 and

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miR-23a-5p, whereas no changes were observed in the luciferase intensity between mutant Runx2

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and miR-23a-5p (Figure 4H), suggesting that Runx2 was a target gene of miR-23a-5p. Therefore, miR-23a-5p could target Runx2 and inhibit its expression, and YAP1 could interact with Runx2 to regulate MT1DP expression.

3.5 Knockdown of MT1DP promoted osteogenic differentiation by regulating FoxA1 and Runx2 To synthetically verify the regulatory mechanism of MT1DP in hFOB1.19 cells, RT-qPCR and Western blot assays were performed. The results revealed that MT1DP knockdown could up-regulate the expression level of FoxA1 (Figure 5A). The expression of FoxA1 and Runx2 were also promoted by MT1DP knockdown (Figure 5B). In function, we found that MT1DP knockdown dramatically accelerated early osteogenesis capacity (Figure 5C). Moreover, we demonstrated that FoxA1 knockdown down-regulated the expression levels of FoxA1 and YAP1 (Figure 5D). FoxA1

Journal Pre-proof knockdown markedly suppressed early osteogenesis capacity (Figure 5E). Therefore, MT1DP

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could downregulate Runx2 by inhibiting FoxA1, thereby affecting osteogenic differentiation.

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4. Discussion Bone remodeling is a process in which bone resorption and bone formation are closely coupled in time and space during bone metabolism[23]. Bone remodeling is a very precise and programmed process, mainly through the regulation between the bone resorption of osteoclast and the bone formation of osteogenesis, so as to generate new bone to replace the old bone in the relevant parts

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of bone injury[5, 24]. In this way, the micro-damage of bone can be repaired or targeted for

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reconstruction, so as to prevent the accumulation of bone tissue fatigue damage, the biomechanical

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function of the bone lesion, and maintain the balance of minerals in the body[25, 26]. Exosomes usually refer to extracellular vesicles of 30 nm~100 nm, which are widely found in organisms.

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Exosomes could participate in the exchange of substances and information between cells, regulate

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cell proliferation and differentiation, and affect the occurrence and de velopment of diseases[27, 28]. In our study, we have extracted exoso mes from RANKL-induced RAW 264.7 cells based on

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previous study[17], and found the extracted exosomes were round with about 50-100 nm in

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diameter. In addition, to identify exosomes, the expression levels of proteins in or outside exosomes

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were examined. We proved that proteins (Hsp70 and TSG101) existed in exosomes could be expressed in exosomes, proteins (TFIIB and LaminA/C) not existed in exosomes could not be expressed in exosomes, suggesting that we have successfully separated osteoclast-derived exosomes. In addition, exosomes, serving as a class of extracellular vesicles, play important regulatory roles in intercellular communication through the transport of lipid, mRNAs, miRNAs, proteins and other bioactive molecules[29]. Exosomes transport their carriers by either direct fusion with the cell membrane of the recipient cells or endocytosis[30]. Exosomes show different levels in pathological conditions or at different stages of cell differentiation, which makes exosomes useful biomarkers for the diagnosis of diseases or the evaluation of cell differentiation[31, 32]. Recent studies have shown

Journal Pre-proof that exosomes from different cell sources play an important regulatory role in bone remodeling[33, 34]. Researches also indicated that exosomes could regulate the proliferation, differentiation and apoptosis of bone marrow mesenchymal stem cells, osteoblasts and osteoclasts in bone to affect bone formation and bone resorption[35, 36]. At the same time, exosomes play a key role in the repair of bone tissue damage such as osteoporosis, femoral head necrosis, fracture, skull defect, cartilage defect and so on[37, 38]. In our previous research, we have found that MALAT1 stimulated the osteoclastic process in hFOB1.19 cells by inhibiting miR-22-5p activity, which could

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repress osteolysis through blocking the VEGF signaling and enhancing RANKL activity [39]. Our

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previous research also proved that osteoclast-derived miR-23a-5p-containing exosomes were

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observably increased in exosomes from RANKL-induced RAW 264.7 cells relative to RAW 264.7

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cells[17]. However, the effect of osteoclast-derived miR-23a-5p-containing exosomes on osteoblast differentiation is still not clear. In our study, we demonstrated for the first time that

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osteoclast-derived miR-23a-5p-containing exosomes could suppress the activity of osteoblast in

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co-cultured cells with hFOB1.19 and human osteoclasts cells, suggesting the major role of

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osteoclast-derived miR-23a-5p-containing exosomes on osteogenic differentiation. Moreover, we found that there was a binding site between miR-23a-5p and Runx2 through

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bioinformatics (Targetscan), suggesting that miR-23a-5p could target Runx2 and affect osteogenesis. We also proved that miR-23a-5p could inhibit Runx2 expression by targeting Runx2. Runt-related protein 2 (Runx2) is the most critical transcription factor that regulates the differentiation and maturation of bone marrow mesenchymal stem cells into osteoblasts during bone development[40]. Runx2 is involved in the regulation of bone metabolism through multiple pathways, which could affect the activities of osteoblast and osteoclast and regulate bone formation and bone resorption[41]. In addition, study has suggested that YAP1 interacted with Runx2, which further acted on lncRNA MT1DP promoter and negatively regulated the expression of MT1DP [42]. And then MT1DP negatively regulated the expression of Runx2 by FoxA1, thus forming an effective feedback regulatory loop[42]. Among them, YAP1 protein, the encoding product of YAP1

Journal Pre-proof gene, is a proline-rich phosphoprotein with 8 isomers and is a key member of Hippo signaling pathway[43]. Studies have shown that YAP1 is involved in biological processes s uch as cell growth, differentiation and apoptosis through Hippo signaling pathway[44, 45]. Recent studies have shown that MT1DP, acts as a long non-coding RNA, could enable cellular defenses against cytotoxicity[46], aggravate oxidative stress[47] and participate in tumor progression[42]. Therefore, we hypothesized that the interaction between YAP1 and Runx2 negatively regulates MT1DP, while MT1DP negatively regulates Runx2 through FoxA1. However, whether this feedback pathway

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could regulate osteogenic differentiation still needs further study. I n our study, we further verified

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that this feedback loop could be regulated by miR-23a-5p in osteoblast after a series of experiments.

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5. Conclusions Our study has illuminated that osteoclast-derived miR-23a-5p-containing exosomes inhibited the activity of osteoblast, and miR-23a-5p-containing exosomes might be the underlying mechanism to suppress

osteogenic

differentiation

by

targeting

Runx2.

Therefore,

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miR-23a-5p-containing exosomes might play essential roles in bone remodeling.

osteoclast-derived

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Acknowledgments This work was supported by Hunan Provincial Natural Science Foundation (Grant No. 2018JJ3865).

Competing interests

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The authors have no commercial or other associations that might pose a conflict of interest.

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Figures legends Figure 1. Identification of exosomes from osteoclasts. (A) Transmission electron microscopy was used to observe the morphology of exosomes, and found that vesicles were about 50-100 nm in diameter. (B) Relative expression levels of TFIIB, LaminA/C, Hsp70 and TSG101 were measured by western blot assay in RAW 264.7 cell lysate and exosomes, respectively. β-actin was used as an internal reference (**P<0.01, ***P<0.001). (C) Flow cytometry analysis was used to determine

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CD63 level in exosomes. (D) RT-qPCR analysis of miR-23a-5p expression in RANKL- induced

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RAW 264.7 cells (**P<0.01). (E) The protein expression level of TRAP was examined by Western

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blot assay in exosomes induced by M-CSF and RANKL (**P<0.01). (F) TRAP staining was performed to analyze the number and distribution of osteoclasts after induction with M-CSF and

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RT-qPCR assay in exosomes (**P<0.01).

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RANKL. (G) After induction with M-CSF and RANKL, miR-23a-5p expression was evaluated by

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Figure 2. Osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity. (A)

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The colocalization of human osteoclasts cell-derived exosomes and MC3T3-E1 was observed by confocal laser scanning microscopy. (B) miR-23a-5p inhibitors or mimics were added to human

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osteoclasts (OC) cells to modulate miR-23a-5p levels in exosomes, and RT-qPCR analysis was used to assess miR-23a-5p expression (*P<0.05, **P<0.01). (C) miR-23a-5p and MT1DP expressions were examined by RT-qPCR assay in OC cells co-cultured with miR-23a-5p mimics or inhibitors-treated exosomes (*P<0.05, **P<0.01). (D) OC cells were co-cultured with the exosomes from cells transfected with miR-23a-5p mimics or inhibitors, Runx2 and ALP expressions were analyzed by RT-qPCR assay (*P<0.05, **P<0.01). (E) Early osteogenesis was detected by ALP staining. Figure 3. Inhibition of exosome release weakened the miR-23a-5p-mediated inhibitory effect of osteoclasts on osteoblasts. OC cells were treated with N-SMase inhibitor GW4869. After 2 days, hFOB1.19 cells were co-cultured with the treated OC cells. qRT-PCR assays were adopted to

Journal Pre-proof analyze the level of miR-23a-5p (A), MT1DP (B), ALP (C) and Runx2 (D) in hFOB1.19 cells (*P<0.05, **P<0.01). (E) Early osteogenesis was detected by ALP staining. (F) After transfection with Rab27a siRNAs in

OC cells, the Rab27a level was measured by Western blot assay in OC

cells (*P<0.05). (G-H) After transfection with Rab27a siRNAs in OC cells, miR-23a-5p and MT1DP expressions were assessed by RT-qPCR assays in hFOB1.19 cells (*P<0.05, **P<0.01). (I-J) After transfection with Rab27a siRNAs in OC cells, Runx2 and ALP expressions were analyzed by RT-qPCR assays in hFOB1.19 cells (*P<0.05, **P<0.01). (K) After transfection with

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Rab27a siRNAs, ALP staining was used to detect the osteogenesis. Figure 4. YAP interacted with Runx2, and inhibited MT1DP expression. (A) The level of

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MT1DP level was analyzed by RT-qPCR assay after YAP1 knockdown in hFOB.19(*P<0.05,

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**P<0.01). (B) The level of MT1DP level was measured by RT-qPCR assay after Runx2

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knockdown in hFOB.19 (*P<0.05, **P<0.01). (C) The expression of YAP1 and Runx2 were detected by Western blot assays after YAP1 knockdown in hFOB.19 (*P<0.05, **P<0.01). (D) The

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expression of YAP1 and Runx2 were detected by Western blot assays after Runx2 knockdown in

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hFOB.19 (*P<0.05). (E) Cells were co-transfected with YAP-Flag and Runx2-HA, and the

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combination of YAP1 and Runx2 was verified by Co-IP assay in hFOB.19. (F) TargetScan was used to predict the binding sequence of miR-23a-5p with Runx2. (G) The regulating effect of miR-23a-5p and Runx2 was measured by Dual-luciferase reporter gene assay (*P<0.05). Figure 5. MT1DP promoted osteogenic differentiation by regulating FoxA1 and Runx2. (A) The expression levels of MT1DP and FoxA1 were analyzed by RT-qPCR assay after MT1DP knockdown (**P<0.01). (B) The expression levels of FoxA1 and Runx2 were measured by Western blot assay after MT1DP knockdown (*P<0.05). (C) After MT1DP knockdown, ALP staining was used to evaluate early osteogenesis. (D) The expression levels of FoxA1, Runx2 and YAP1 were measured by Western blot assay after FoxA1 knockdown (*P<0.05, **P<0.01). (E) After FoxA1 knockdown, ALP staining was used to evaluate early osteogenesis.

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Author contributions

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Xu-Cheng Yang: Conceptualization, Project administration, Writing - review & editing; Peng Xie, Data curation, Investigation, Resources; Jun-Xiao Yang: Formal analysis, Investigation, Funding acquisition, Methodology, Supervision; Ting Wen: Software, Visualization, Writing - original draft; Yu-Sheng Li: Validation, Writing - original draft.

Journal Pre-proof Highlight: 1. Identification of osteoclast-derived miR-23a-5p-containing exosomes 2. Osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity 3. Inhibition of exosome secretion could weaken osteoblast dysfunction 4. YAP/Runx2 axis inhibited MT1DP expression

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5. Knockdown of MT1DP promoted osteogenic differentiation by FoxA1 and Runx2