Exosomal miR‐130a‐3p regulates osteogenic differentiation of Human Adipose‐Derived stem cells through mediating SIRT7/Wnt/β‐catenin axis

Abstract Objectives It is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose‐derived stem cells (ADSCs) will be probably promoted by exosomes. Materials and Methods By means of lentiviral transfection, miR‐130a‐3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis‐related genes, to verify the effect of miR‐130a‐3p on the osteogenic differentiation of ADSCs. CCK‐8 was used to detect the effect of miR‐130a‐3p on the proliferation of ADSCs. The target binding between miR‐130a‐3p and SIRT7 was verified by dual‐luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR‐130a‐3p was further verified by detecting osteogenic‐related genes and proteins and alkaline phosphatase activity. Results (a) Overexpression of miR‐130a‐3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR‐130a‐3p. (b) Our study further found that overexpression of miR‐130a‐3p leads to down‐regulation of SIRT7 expression with up‐regulation of Wnt signalling pathway‐associated protein. (c) Overexpression of miR‐130a‐3p inhibited proliferation of ADSCs, while knockdown promoted it. Conclusions The obtained findings indicate that exosomal miR‐130a‐3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/β‐catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.

from adipose tissue. 1,2 Compared with other adult stem cells, ADSCs can not only differentiate into adipocytes, chondrocytes and osteoblast, but also have the advantages of abundant storage in vivo, easy acquisition and expansion. 3 In recent years, the application of ADSCs in the field of bone regeneration has received extensive attention.
Achieving directional osteogenic differentiation of stem cells is the key to bone regeneration, and 'inducing factors' play a critical role in this process. Our previous studies have confirmed that the exosomes derived from osteogenically differentiated ADSCs can be used as an effective 'inducing factor' to promote osteogenic differentiation of ADSCs. 4 However, the molecular mechanism of exosomes in the osteogenic differentiation of ADSCs remains elusive. MicroRNAs (miRNAs) are endogenous non-protein-coding RNA with a length of about 22 nt, which is a quintessential post-transcriptional regulator and it can regulate the expression of target genes mainly through specific binding with 3'UTR of target genes. 5 As an important 'cargo' of exosomes, miRNAs have been demonstrated to regulate bone regeneration. [6][7][8] In our previous studies, we intended to figure out how miRNAs regulate bone regeneration, so we used microarray assays to analyse the different expression of exosomal miRNAs, and found that miR-130a-3p has the highest fold change, which means that miR-130a-3p may be the main influence. 4 So how does miR-130a-3p play a part as a regulator? Xiao F et al found that miR-130a-3p can significantly alleviate fatty liver, which mainly regulates liver lipid metabolism by regulating FAS expression. 9 In addition, it was also noted that the treatment of 3T3-L1 cells with miR-130a-3p could reduce lipid production. 10 Up to this date, other researches on miR-130a-3p are more focused on the field of cancer. 11,12 However, the role of miR-130a-3p in osteogenic differentiation of mesenchymal stem cells (MSCs) is rarely known.
As a member of the sirtuin family of NAD + -dependent deacetylase, 13 SIRT7 interacts with various substrate proteins through its deacetylation activity and participates in the regulation of cell proliferation, senescence, apoptosis, metabolism and other processes. 14,15 Currently, an increasing number of studies have indicated that sirtuin family is closely related to osteogenic differentiation. [16][17][18] According to previous studies, SIRT7 has been shown to promote adipogenesis, 19 while inhibiting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). 20 Moreover, as mentioned before, miRNAs are considered to be an endogenous regulator of SIRT7, which binds to 3'UTR of SIRT7 to down-regulate its expression. 21 In the past few years, many members of the sirtuin family have been confirmed to relate to Wnt signalling pathway closely. 20,22,23 Wnt signalling pathway has been widely confirmed to play an important role in the osteogenic differentiation of MSCs. Among them, the canonical Wnt signalling pathway is characterized by stable expression of β-catenin and its transfer into the nucleus. When in the absence of Wnt ligands, β-catenin interacts with Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK-3β) to form 'APC-Axin-GSK-3β' complex. As a result, β-catenin is degraded and cannot enter the nucleus, forming an 'off-state' pathway. When the expression of Wnt ligands is activated, Wnt interacts with lipoprotein receptor-related protein (LRP) and Frizzled to form receptor complex, and loose protein (Dsh) in the cytoplasm gathers under the cell membrane, leading to GSK-3β phosphorylation and preventing 'APC-Axin-GSK-3β' complex from formation. Thus, the degradation process of β-catenin is blocked, and a large amount of free β-catenin enters the nucleus, which is to say, Wnt signalling pathway is now 'on-state'. 24 In this study, we confirmed the vital role of exosomal miR-130a-3p in the osteogenic differentiation of ADSCs, as well as clarified the targeting relationship between miR-130a-3p and SIRT7.
Furthermore, we explored the modulation of Wnt signalling pathway in this process, so as to confirm the important significance of miR-130a-3p/SIRT7/Wnt/β-catenin axis in regulating the osteogenic differentiation process of ADSCs, providing a theoretical basis for bone regeneration.

| Isolation, culture and characterization of ADSCs
According to methods previously reported, 25 Multi-lineage potential assay and flow cytometry analysis were performed to identify characteristics of ADSCs, as we have done in the past. 4 The antibodies including anti-CD34-FITC, anti-CD45-PE, anti-CD44-FITC, anti-CD73-PE and anti-CD105-PE were purchased from BD biosciences (USA).

| Extraction and Identification of ADSCsderived exosomes
ADSCs-derived exosomes used in this study were obtained by Differential Ultracentrifugation, as described previously by Théry C et al 27 In order to define the exosomes, transmission electron microscopy [TEM, (HITACHI, JAPAN)] was used to observe their structures. The characteristic markers including TSG101, calnexin and CD9 were analysed by Western blot, as described in 2.8.

| Nanoparticle tracking analysis (NTA)
The exosomes sample pool was washed with deionized water.
ZetaView Particle Metrix (Particle Metrix, Germany) is calibrated with polystyrene microspheres with a size of 110 nm. Then, the sample pool was washed with 1 × PBS. Finally, the exosomes sample was diluted by 1 × PBS buffer (Biological Industries, Israel) to for testing.

| Lentiviral transfection
The lentivirus particles used in this study were purchased from GenePharma company (China). First, 1 × 10 5 ADSCs were plated in a 6-well plate and incubated overnight in conventional growth medium under the condition of 37°C/5% CO2 and saturated humidity.
Next, cells were incubated with lentiviral particles and 5 μg/mL polybrene in conventional growth medium at a multiplicity of infection (MOI) of 50. After 24 hours, the results were observed under a light microscope (OLYMPUS, Japan) and an inverted fluorescence microscope (OLYMPUS, Japan). Two days later, the expression of miR-130a-3p was measured by qPCR to see whether transfection is successful or not. It was illustrated more in Materials and Methods 2.7.

| Osteogenic induction of ADSCs and Alizarin Red S (ARS) assay
When the cells reached to 90% confluence, the conventional growth medium was replaced with osteogenic differentiation medium, which was prepared by 15 mL 15% foetal bovine serum [FBS

| Alkaline phosphatase (ALP) activity assay
The detected cells were lysed by Cell lysis buffer for Western and IP without inhibitors (Beyotime Biotechnology, China), and the supernatant was collected for semi-quantitative analysis of ALP using an Alkaline Phosphatase Assay Kit (Beyotime Biotechnology, China) according to the manufacturers' instructions. Para-nitrophenol (pnitrophenol), as a common substrate for phosphatase activity, can produce yellow products under alkaline conditions. Optical density (OD) values were measured using a microplate reader (SpectraMax Plus384, Molecular Devices, USA) at 405 nm. The expression level of ALP was standardized to the total protein content of cells to obtain the absorbance index.

| RNA isolation and Quantitative real-time PCR (qPCR)
In accordance with the manufacturer's instructions, the exosomal RNA was isolated using SeraMir Exosome RNA Purification kit (System Biosciences, USA). TRIzol ™ Reagent (Invitrogen, USA) and PrimeScript ™ RT Master Mix (TAKARA, Japan) were used to extract total RNA and synthesize cDNA from cells, respectively. The cycle was as follows: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds; and extension at 60°C for 30 seconds.
The cycle was done for 40 rounds. All primers were synthesized by GenePharma (China). The primer sequences are given in Table 1.
Moreover, the details of primers are provided in Supplementary file 1. GAPDH was used for mRNA normalization. U6 was used for miRNA normalization. The relative expression levels of target genes were calculated using the 2 −ΔΔCt method.  Firefly/Ranilla Luciferase activity was used for internal control, and all assays were repeated three times.

| Statistical analysis
In this study, SPSS 17.0 and GraphPad Prism 7.0 were used for statistical analysis. Besides, all experiments were repeated at least three times. P value < .05 is considered as statistically significant differences.

| Identification of ADSCs
To identify characteristics of ADSCs, flow cytometry analysis and multi-lineage potential assay were performed. As shown in

| Overexpression and knockdown of miR-130a-3p in ADSCs
To further explore the roles of miR-130a-3p, which was highly ex-

| Overexpression of miR-130a-3p inhibits proliferation but promotes osteogenic differentiation of ADSCs, while miR-130a-3p knockdown promotes proliferation
To investigate whether miR-130a-3p can affect the proliferation of ADSCs, we carried out cell viability assay using CCK8. As the result shown in Figure 4

| MiR-130a-3p can target SIRT7 mRNA in the 3'UTR
In order to explore how miR-130a-3p regulates osteogenic differentiation of ADSCs, we searched for its potential downstream target genes through bioinformatics analysis. TargetScan tool predicted that miR-130a-3p can bind to the 3'UTR of SIRT7 [ Figure 5

| Exosomal miR-130a-3p regulates osteogenic differentiation of ADSCs by mediating Wnt signalling pathway
To further clarify the specific mechanism by which miR-130a-3p

F I G U R E 2
Identification of exosomes derived from ADSCs and the expression of miR-130a-3p in ADSCs-derived exosomes. A, The size and morphology of exosomes observed using TEM. Scale bar: 100 nm. B, Specific markers of exosomes detected by Western blot. C, Nanoparticle tracking analysis of exosomes. D, qPCR analyses expression of miR-130a-3p in Exos_D0 and Exos_D14. *represents significant differences between Exos_D0 and Exos_D14. *P < .05 As the result shown in Figure 6(E,F), compared with control and lenti-control groups, ALP activity on 3rd day and 7th day was significantly increased in overexpression group without DKK1. Compare with control group, ALP activity on 3rd day was significantly decreased. ALP activity on 7th day was significantly decreased, compared with control and lenti-control groups. Compared with no DKK1 treatment groups, ALP activity in groups with DKK1 treatment was significantly decreased. ALP activity in the overexpression group was most significantly decreased. The diagram of the mechanism that exosomal miR-130a-3p regulates osteogenic differentiation of ADSCs by mediating Wnt signalling pathway was presented in Figure 6(G).
In general, these results confirmed that blocking Wnt signalling pathway inhibits osteogenesis and further proved that miR-130a-3p promotes osteogenic differentiation of ADSCs by activating Wnt signalling pathway.

| D ISCUSS I ON
It is an urgent issue in the field of bone regeneration to find an efficient way to induce osteogenic differentiation of stem cells. Our previous study has confirmed that only osteogenically differentiated ADSC-derived exosomes can promote osteogenic differentiation of ADSCs, which is expected to be an effective osteogenic induction method. 4 To explore how exosomes worked, we selected miR-130a-3p, which is highly expressed in exosomes derived from osteogenically differentiated ADSC, for subsequent study. Seenprachwwong et al have proven that miR-130a and miR-27b can enhance hBMSCs via specific down-regulation of PPAR-γ (a major transcription factor of adipogenesis). 28 It suggested that the osteogenic differentiation of MSCs is negatively correlated with adipogenesis. Hence, osteogenic potential of MSCs could be enhanced by inhibiting their adipogenesis. In the current study, we clarified the underlying mechanism F I G U R E 3 Construction of miR-130a-3p overexpressing ADSCs, miR-130a-3p knockdown ADSCs and lenti-control ADSCs. A, 24 h after transfection, ADSCs observed under a normal microscope and an inverted fluorescence microscope (magnification, ×4). B, The expression of miR-130a-3p quantified by qPCR. Notes: *represents significant differences between the control group and other groups; #represents significant differences between lenti-control and overexpression groups, or lenti-control and knockdown groups. *P < .05; #P < .05 of miR-130a-3p in regulating proliferation and osteogenic differentiation of ADSCs by altering its expression. Importantly, exosomes may modulate proliferation and osteogenic differentiation of ADSCs by delivering miR-130a-3p.
The fact that exosomes carry miRNAs which serve as important mediators of intercellular communication, has been proven to promote osteogenic differentiation of stem cells. 4,29,30 MiRNAs play a crucial role in bone development and homoeostasis. 8,31 Our findings revealed that overexpression of miR-130a-3p can promoted osteogenic differentiation but inhibited proliferation of ADSCs. Knockdown of miR-130a-3p can promoted proliferation without significantly inhibiting osteogenic differentiation of ADSCs. Consistently, it has been reported that miR-130a-3p inhibited cell proliferation in nasopharyngeal carcinoma. 32 Additionally, lipid production was decreased due to 3T3-L1 cells being treated with miR-130a-3p. 10 To our knowledge, this is Furthermore, our results also indicated that miR-130a-3p can target SIRT7 by dual-luciferase reporter gene assay. Knockdown of SIRT7 using lentiviral transfection is now confirmed to promote osteogenic differentiation of bone marrow mesenchymal stem cells. 20 However, there are safety risks in the clinical application of lentiviral modified cells. In this study, we explored the possibility of miR-130a-3p enriched in safer exosomes as SIRT7 targeting suppressor, to achieve the similar effect of promoting osteogenic differentiation of ADSCs, which is more significant for clinical application of promoting bone regeneration.
In this study, we found that miR-130a-3p overexpression can dra- *represents significant differences between control and other groups; #represents significant differences between lenti-control and overexpression groups, or lenti-control and knockdown groups; and *represents significant differences between NC and hsa-miR-130a-3p groups. *P < .05, #P < .05, *P < .05 F I G U R E 6 The mechanism of miR-130a-3p promoting osteogenic differentiation of ADSCs through Wnt signalling pathway. A-B, The expression of osteogenic-related protein and key protein of Wnt signalling pathway analysed by Western blot on 3rd and 7th day. C-D, The expression of ALP and RUNX2 mRNA detected by qPCR. E-F, ALP activity detected by ALP assay kit. G, The diagram of the mechanism that exosomal miR-130a-3p regulates osteogenic differentiation of ADSCs by mediating Wnt signalling pathway. Notes:*represents significant differences between control and other groups without DKK1 treatment; *represents significant differences between no treatment and DKK1 treatment group; and #represents significant differences between lenti-control and overexpression groups, or lenti-control and knockdown groups without DKK1 treatment. *P < .05; *P < .05; #P < .05 More and more studies have confirmed that exosome plays an important role in osteogenesis. 37,38 In our present study, for the first time, we confirmed the effect of exosomal miR-130a-3p on proliferation and osteogenic differentiation of ADSCs. These findings indicated that exosomal miR-130a-3p may be a new target for bone regeneration. Other potential targets of miR-130a-3p and signalling pathways involved in regulating osteogenic differentiation by exosomal miR-130a-3p should be explored in the future. Additionally, the function of more miRNAs delivered through exosomes which is related to osteogenesis remains to be elucidated.
Long-term vision, the real achieve clinical application of the method by exosomal miRNAs to treat bone defects, there is still a long way to go. The development of bone tissue engineering provides a shortcut. Bone tissue engineering is mainly composed of three elements: seed cells, inducible factors and scaffold materials.
In research experiments, we confirmed that exosomal miR-130a-3p can be used as a highly effective osteogenic induction factor in the construction of tissue engineering bone. At the same time, we need a scaffold material can provide 'matrix' support to solve the problem that it cannot act alone on local bone defects. Therefore, the preparation of scaffold materials with good structure, strength and other properties that can be used in vivo is an urgent problem to be solved at present. It will be the focus of future research, which will also greatly promote the development of clinical treatment of bone defects.

| CON CLUS IONS
In conclusion, our results showed that exosomal miR-130a-3p can regulate proliferation and osteogenic differentiation of ADSCs.
The overexpression of miR-130a-3p promoted osteogenic differentiation but inhibited proliferation, while knockdown only promoted proliferation of ADSCs. Thus, exosomes rich in miR-130a-3p may become a novel 'inducible factor' to promote regeneration and repair in the future.

ACK N OWLED G EM ENTS
The work was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
No conflicts of interest exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data obtained or analysed in this study are included in this paper.