MiR-137-mediated negative relationship between LGR4 and RANKL modulated osteogenic differentiation of human adipose-derived mesenchymal stem cells

ABSTRACT

MicroRNA-137 (miR-137) has recently emerged as an osteogenic regulator in several cell lines. This study aimed to identify the function of miR-137 on the crosstalk between leucine rich repeat containing G protein-coupled receptor 4 (LGR4) and receptor activator of nuclear factor-κB ligand (RANKL), thus unveiling the critical role of LGR4-RANKL interplay in the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASCs). By examining the osteogenic capacity and possible downstream genes expression with miR-137 overexpression/knockdown, we found that miR-137 downregulated LGR4 while upregulating RANKL. According to the results of dual-luciferase reporter assay, LGR4 was validated as a direct target of miR-137. Surprisingly, a negative relationship between LGR4 and RANKL was confirmed by the knockdown of these two genes. Furthermore, RANKL inhibitor could alleviate or reverse the inhibitory effects on osteogenesis generated by LGR4 knockdown. Collectively, this study indicated that miR-137-induced a negative crosstalk between LGR4 and RANKL that could contribute to the osteogenic regulation of hASCs and provide more systematic and in-depth understanding of epigenetic modulation by miR-137.

Keywords
MicroRNA; LGR4; osteogenic differentiation; human adipose-derived mesenchymal stem cells; RANKL

Introduction

As an ideal stem cell source, human adipose-derived mesenchymal stem cells (hASCs) are widely used in bone tissue engineering due to their accessibility, multipotency and low immunogenicity (Dinescu et al., 2021). MicroRNAs (miRNAs), a class of small non-coding RNAs ranging from 19-25 nucleotides in size, regulate protein-coding genes post-transcriptionally and play crucial roles in various biological activities, including the proliferation and osteogenic differentiation of hASCs (Ranganathan and Sivasankar, 2014; Fan et al., 2021). It has been demonstrated that miR-137 acts as a key regulator in various kinds of malignancies (Li et al., 2017; Liu et al., 2017; Qin et al., 2017; Luo et al., 2018; Zhang et al., 2018) and neural development (Silber et al., 2008; Szulwach et al., 2010; Tarantino et al., 2010; Sun et al., 2011; Jiang et al.,2013). But up to present, only a few studies investigated the effects of miR-137 on osteogenesis (Zheng et al., 2019; Kong et al., 2020; Ma et al., 2020; Yu et al., 2020; Fan et al., 2021), and the mechanisms of osteogenic modulation by miR-137 are still not fully understood, especially considering the diverse biological properties of different cell types.

The gene Leucine rich repeat containing G protein-coupled receptor 4 (LGR4) has been implicated in various biological processes, such as bone mineralization and remodeling, innate immune responses, intestinal stem cell metabolism and energy metabolism (Zhou et al., 2017). Some studies have confirmed the direct binding of LGR4 by miR-137 in prostate cancer cells (Zhang et al., 2020), U-2 and MC3T3 cells (Liu and Xu, 2018). However, whether LGR4 is a target of miR-137 in hASCs needs to be identified. Moreover, LGR4 is found to be engaged in the regulation of osteogenesis, and its inhibition leads to impaired bone formation in several lineages of mesenchymal stem cells (Zhang et al., 2017; Zhou et al., 2017; Sun et al., 2019). Based on these findings, we conjectured that miR-137 could regulate the osteogenic differentiation of hASCs through LGR4.

The gene product of Receptor activator of nuclear factor-κB ligand (RANKL) is membrane-bound or secreted by either proteolytic cleavage or alternative splicing (Chen et al., 2018a). After binding with Receptor activator of nuclear factor-κB (RANK), RANKL activates the downstream signaling pathways related with osteoclastic proliferation and differentiation (Boyle et al., 2003). Besides, RANKL can positively regulate the osteoblastic differentiation of vascular smooth muscle cells (VSMCs) (Davenport et al., 2016), while negatively regulating the osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) (Chen et al., 2018b; Elango et al., 2019), suggesting that RANKL could have effects on the osteogenic differentiation of hASCs.

Recent studies have shown that LGR4 is a novel receptor of RANKL and negatively regulates osteoclast differentiation by the competition with RANK to bind to RANKL (Luo et al., 2016; Renema et al., 2016). But the silencing of LGR4 in VSMCs can prevent parathyroid hormone (PTH)-induced vascular calcification without changes in RANKL and OPG expression (Carrillo-López et al., 2021). However, how the interplay of LGR4-RNAKL influences the osteogenic differentiation of hASCs is not clear so far. Therefore, we proposed as a hypothesis that miR-137 regulates the osteogenesis of hASCs by mediating the interplay between LGR4 and RANKL.

In this study, we confirmed that LGR4 was a target gene of miR-137 in hASCs and the crosstalk between LGR4 and RANKL participated in the osteogenic control of miR-137. The revelation of epigenetic mechanisms on miR-137-modulated osteogenesis is valuable for miRNA-targeted therapy of bone defect, and the findings of LGR4-RANKL contribution in the osteogenic differentiation has implications for future clinical management of bone disorders.

Material and Methods

Cell culture and osteogenic induction

The hASCs collected from three different donors were obtained at ScienCell Research Laboratory (Carlsbad, CA) and cultured in proliferation medium (PM), which contained Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Rockford, IL), 10% (v/v) fetal bovine serum (ExCell Bio, Shanghai, China) and 1% (v/v) penicillin/streptomycin (Thermo Fisher Scientific). For the osteoinduction of hASCs, cells were cultured in osteogenic medium (OM), composed of PM, 100 nM dexamethasone (Sigma‐Aldrich, St. Louis, MO), 10 mM β‐glycerophosphate (Sigma-Aldrich) and 0.2 mM L‐ascorbic acid (Sigma-Aldrich). The hASCs were incubated at 37 °C with 5% CO₂ and 100% humidity. All the in vitro cell experiments applied the third passage (P3) hASCs and were repeated in triplicate at least.

Lentiviral transfection

GenePharma (Suzhou, China) synthesized and packaged the recombinant lentiviruses containing green fluorescent protein (GFP)‐labeled plasmid vectors. The plasmids of negative control (NC), miR‐137 overexpression (miR‐137), miR‐137 knockdown (anti‐miR‐137), LGR4 shRNA (anti-LGR4) and RANKL shRNA (anti-RANKL) were separately employed to generate the corresponding lentiviruses. The hASCs were transfected by respective lentiviruses as previously described (Ma et al., 2020;Fan et al., 2021). After being immersed in the viral supernatant (multiplicity of infection = 100) with 5 mg/mL polybrene (Sigma-Aldrich) for 24 h, the cells were then selected by 1 μg/ml puromycin (Sigma-Aldrich) and cultured in fresh PM. The transfected rates were computed by counting the number of GFP-marked cells and total cells under an inverted fluorescence microscope (TE2000-U, Nikon, Tokyo, Japan). The hASCs transfected with anti-LGR4 lentiviruses were seeded in 96-well plates (1 × 10⁴ cells/well) and treated with RANKL inhibitor (denosumab; Amgen, Thousand Oaks, CA) at a concentration of 5 μg/ml before the examination of osteogenic ability.

Alkaline phosphatase (ALP) staining and quantification

The transfected hASCs were seeded at a density of 2 × 10⁵ cells/well in 24-well plates and cultured in PM or OM for seven days. ALP staining was performed following the instructions of BCIP/NBT staining kit (Beyotime, Shanghai, China). As for the quantification of ALP activities, cells were rinsed with phosphate buffer saline (PBS) and 1% Triton X-100 (Solarbio, Beijing, China) three times, then scraped into milli-Q water and underwent three freeze-thaw cycles. By applying the pierce BCA protein assay kit (Thermo Fisher Scientific), total protein was read at 562 nm and calculated with a bovine serum albumin standard curve according to the manufacturer’s protocol. Finally, ALP activity was tested at 520 nm using an alkaline phosphatase assay kit (Jiancheng, Nanjing, Jiangsu, China) and calculated after normalization to the total protein concentrations.

Mineralization assays

The extracellular mineralization level of hASCs were tested after a 14-day in vitro culture with PM or OM. After being fixed with 95% ethanol for 30 min, the cells were steeped in 1% alizarin red s (ARS) staining solution (pH 4.2; Sigma-Aldrich) at room temperature for 30 min. To quantify the degree of matrix calcification, ARS-stained plates were separately dissolved by 100 mM cetylpyridinium chloride (Sigma-Aldrich) for 1 h and then the absorbances were detected with an EnSpire multimode plate reader (PerkinElmer, Waltham, MA) at 562 nm. Finally, the ARS relative intensity was calculated after normalization to the total protein concentrations.

Quantitative real-time polymerase chain reaction (qRT-PCR)

The total RNA of hASCs was lysed in TRIzol (Invitrogen, Carlsbad, CA) and complementary DNA was synthesized using a reverse transcription system (Takara, Tokyo, Japan). The quantification of miR-137 and gene transcripts were respectively examined using the miScript SYBR Green PCR kit (Qiagen, Frankfurt, Germany) and FastStart universal SYBR green master (ROX) (Roche, Indianapolis, IN) on a 7500 real-time PCR detection system (Applied Biosystems, Foster City, CA). Correspondingly, the expression levels of miR-137 and mRNA were calculated relative to U6 small nuclear RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The 2^−ΔΔCt method was adopted to analyze the fold-changes. The sequences of the primers were as follows: U6 (forward, 5’-CTCGCTTCGGCAGCACA-3’; reverse, 5’-AACGCTTCACGAATTTGCGT-3’), GAPDH (forward, 5’-GGAGCGAGATCCCTCCAAAAT-3’; reverse, 5’-GGCTGTTGTCATACTTCTCATGG-3’), miR-137 (forward, 5’-TATTGCTTAAGAATACGCGTAG-3’; reverse, 5’-AACTCCAGCAGGACCATGTGAT-3’), LGR4 (forward, 5’-CTTTGTTTGCCATTTCCTA-3’; reverse, 5’-CTAGTGAGTTTAATAGCACTAA-3’), RANKL (forward, 5’-GCCAGTGGGAGATGTTAG-3’; reverse, 5’-TTAGCTGCAAGTTTTCCC-3’), OPG (forward, 5’-CATGAGGTTCCTGCACAGCTTC-3’; reverse, 5’-ACAGCCCAGTGACCATTCCTAGTTA-3’) and runt-related transcription factor 2 (RUNX2) (forward, 5’-TGGTTACTGTCATGGCGGGTA-3’; reverse, 5’-TCTCAGATCGTTGAACCTTGCTA-3’).

Western blotting

Total protein was lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma‐Aldrich) and 2% protease inhibitor cocktail (Roche). The protein concentration was determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific). Equal amount of protein sample (50 μg) was loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to the polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat dry milk (BioRuler, Danbury, CT) and then incubated overnight at 4 °C with primary antibodies (1:1000) against GAPDH (Cell Signaling Technology, Beverly, MA; 5174S), LGR4 (Abcam, Cambridge, UK; ab75501), RANKL (Santa Cruz Biotechnology, Dallas, Texas; sc-377079), OPG (Santa Cruz Biotechnology; sc-11383) and RUNX2 (Cell Signaling Technology; 12556). After being incubated with the secondary antibodies (1:10,000) of horseradish peroxidase‐conjugated goat anti‐rabbit (Abcam; ab6721) at room temperature for 1 h, the protein bands were visualized by the pierce ECL plus western blotting substrate (Thermo Fisher Scientific). The optical density of the bands was analyzed with the ImageJ software (National Institutes of Health, Bethesda, Maryland) and the relative expression of protein was measured using GAPDH as an internal control.

Dual-luciferase reporter assay

The alignments of the target region in LGR4 were predicted by PicTar prediction software. The 3′ untranslated region (3′ UTR) of LGR4 mRNA, including the possible binding sites to miR-137, was amplified by PCR and cloned into pEZX‐MT06 vector (GeneCopoeia, Guangzhou, China) to generate wild-type LGR4 (LGR4‐WT) luciferase reporter plasmids. The mutant-type reporter vectors of LGR4 (LGR4‐MT) were formed by using a site-directed mutagenesis kit (SBS Genetech, Beijing, China). As mentioned before (Fan et al., 2016, 2021; Ma et al., 2020), the luciferase assay was performed by the co-transfection of LGR4-WT or LGR4-MT plasmids, NC or miR-137 mimics and lipofectamine 3000 (Invitrogen). At 48 h after transfection, the luciferase activity was examined by the dual-luciferase reporter assay system (Promega, Madison, WI) and normalized to Renilla luciferase activity.

Statistical analysis

Data were shown as mean ± standard deviation (SD) of three individual experiments and analyzed with the SPSS Statistics 20.0 software (IBM, Armonk, NY). Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s test was used to compare the differences of two or multiple groups, respectively. A P value < 0.05 was considered as statistically significant.

Results

MiR-137 played a negative role in the osteogenic differentiation of hASCs in vitro

In order to detect the expression profile of key factors in our signaling network model during the osteogenic induction of hASCs, we detected the expression of miR-137, LGR4 and RANKL at different time points (0 d, 3 d, 7 d and 14 d). We observed that the expression of miR-137 and RANKL decreased along with the osteogenesis of hASCs while LGR4 increased (Figure 1). To confirm the role of miR-137 in osteogenisis, hASCs were transfected with lentivirus constructions for NC and for overexpressing or downregulating miR-137 in a transfection rate over 90% (^{Figure S1A}). The qRT-PCR analysis showed a more than 10-fold increase of miR-137 in the miR-137 overexpression group while a decrease of about 90% in the miR-137 knockdown group (^{Figure S1B}). After a 7-day culturing in PM or OM, the ALP activity assay of transfected hASCs displayed that miR-137 overexpression weakened the ALP activity, but the knockdown of miR-137 enhanced it (^{Figure S2 A} and ^B). Similarly, after a 14-day culturing in PM or OM, the ARS test of transfected hASCs manifested that miR-137 overexpression reduced the mineralization level of extracellular matrix whereas inhibition of miR-137 significantly promoted the formation of calcified nodules (^{Figure S2C} and ^D). These data authenticated that miR-137 negatively regulated the osteogenesis of hASCs in vitro.

Figure 1 -
The expression characteristics of key factors in our signaling network model during the osteogenic induction of hASCs. (A) qRT-PCR analyses (0 d, 3 d, 7 d and 14 d) of miR-137, LGR4 and RANKL relative mRNA expression during the osteogenic differentiation of hASCs. (B) Western blotting analyses (0 d, 3 d, 7 d and 14 d) of LGR4 and RANKL protein expression during the osteogenic differentiation of hASCs. All experiments were performed in triplicate. Data are presented as mean ± SD. *P<0.05, **P<0.01.

MiR-137 downregulated LGR4 while upregulated RANKL during osteogenic inhibition

By overexpressing or knocking down miR-137 in hASCs, we first investigated the effects of miR-137 on LGR4 expression. Both the qRT-PCR (3 d, 7 d and 14 d) and western blotting (7 d) results showed that LGR4 was suppressed by miR-137 overexpression but activated by miR-137 knockdown. Then we further identified the impacts of miR-137 on RANKL and found that miR-137 positively regulated the expression of RANKL at mRNA and protein levels. As critical osteogenesis-related genes, both OPG and RUNX2 presented opposite trends with the alterations of miR-137 (Figure 2), which was accordant with the negative effects of miR-137 on osteogenesis. These results suggested that miR-137 could suppress LGR4 but induce RANKL in osteogenic impairment.

Figure 2 -
MiR-137 downregulated LGR4 while upregulating RANKL during osteogenic inhibition. (A) qRT-PCR analyses (3 d, 7 d and 14 d) of LGR4, RANKL, OPG and RUNX2 relative mRNA expression with miR-137 overexpression or knockdown. (B) Western blotting analyses (7 d) of LGR4, RANKL, OPG and RUNX2 protein expression with miR-137 overexpression or knockdown. All experiments were performed in triplicate. Data are presented as mean ± SD. *P<0.05, **P<0.01.

MiR-137 directly targeted LGR4 in hASCs

To confirm whether LGR4 is a direct target of miR-137, we performed the dual-luciferase reporter assay. The assumed targeting sites of miR-137 in the 3′ UTR of LGR4 were predicted by PicTar software (Figure 3 A). As shown in Figure 3 B, the relative luciferase activity was significantly reduced by miR-137 mimics in the LGR4-WT group 48 h after transfection, whereas it was not affected in the LGR4-MT group. These findings support that miR-137 can target LGR4 and downregulate its expression in hASCs.

Figure 3 -
MiR-137 directly targeted LGR4 in hASCs. (A) PicTar software predicted the binding sites of miR-137 in the 3′ UTR of LGR4-WT (the red sections denoted the mutated bases in LGR4-MT). (B) Analyses of the relative luciferase activities in the LGR4-WT and LGR4-MT group 48 h after transfection. All experiments were performed in triplicate. Data are presented as mean ± SD. **P<0.01.

LGR4 knockdown attenuated osteogenesis by activating RANKL

To further clarify the involvement of LGR4 in the osteogenic regulation of miR-137, we transfected hASCs with NC and LGR4 knockdown lentiviruses and examined the osteogenic potential and RANKL expression. The ALP staining and quantification on 7 d showed that LGR4 knockdown reduced the osteogenic ability, suggesting that LGR4 was a positive regulator in the osteogenesis of hASCs (Figure 4 A and B). After confirming the effective silencing of LGR4, we observed that LGR4 knockdown prominently elevated the mRNA (3 d, 7 d and 14 d) and protein (7 d) levels of RANKL. Moreover, the downregulation of OPG and RUNX2 caused by LGR4 knockdown also confirmed the inhibitory effects of LGR4 knockdown on the osteogenic differentiation (Figure 4 C and D).

To verify whether LGR4 could regulate osteogenesis depending on RANKL, we applied denosumab (RANKL inhibitor) to the LGR4 knockdown group and examined the osteogenic capacity and expression of genes involved in our hypothetical signaling network (LGR4, RANKL, OPG and RUNX2). Both the ALP activity and expression of related genes showed that denosumab alleviated or even reversed the effects of LGR4 knockdown caused in hASCs (Figure 4). Taken together, the results showed that LGR4 knockdown impaired osteogenesis through RANKL stimulation and indicated the negative relationship between LGR4 and RANKL during the osteogenic regulation of miR-137.

Figure 4 -
LGR4 knockdown attenuated osteogenesis by activating RANKL. ALP staining (A) and quantification (B) of hASCs transfected with NC, LGR4 knockdown lentiviruses or simultaneously treated with denosumab (RANKL inhibitor) for 7 days. (C) qRT-PCR analyses (3 d, 7 d and 14 d) of LGR4, RANKL, OPG and RUNX2 relative mRNA expression with LGR4 knockdown or simultaneously treated with denosumab. (D) Western blotting analyses (7 d) of LGR4, RANKL, OPG and RUNX2 protein expression with LGR4 knockdown or simultaneously treated with denosumab. All experiments were performed in triplicate. Data are presented as mean ± SD. *P<0.05, **P<0.01.

RANKL knockdown promoted osteogenesis by inducing LGR4

To gain further insights into the crosstalk between LGR4 and RANKL, we knocked down RANKL and tested whether LGR4 and osteogenic ability were affected. Our results showed that RANKL knockdown led to enhanced osteogenic potential of hASCs, which was proved by the ALP activity assay on 7 d (Figure 5 A and B). After examining the expression at mRNA (3 d, 7 d and 14 d) and protein (7 d) levels, we found that knockdown of RANKL remarkably stimulated LGR4, OPG and RUNX2 (Figure 5 C and D). Therefore, we verified the negative role of RANKL in LGR4 and osteogenic differentiation of hASCs. Additionally, we further investigated whether RANKL knockdown had impacts on miR-137 and observed that the expression of miR-137 displayed a remarkable decline on 3 d, 7 d and 14 d (^{Figure S3}). These findings, coupled with the facts that miR-137 positively regulated RANKL, prompted that miR-137 and RANKL formed a positive feedback circuit. In the light of the above results, we thoroughly corroborated that miR-137-induced negative reciprocal action between LGR4 and RANKL could regulate the osteogenesis of hASCs.

Figure 5 -
RANKL knockdown promoted osteogenesis by inducing LGR4. ALP staining (A) and quantification (B) of hASCs transfected with NC or RANKL knockdown lentiviruses for 7 days. (C) qRT-PCR analyses (3 d, 7 d and 14 d) of RANKL, LGR4, OPG and RUNX2 relative mRNA expression with RANKL knockdown. (D) Western blotting analyses (7 d) of RANKL, LGR4, OPG and RUNX2 protein expression with RANKL knockdown. All experiments were performed in triplicate. Data are presented as mean ± SD. *P<0.05, **P<0.01.

Discussion

MiR-137 has been reported to participate in the progression of various cancers (Li et al., 2017; Liu et al., 2017; Qin et al., 2017; Luo et al., 2018; Zhang et al., 2018), development and maturation of neurons (Silber et al., 2008; Szulwach et al., 2010; Tarantino et al., 2010; Sun et al., 2011; Jiang et al., 2013) and bone metabolism (Zheng et al.,2019; Kong et al., 2020; Ma et al., 2020; Yu et al., 2020; Fan et al., 2021). But compared with the previous two research fields, miR-137-modulated osteogenic differentiation is still unclear. In our previous studies, we have shown that the signaling network composed of NOTCH1-HES1, LSD1 and BMP2-SMADs pathways contributed to the osteogenic control of hASCs by miR-137 (Ma et al., 2020; Fan et al.,2021). However, since the osteogenic differentiation is a complex biological process which involves numerous signaling molecules, it is necessary to fully understand the underlying mechanisms of miR-137 on osteogenesis in order to develop miRNA-targeted therapy for the treatment of bone diseases. In this work, we validated that LGR4 is a target of miR-137 in hASCs and the negative interplay of LGR4 and RANKL induced by miR-137 is engaged in the osteogenic differentiation of hASCs.

Firstly, we observed the decreased expression of miR-137 and RANKL but increased expression of LGR4 during the osteogenic culture of hASCs. The different expression profile of these genes suggested a potential contribution for the osteogenesis in hASCs. LGR4 is reported to affect the osteogenic differentiation of several types of mesenchymal stem cells. By impairing the Wnt/β-catenin pathway, inhibition of LGR4 suppresses the proliferation, migration, and odonto/osteogenic differentiation of stem cells from apical papillae (Zhou et al., 2017). Deletion of LGR4 decreases bone mass through inhibiting BMSCs differentiation to osteoblasts and delays fracture healing following by BMSCs transplantation therapy (Sun et al., 2019). In hASCs, LGR4 silencing inhibits the activity of ERK signaling and blocks the osteogenesis (Zhang et al., 2017). After revalidating the inhibitory effects of miR-137 on the osteogenic differentiation of hASCs in vitro, we found that miR-137 was a negative regulator of LGR4 as well. Importantly, the dual-luciferase reporter assay determined that miR-137 directly bound to the 3′ UTR of LGR4. So far, only two studies have reported the binding of LGR4 by miR-137 in prostate cancer cells (Zhang et al., 2020), U-2 and MC3T3 cells (Liu and Xu, 2018). Here in hASCs, we validated that LGR4 is also a target gene of miR-137 in hASCs. To clarify whether miR-137-regulated osteogenesis relied on the direct suppression of LGR4, we knocked down LGR4 and observed that both the ALP activity and osteogenic markers (OPG and RUNX2) expression were restrained while RANKL was induced. These findings demonstrated that miR-137 could attenuate osteogenesis of hASCs by directly targeting LGR4.

RANK-RANKL signaling is a canonical pathway in osteoclast proliferation and differentiation. Besides having impacts on osteoclastogenesis, RANKL is found to be involved in osteogenesis as well. RANKL promotes osteoblastic activity in human aortic smooth muscle cells by upregulating BMP2 release from human aortic endothelial cells (Davenport et al., 2016). Whereas in BMSCs, RANKL regulates bone homeostasis by inhibition of osteogenic differentiation and promotion of osteoclast differentiation (Chen et al., 2018b; Elango et al., 2019). By overexpressing or knocking down miR-137, we found that RANKL was positively regulated by miR-137. To further elucidate the role of RANKL in the osteogenesis of hASCs, we examined relative ALP activity and expression of OPG and RUNX2 to assess the osteogenic capability with RANKL knockdown. Our results showed that RANKL inhibition markedly increased ALP activity and osteogenesis-related genes expression, which were accordant with the above results that miR-137 knockdown promoted osteogenesis while downregulating RANKL. Consequently, RANKL served as a negative regulator in the osteogenic differentiation of hASCs.

Based on the above conclusions that LGR4 and RANKL separately had positive and negative effects on the osteogenesis of hASCs, we further investigated the relationship between these two proteins. Recently, as a novel RANKL receptor, LGR4 is found to compete with RANK to bind RANKL and activate the Gαq/GS3K-β signaling pathway, resulting in the blockage of osteoclastogenesis (Luo et al., 2016). Additionally, LGR4 is a transcriptional target of the canonical RANKL-nuclear factor of activated T cells 1 (NFATc1), showing that LGR4 acts as the feedback loop controlling RANKL activities (Renema et al., 2016). Nevertheless, LGR4 silencing can prevent PTH-induced vascular calcification without changing RANKL and OPG expression in VSMCs (Carrillo-López et al., 2021). So far, this is the first study to evaluate the crosstalk between LGR4 and RANKL during the osteogenesis of hASCs. We have observed marked expression of RANKL by LGR4 knockdown, and that the inhibition of RANKL relieved or even reversed the outcomes produced by LGR4 deficiency. Therefore, these results suggest that LGR4 knockdown attenuated the osteogenesis of hASCs by stimulating RANKL. Moreover, RANKL knockdown dramatically activated LGR4 and osteogenic differentiation, indicating that RANKL had negative effects on LGR4 and osteogenesis of hASCs. Interestingly, RANKL silencing led to declined expression of miR-137 and suggested that RANKL had positive feedback effects on miR-137, and that the downregulation of miR-137 induced by RANKL knockdown might further enhance LGR4 stimulation. The involvement of miR-137-RANKL positive feedforward loop could partly explain the negative interrelationship between LGR4 and RANKL, though the synergistic effects of LGR4 and RANKL were reported in osteoclastic lineage (Luo et al., 2016; Renema et al., 2016). Also, given the diversity of biological characteristics of different cell lines, we speculated other signals might participate in the mediation of LGR4-RANKL crosstalk and further investigations need to be conducted.

Taken together, our study revealed that miR-137 negatively regulated the osteogenic differentiation of hASCs by direct binding to LGR4 and established a molecular mechanism model elaborating the critical role of LGR4-RANKL negative interplay which was mediated by miR-137 (Figure 6). These findings provide important insights for the future of miRNA-based therapeutics in bone metabolism disorder.

Figure 6 -
A molecular mechanism model involving the miR-137-mediated negative crosstalk between LGR4 and RANKL during the osteogenic differentiation of hASCs. In brief, miR-137 directly targeted LGR4 while positively regulated RANKL. Remarkably, negative interplay existed between LGR4 and RANKL and could be synergistic reinforced by miR-137. The green solid lines indicated positive regulation and the red dotted lines indicated negative regulation.

Acknowledgements

This study was financially granted by grants from the National Natural Science Foundation of China (No. 81700937).

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Supplementary material

The following online material is available for this article:

Figure S1 - The efficiency and effects of lentiviral transfection.

Figure S2 - MiR-137 played a negative role in the osteogenic differentiation of hASCs in vitro.

Figure S3 - RANKL knockdown suppressed the expression of miR-137.

Edited by

Associate Editor:

Ricardo G. Correa

Publication Dates

• Publication in this collection
  19 Sept 2022
• Date of issue
  2022

History

• Received
  16 Oct 2021
• Accepted
  24 July 2022