IL‐10 induces MC3T3‐E1 cells differentiation towards osteoblastic fate in murine model

Abstract Interleukin‐10 (IL‐10) displays well‐documented anti‐inflammatory effects, but its effects on osteoblast differentiation have not been investigated. In this study, we found IL‐10 negatively regulates microRNA‐7025‐5p (miR‐7025‐5p), the down‐regulation of which enhances osteoblast differentiation. Furthermore, through luciferase reporter assays, we found evidence that insulin‐like growth factor 1 receptor (IGF1R) is a miR‐7025‐5p target gene that positively regulates osteoblast differentiation. In vivo studies indicated that the pre‐injection of IL‐10 leads to increased bone formation, while agomiR‐7025‐5p injection delays fracture healing. Taken together, these results indicate that IL‐10 induces osteoblast differentiation via regulation of the miR‐7025‐5p/IGF1R axis. IL‐10 therefore represents a promising therapeutic strategy to promote fracture healing.

are established from a C57BL/6 mouse calvaria and selected on the basis of high alkaline phosphatase (ALP) activity in the resting state. 4,5 In a previous study, it is reported that MC3T3-E1 cells secrete collagen and express murine leukaemia inhibitory factor (mLIF) in RNA. 6 However, the lack of knowledge on the mechanisms underlying MC3T3-E1 differentiation continues to hinder the progress of MC3T3-E1-based therapies for bone regeneration. IL-10 has profound and indispensable functional effects on infection, inflammation, tissue homeostasis, autoimmunity and cancer. [7][8][9][10][11] In recent years, the IL-10 family have been shown to regulate arthritis, 12,13 suggesting potential effects on osteoblast differentiation. Similarly, a recent study had reported that overexpression of IL-10 could disrupt osteoclast differentiation via NF-ĸB signalling. 13 However, the underlying mechanism of IL-10 in the regulation of osteogenic differentiation is still elusive. Herein, it is necessary to catch a better understanding of the osteoblastic functionality of IL-10. microRNAs (miRNAs) are short (20-24 nt) non-coding RNAs involved in the post-transcriptional regulation of gene expression in multicellular organisms by affecting both mRNA stability and translation. 14,15 During recent decades, suppressive miRNAs have been shown to exert regulatory functional effects through targeting osteogenic genes involved in osteoblast growth, osteoclast-mediated bone absorption processes and bone balance. [16][17][18][19] To investigate the association between miRNAs and osteogenic differentiation, we obtained data sets (GSE76197) related to bone formation from the Gene Expression Omnibus (GEO) (https :// www.ncbi.nlm.nih.gov/geo/). We identified the down-regulation of miR-7025-5p in six mice (three intact control samples and three post-fracture samples) that implicated its regulatory role during osteoblast differentiation.
In this study, we investigated the role of IL-10 in the modulation of osteoblast differentiation both in vitro and in vivo. We demonstrate that IL-10 positively influences osteoblast differentiation through its ability to down-regulate miR-7025-5p. The down-regulation of miR-7025-5p could induce IGFIR mRNA expression and accelerate fracture healing. Taken together, these results demonstrate that IL-10 promotes osteoblast differentiation through regulation of the miR-7025-5p/IGF1R axis.

| Radiographic images
On days 7, 14 and 21 post-injury, fractured femurs were scanned using the FX PRO imaging system with a 10-seconds exposure time.

| Micro-CT analysis
The fracture site was scanned using a BRUKER SkyScan 1276 scanner microCT system (BRUKER) to provide images at 2400 views, 5 frames/view, 37 kV and 121 mA. Three-dimensional images were rendered and evaluated using CT-Vox2.1 version (BRUKER Minimal Intensity Projection Software). Soft tissues were thoroughly cleaned. After scanning, calluses were preserved at −80°C for miRNA extraction and PCR and Western blot. Measurement parameters were as follows: bone volume (BV), total volume (TV) and BV/TV.

| Quantitative real-time PCR
qRT-PCR assays were performed as previously described. 20 Briefly, total RNA was prepared using TRIzol reagent (Thermo Fisher Scientific, #L15596026). First-strand cDNA was synthesized using the qPCR RT Master Mix (Toyobo). Relative gene expression levels of mRNA were calculated using the 2 −ΔΔCt method (Ct of GAPDH minus the Ct of the target genes) and the relative expression of miRNA was normalized against U6 and determined as the 2 −ΔΔCt . Primer sequences are listed in Table 1.

| Western blot
Cell lysates were prepared using NETN buffer (20 mmol/L Tris HCl, pH 8.0, 100 mmol/L NaCl, 1 mmol/L EDTA and 0.5% Nonidet P-40) and were resolved by SDS-PAGE. Proteins were transferred to PVDF membranes and blocked in 5% skimmed milk at 4℃overnight. Membranes were probed with primary antibodies and labelled with HRP-conjugated secondary antibodies (Aspen, #AS1058). Chemiluminescence detection systems (Canon, #LiDE110) were used to visualize protein bands.

| Alkaline Phosphatase staining
A BCIP/NBT alkaline phosphatase (ALP) colour development kit (Beyotime, #C3206) was used based on provided directions to assess ALP staining. Briefly, after washing twice with PBS, cells were fixed with 10% formalin for 15 minutes. The BCIP/NBT liquid substrate was then applied to cells for 24 hours. Samples were prepared in the dark at room temperature. Colour changes were obtained under a charge-coupled device (CCD) microscope, and the stained cell cultures were imaged using a scanner (EPSON V600). Samples were analysed in triplicate.

| Alizarin red staining
MC3T3-E1 cells were grown in osteogenic media supplemented with 100 nmol/L dexamethasone, 50 mmol/L ascorbic acid and 10 mmol/L b-glycerophosphate (Cyagen, #HUXMA-90021) in 6-well plates to induce osteoblast mineralization. Cells were fixed in formalin (10%) at room temperature for 15 minutes, washed in 2 mL PBS and stained using 1 mL 0.5% alizarin red staining solution at room temperature for 15 minutes. Cells were rinsed with distilled water for 5 minutes with shaking, and red mineralized nodules were assessed via a CCD microscope and scanned (EPSON V600). All staining data were repeated three times.

| Stimulation of IL-10/agomiR-7025-5p in fracture models
Mice were injected locally at the fracture sites. A volume of 0.1 mL per injection was administered on days 1, 3 and 7 following fracture induction. IL-10 (Cyagen, #MEILP-1001) or agomiR-7025-5p (GenePharma) was directly injected in local sites. Following experimental completion, animals were euthanized, and bones and calluses were isolated. qRT-PCR and Western blot analysis were performed.

| Statistical analysis
Data are presented as the means ± SD, and GraphPad Prism 8.0 (GraphPad Software, Inc) was used for all analyses unless otherwise stated. A one-way analysis of variance using a Tukey's post-hoc test TA B L E 1 miRNAs and mRNA primer sequence microRNAs or gene name Primer sequence (5′ to 3′) was applied to compare three or more groups. A two-tailed Student's test was applied for data comparisons between the groups. P < .05 was deemed statistically significant.

| IL-10 accelerates fracture healing in vivo
To demonstrate the effects of IL-10 on fracture healing, we admin- These results indicate that IL-10 promotes fracture healing in vivo.

| IL-10 promotes osteoblast differentiation in vitro
To explore the association between IL-10 and osteoblast differentiation, MC3T3-E1 cells were treated with various concentrations of IL-10 (0, 10, 30, 50 and 100 ng/mL) for 24 hours, and osteoblastsogenesis alpha-1 type I collagen (Col 1a1), alkaline phosphatase (ALP), osteocalcin (OCN) and runt-related transcription factor 2 (RunX2) were determined via qRT-PCR and Western blot analysis. As shown in Figure 2A,B, IL-10 promoted osteoblast differentiation and the levels of osteogenic markers in the IL-10-treated groups showed a concentration-dependent increase. The effects of IL-10 on extracellular matrix mineralization in MC3T3-E1 cells were investigated through the treatment of various concentrations of IL-10 (0, 10, 30, 50 and 100 ng/ mL) and after continuous culture, higher mineral deposition was observed in IL-10-treated groups relative to controls ( Figure 2C,D).
These results demonstrated that IL-10 positively regulates osteoblastsogenesis and activates ALP activity and mineralization.

| IL-10 promotes osteoblast differentiation through the inhibition of miR-7025-5p
We next compared the levels of miR-7025-5p in response to IL-10. We observed a significant increase in the expression of all osteogenic markers in IL-10-treated groups, which was inhibited through the expression of the miR-7025-5P mimic ( Figure 3B). Moreover, when transfected cells were assessed for extracellular matrix mineralization after continuous culturing, higher mineral deposition was obvious in the IL-10 group relative to other groups ( Figure 3C,D). Collectively, these data suggest that IL-10 positively regulates osteoblastsogenesis through the inhibition of miR-7025-5p.

| Negative effects of miR-7025-5p on fracture healing in vivo
To explore the relationship between miR-7025-5p and bone formation, we calculated the relative levels of miR-7025-5p in mice fracture gene chips ( Figure 4A). Calluses from the fracture sites in mouse models were also collected for the assessment of miR-7025-5p levels ( Figure 4B). We found that miR-7025-5p expression markedly decreased during the early stages of fracture healing. To further investigate the role of miR-7025-5p in vivo, mice fracture models were randomly divided into two groups (control and receiving agomiR-7025-5p). In the agomiR-7025-5p group, all animals received local injection of fluorescent miR-7025-5p into the fracture sites on days 0, 4 and 7 post-injury, and animals were imaged to assess the levels of miR-7025-5p in the fracture sites ( Figure 4C). High levels of miR-7025-5p were found in the calluses of agomiR-7025-5p animals on days 4, 7, 14 and 21 by qRT-PCR ( Figure 4D). Moreover, when X-rays and CT examinations were performed to compare the speed and quality of fracture healing, mice treated with agomiR-7025-5p exhibited a smaller callus volume and larger fracture gap relative to control animals ( Figure 4E,F). Additionally, in the agomiR-7025-5p animals, there was a smaller total bone callus volume relative to control animals on post-fracture day 14, and the difference remained significant between agomiR-7025-5p and control animals on postfracture day 21 ( Figure 4G).These results indicate that miR-7025-5p acts as a negative regulator of fracture healing.  Figure 5B). When the influence of miR-7025-5p on extracellular matrix mineralization was assessed, higher mineral deposition was observed in the antagomiR-7025-5p group, particularly when compared to agomiR-7025-5p groups ( Figure 5C,D). Taken together, these results reveal that miR-7025-5p negatively regulates osteoblastsogenesis and thereby suppressing ALP activity and mineralization.
Using these constructs, we found that agomiR-7025-5p substantially attenuated WT IGF1R 3ʹ UTR reporter activity ( Figure 6A), but failed to influence the activity of the mutated 3ʹ UTR IGF1R reporter ( Figure 6B). IGF1R was continuously detected for 14 days during the early stages of fracture and increased during these stages ( Figure 6C). In vivo, calluses were collected from the two groups (control and agomiR-7025-5p) on days 4, 7, 14 and 21 to detect the levels of IGF1R by PCR analysis. Lower levels of IGF1R mRNA were detected in the agomiR-7025-5p group compared with the control group on each day ( Figure 6D). Furthermore, calluses of the fracture sites were harvested from animals in the two groups on days 14 and 21, and Western blot analysis showed that levels of IGF1R were significant lower in agomiR-7025-5p compared with control groups ( Figure 6E). In vitro, MC3T3-E1 cells were transfected with transfection reagent (control), antagomiR-negative control (agomiR-NC), antagomiR-7025-5p, agomiR-negative control (antagomiR-NC), or agomiR-7025-5p and IGF1R mRNA levels were measured by qRT-PCR ( Figure 6F) and Western blot ( Figure 6G). We observed higher relative IGF1R mRNA levels in the antagomiR-7025-5p group compared with the other groups. In addition, to investigate whether osteoblast differentiation is IGF1R-dependent, we assessed whether antagomiR-7025-5p can rescue the negative effects of siRNA-IGF1R on osteogenic differentiation. Western blot and qRT-PCR analysis revealed that antagomiR-7025-5p could restore IGF1R dependent gene expression during osteogenesis, including collagen I, ALP, OCN and RunX2 ( Figure 6H-I). These results demonstrate that miR-7025-5p directly targets the IGF1R 3ʹ UTR during osteoblast differentiation.

| D ISCUSS I ON
In this study, we evaluated the role of IL-10 during the process of osteogenic differentiation. Our results suggested that IL-10 positively

F I G U R E 3 MiR-7025-5p is Downregulated and Osteogenic Differentiation
Increases in IL-10-Treated Groups. A, MC3T3-E1 cells were treated with various concentrations of IL-10 (0, 10, 30, 50 and 100 ng/mL) for 24 hours, and miR-7025-5p levels in the IL-10-treated groups were significantly lower than the control group. B, MC3T3-E1 cells were treated with lipofectamine 3000 and IL-10, or IL-10 + agomiR-7025-5p. A significant increase in the expression of osteogenic markers in the IL-10 group relative to the other groups was observed by qRT-PCR analysis. C, ALP staining from (B). D, Alizarin red-mediated calcium staining in (B). Scale bar = 10 mm. Data are means ± SD of triplicate experiments. *P < .05, **P < .01, ***P < .001 regulates osteogenic processes both in vitro and in vivo through regulation of the miR-7025-5p/IGF1R axis (Figure 7). In addition, we   lower affinity. Activated IGF1R is involved in cell growth and survival and influences an array of physiological and pathological conditions. [29][30][31] For example, IGF1R inhibition is an escape mechanism in Ewing sarcoma and IGF1R inhibitors are candidate therapies for this disease. 32 In this study, we found that IGF1R interacts with miR- In summary, we demonstrate that IL-10 positively regulates fracture healing through regulation of the miR-7025-5p/IGF1R axis.
IL-10 therefore represents a potential therapeutic strategy to promote fracture healing in the clinic.