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β-catenin signaling pathway
Journal of Pharmacological Sciences 145 (2021) 69e78
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MiR-23b-3p promotes postmenopausal osteoporosis by targeting MRC2 and regulating the Wnt/b-catenin signaling pathway Ran Li 1, Qing Ruan 1, Fei Yin, Kunchi Zhao* Department of Orthopedics, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2020 Received in revised form 26 October 2020 Accepted 9 November 2020 Available online 11 November 2020
Postmenopausal osteoporosis (PMOP) is one of the most common metabolic bone diseases in postmenopausal women. Increasing evidence has indicated that microRNAs (miRNAs) play vital regulatory roles during osteoporosis progression. This study aimed to investigate the potential function of miR-23b3p in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). PMOP was induced in mice by bilateral ovariectomy. X-ray absorptiometry was applied to detect BMD and BMC in PMOP mice. Luciferase reporter assay and RIP assay were utilized to investigate the relationship between miR-23b-3p and MRC2. We found the upregulation of miR-23b-3p in bone tissues of PMOP mice. Silencing of miR-23b-3p relieved PMOP in mice. Moreover, miR-23b-3p knockdown facilitated the osteogenic differentiation of hMSCs by increasing the expression of Runx2, OCN, Osterix and promoting ALP activity. Mechanistically, MRC2 is a downstream target gene of miR-23b-3p. MRC2 knockdown significantly rescued the promoting effect of lenti-miR-23b-3p inhibitor on osteogenic differentiation of hMSCs. Furthermore, miR-23b-3p targeted MRC2 to inhibit the Wnt/b-catenin pathway during the osteogenic differentiation of hMSCs. In summary, inhibition of miR-23b-3p alleviates PMOP by targeting MRC2 to inhibit the Wnt/b-catenin signaling, which may provide a novel molecular insight for osteoporosis therapy. © 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: miR-23b-3p MRC2 Wnt/b-catenin pathway Postmenopausal osteoporosis
1. Introduction Postmenopausal osteoporosis (PMOP) is a prevalent metabolic bone disease induced by ovarian failure and decreased estrogen.1e3 The abnormal bone loss and structural destruction increase the risk of bone fracture in postmenopausal women.4e6 Great efforts have been made to improve the treatment of PMOP.7,8 However, huge challenges including the side effects of drug and serious complications limit the effects of clinical therapy of PMOP. Low bone mass and degenerative bone tissues may increase the risk of fracture during the progression of PMOP.9,10 Nowadays, strategies for osteoporosis treatment including raloxifene, hormone replacement, calcium supplement mainly focus on repressing bone breakdown rather than stimulating the formation of new bone.11e13 Therefore,
* Corresponding author. China-Japan Union Hospital of Jilin University, No.126 Xiantai Street, Changchun, Jilin, China. E-mail address: [email protected] (K. Zhao). Peer review under responsibility of Japanese Pharmacological Society. 1 These authors contributed equally to this work.
to identify the underlying mechanism of new bone formation is needed to improve the clinical treatment of PMOP. Human mesenchymal stem cells (hMSCs) can be isolated from bone marrow and are potential to differentiate into multiple cell types.14e16 Under the appropriate stimuli, MSCs can undergo osteoblastic or adipocytic differentiation.17,18 Bone homeostasis depends on the balance between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs). Disruption of the normal homeostasis may lead to the decline of bone mass and induce the development of OP.19,20 hMSCs are characterized by abundant sources, strong differentiation potential and easy access, and the mechanisms underlying hMSC osteoblastic differentiation have become a hot topic in bone-related research.21 MicroRNAs (miRNAs) are small non-coding RNAs with approximately 22 nucleotides in length. Accumulating evidence has indicated the importance of miRNAs in bone-related diseases. For example, miR-27b-3p plays a protective role in rheumatoid arthritis by repressing the apoptosis of chondrocytes.22 MiR-26a ameliorates the development of steroid-induced femoral head necrosis by reducing the apoptosis of osteocytes and increasing the
https://doi.org/10.1016/j.jphs.2020.11.004 1347-8613/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Journal of Pharmacological Sciences 145 (2021) 69e78
proliferation of osteoblasts.23 MiR-210 positively regulates VEGF expression and promotes osteoblast differentiation in PMOP.24 Moreover, miRNAs are closely associated with the differentiation of stem cells. For instance, miR-181a-3p targets BMP10 to negatively regulate the osteogenic differentiation of bone marrowderived MSCs.25 MiR-146a suppresses bone regeneration and the osteogenesis of adipose-derived MSCs.26 In addition, a recent study demonstrated that serum miR-23b-3p may serve as a potential biomarker for osteoporosis in postmenopausal women.27 However, the specific function of miR-23b-3p in the pathogenesis of PMOP remains to be further investigated. The recent study focused on the role and underlying regulatory mechanism of miR-23b-3p in osteogenic differentiation of hMSCs, which might shed a new insight into PMOP development.
was used for relative quantification. Relative sequences of primers are shown in Supplementary Table. 2.5. Hematoxylin and eosin (H&E) staining The morphology of the tibia bone tissues was evaluated by H&E staining using a light microscope. Briefly, tissues were mixed with 10% formaldehyde, followed by dehydrating, paraffin embedding and sectioned at 4 mm. H&E staining was performed after rehydration following the protocols from Beijing Solarbio Science & Technology (China). Sections were differentiated in hydrochloric acid ethanol, rinsed in water, recovered in ammonia water, and then stained with eosin. 2.6. Alizarin Red S staining
2. Materials and methods Cells were fixed with 95% cold ethanol for 20 min and air dried. 40 mmol/L of Alizarin Red S (SigmaeAldrich, USA) was dissolved in dH2O. Then, samples were stained by Alizarin Red S solution at 25 C for 30 min. Next, 10% (w/v) cetylpyridinium chloride (Sigma, USA) was prepared and used for destaining. Finally, Alizarin Red S in samples was analyzed by measuring absorbance at 560 nm.
2.1. Blood collection The peripheral blood from PMOP patients (n ¼ 30) and healthy volunteers (n ¼ 30) were collected in ChinaeJapan Union Hospital of Jilin University (Jilin, China). The participants had no bedridden history within six months before the blood collection. The written informed consents have been signed by all participants. This study was approved by the Ethical Review Committee of ChinaeJapan Union Hospital of Jilin University (Jilin, China).
2.7. Alkaline phosphatase (ALP) activity An ALP Detection Kit (Nanjing Jiancheng Bioengineering, Nanjing, China) was applied to evaluate ALP activity according to provided protocols. Before detection, cells were frozen and thawed to 25 C four times. Then, cell lysates were combined with the ALP substrate at 37 C for 30 min in 96-well plates. Finally, ALP activity was assessed by measuring absorbance at 405 nm via a microplate reader (Biorad, USA).
2.2. Cell culture and osteogenic differentiation The hMSCs (HUXMA-01001) were obtained from Cyagen Biosciences (China) and cultured in oriCellTM human Mesenchymal Stem Cell growth media (HUXMA-90011, Cyagen Biosciences) containing 10% fetal bovine serum (FBS; Gibco, USA) at the density of 5 104 cells/cm2. Afterwards, hMSCs were cultured in the humidified atmosphere with 5% CO2 at 37 C. Cells were detached by 0.25% trypsineEDTA (Gibco, USA) every 3 days and then passaged. The 3rd passage cells were used for subsequent experiments. When cells reached 70% confluency, they were treated with 50 mmol/L of ascorbic acid, 100 nM of dexamethasone and 10 mmol/L of b-glycerophosphate (SigmaeAldrich, St. Louis, USA) for 20 days to induce osteogenic differentiation. The media was changed every 3 days.
2.8. Western blot analysis Cells were lysed using RIPA buffer (Beyotime, Haimen, China) containing phenylmethylsulfonyl fluoride. A bicinchoninic acid protein assay kit (Beyotime, Haimen, China) was used to measure protein concentration. Equal amounts of proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to the PVDF membranes (Millipore, USA). Then, 5% skim milk was utilized to block membranes for 2 h at 25 C. Next, the membranes were incubated at 4 C overnight with the primary antibodies as described below (Abcam, Shanghai, China): Runx2 (ab192256), Osterix (ab209484), Ocn (ab13420), MRC2 (ab70132), Wnt 1 (ab15251), b-catenin (ab16051), c-Myc (ab32072), Cyclin D1 (ab40754), Survivin (ab76424), and b-actin (ab179467). Subsequently, blots were cultured at 25 C for 2 h with secondary antibodies. Finally, blots were visualized using an Enhanced Chemiluminescence Substrate kit (Millipore, USA). The ImageJ software was used for densitometry analysis of the band intensity.
2.3. Cell infection Lentivirus containing miR-23b-3p mimics (lenti-miR-23b-3p mimics), miR-23b-3p inhibitor (lenti-miR-23b-3p inhibitor), shMRC2 (lenti-sh-MRC2) and empty lentivirus vectors (lenti-mock) were purchased from Hanbio Biotechnology (Shanghai, China). hMSCs were infected with the lentivirus vectors according to a previous study.28 2.4. RNA extraction and reverse-transcription quantitative PCR (RTqPCR)
2.9. Luciferase reporter assay
TRIzol (Invitrogen, USA) was applied for total RNA extraction according to the provided protocols. Then, isolated RNAs were reverse transcribed into cDNA using the PrimeScript® RT Master Mix reagent kit (TaKaRa, Japan). To quantify miR-23b-3p expression, a stem-loop real-time PCR miRNA kit (Ribobio, China) was used. U6 served as an internal control for miR-23b-3p. For mRNA evaluation, GAPDH acted as an internal control. RT-qPCR reactions were performed using a SYBR Premix Ex Taq kit (TOYOBO, Japan) with a 7500 Real-Time PCR System (ABI, USA). The 2DDCt method
The wild type MRC2 30 untranslated region (UTR) containing the complementary binding site of miR-23b-3p (or the mutant 30 UTR) was inserted to the pmirGLO vectors to construct pmirGLO-MRC2Wt (or pmirGLO-MRC2-Mut) vectors. These vectors were then transfected into hMSCs infected with lenti-miR-23b-3p mimics or lenti-mock. For detection of the Wnt/b-catenin pathway, TOP flash or control FOP flash vectors were respectively transfected into Day 20 fully differentiated hMSCs infected with lenti-mock or lentimiR-216b-5p mimics. A luciferase Reporter Assay System 70
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3.2. MiR-23b-3p inhibition facilitates hMSC osteogenic differentiation
(Promega) was utilized to detect the relative luciferase activities 48 h later.
Based on the above investigations, we aimed to probe the potential function of miR-23b-3p in the osteogenic differentiation of hMSCs. As presented in Fig. 2A, miR-23b-3p expression was gradually decreased during hMSC osteogenic differentiation. RT-qPCR analysis suggested that miR-23b-3p expression was reduced by infection of lenti-miR-23b-3p inhibitor in hMSCs (Fig. 2B). Next, osteogenesis in lenti-miR-23b-3p inhibitor-infected hMSCs was evaluated on day 0, day 10, and day 20 after the addition of osteogenic medium. According to the results of Alizarin Red S staining, lenti-miR-23b-3p inhibitor-infected cells showed more intense staining consistent with increased mineralization on day 10 and day 20 (Fig. 2C). Subsequently, we detected the levels of osteogenic markers (Runx2, Osterix and Ocn) in infected hMSCs. The results demonstrated that inhibition of miR-23b-3p induced the upregulated expression of Runx2, Osterix and Ocn on both day 10 and day 20 (Fig. 2DeF). As shown in Fig. 2G, the same tendency was found in the protein expression of Runx2, Osterix and Ocn in hMSCs infected with lenti-miR-23b-3p inhibitor. Furthermore, we observed that lenti-miR-23b-3p inhibitor significantly increased ALP activity in hMSCs on day 10 and day 20 (Fig. 2H).
2.10. RNA immunoprecipitation (RIP) assay RIP assay was performed using an EZ-Magna RIPA Kit (Millipore, USA). The hMSCs lysate was cultured with RIPA buffer containing magnetic beads conjugated with Ago2 antibodies (Abcam, Shanghai, China). IgG was used as internal interference. Then, the immunoprecipitated RNAs were isolated using Proteinase K and the purified RNAs were assessed by RT-qPCR. 2.11. Ovariectomized osteoporosis model A total of 16 female mice (18e22 g, 5 weeks) were purchased from Vital River Co. Ltd. (Beijing, China). All procedures were approved by the ethics committee of ChinaeJapan Union Hospital of Jilin University. Mice were housed in pathogen-free laboratory animal room at 26 C with a 12 h light/dark cycle (relative humidity 50e60%). Mice could adapt to the environment for a week, and then bilateral ovariectomy was conducted in the mice to induce PMOP. Mice were randomly divided into OVX group and sham group. Mice in OVX group were anaesthetized by injecting 5% chloral hydrate (400 mg/kg animal body weight) into abdominal cavity and extirpated both sides of the ovaries. Mice in sham group received the same operation without bilateral ovariectomy. Four weeks later, OVX mice were injected with lenti-mock or lenti-miR-23b-3p mimics via caudal vein on days 1e3 of the first and fourth week, respectively. Six weeks after the first injection, the left proximal femurs were used for the detection of bone mineral density (BMD), bone mineral content (BMC), thickness of trabeculae (Tb.Th), number of trabeculae (Tb.N), and separating degree of trabeculae (Tb.Sp). BMD and BMC were measured by dual-energy X-ray absorptiometry with a PIXImus II densitometer (Skyscan, Antwerp, Belgium). Tb.Th, Tb.N and Tb.Sp were measured by a microcomputed tomography (mCT) system (Skyscan).
3.3. MRC2 is a direct target of miR-23b-3p Subsequently, downstream targets of miR-23b-3p were searched. Eight potential target genes were screened using starBase tool,29 as shown by the Venn diagram in Fig. 3A. As presented by RT-qPCR, miR-23b-3p expression was increased in hMSCs infected with lenti-miR-23b-3p mimics (Fig. 3B). Then, we assessed the levels of 8 candidate mRNAs in infected hMSCs and discovered that, compared to other 7 targets, MRC2 expression exhibited the significant downregulation by lenti-miR-23b-3p mimics (Fig. 3C). Therefore, we aimed to perform subsequent experiments with MRC2. As revealed in Fig. 3D, expression of MRC2 was higher in OVX group, than in sham group of the mice. Fig. 3E indicated that the expression of MRC2 was gradually increased during the osteogenic differentiation of hMSCs. As indicated in Fig. 3F, the 30 UTR of MRC2 contains a site complementary to the seed region of miR23b-3p, which is predicted from the starBase tool. In addition, lenti-miR-23b-3p mimics significantly reduced the luciferase activity of pmirGLO-MRC2-Wt vectors, while no evident change was found on mutant reporters. RIP assay revealed that both miR-23b3p and MRC2 were enriched in Ago2-conjugated beads rather than in IgG-conjugated beads (Fig. 3G). Furthermore, the protein level of MRC2 was significantly reduced by miR-23b-3p upregulation during hMSC osteogenic differentiation (Fig. 3H).
2.12. Statistical analysis Data are expressed as the mean ± standard deviation and analyzed by SPSS (v16.0; SPSS, USA). Comparisons between different groups were performed via the unpaired Student's t test or ANOVA, with p < 0.05 as the significant threshold. 3. Results 3.1. MiR-23b-3p inhibition alleviates the development of PMOP in mice To figure out the potential effect of miR-23b-3p on PMOP, RTqPCR analysis was conducted to measure miR-23b-3p expression in the peripheral blood of healthy individuals and PMOP patients. As shown in Fig. 1A, miR-23b-3p was significantly upregulated in peripheral blood of PMOP patients. Next, we performed OVX experiments on mice. H&E staining was carried out to determine the pathological changes of tibia bone tissues in mice. As revealed in Fig. 1B, tibia bone tissues exerted loosely arranged bone trabeculae structure in OVX group compared with sham group. Moreover, miR-23b-3p was highly expressed in tibia bone tissues of OVX mice than in sham group (Fig. 1C). As presented in Fig. 1DeH, OVX mice exhibited lower BMD, BMC, Tb.Th and Tb.N, and higher Tb.Sp than sham mice. However, the injection of lenti-miR-23b-3p inhibitor rescued these effects.
3.4. MiR-23b-3p suppresses hMSC osteogenic differentiation by regulating MRC2 We next explored whether miR-23b-3p participates in hMSC osteogenic differentiation via regulating MRC2. First, MRC2 was knocked down in hMSCs by infection of lenti-sh-MRC2 (Fig. 4A). As illustrated by Alizarin Red S staining in Fig. 4B, MRC2 knockdown counteracted the promotive effects of miR-23b-3p inhibition on matrix mineralization. Compared with the lenti-miR-23b-3p inhibitor group, MRC2 knockdown significantly decreased the mRNA and protein levels of Runx2, Osterix and Ocn during hMSC osteogenic differentiation (Fig. 4CeF). In addition, inhibited miR-23b-3pmediated increase in ALP activity on both day 10 and day 20 were partially recovered by MRC2 knockdown (Fig. 4G). 71
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Fig. 1. MiR-23b-3p inhibition alleviates the development of PMOP. (A) The levels of miR-23b-3p in blood samples from PMOP patients (n ¼ 30) and healthy controls (n ¼ 30) were measured by RT-qPCR. (B) H&E staining (scale bar: 50 mm) was performed to reveal the pathological changes of tibia bone tissues in mice. (C) RT-qPCR analysis was conducted to evaluate the expression of miR-23b-3p in tibia bone tissues (n ¼ 8) with U6 as an internal control. (DeH) Bone mineral density (BMD), bone mineral content (BMC), thickness of trabeculae (Tb.Th), number of trabeculae (Tb.N) and separating degree of trabeculae (Tb.Sp) were detected in mice (n ¼ 8). *p < 0.05, **p < 0.01, ***p < 0.001.
3.5. MiR-23b-3p inhibits the Wnt/b-catenin pathway during osteogenic differentiation of hMSCs via targeting MRC2
4. Discussion MiRNAs are a class of highly conserved single chain small noncoding RNAs, which can combine with the 30 -untranslated region of target mRNA sequences.32 It has been reported that miRNAs exert crucial functions in the occurrence and development of many diseases, including PMOP.33e36 MiR-23b-3p has been found to participate in the progression of degenerative joint disease.37 Upregulated serum level of miR-23b-3p has been considered to associate with osteoporosis in postmenopausal women.27 Consistent with the previous study, we revealed that miR-23b-3p expression was upregulated in the peripheral blood of PMOP patients compared with healthy individuals. Moreover, our findings indicated that miR-23b-3p was highly expressed in PMOP mice bone tissues and miR-23b-3p inhibition alleviated PMOP development. MSCs osteogenic differentiation is closely correlated with several osteogenic factors, including Runx2, Osterix, OCN and
The Wnt/b-catenin singling pathway has been found to play crucial roles during osteogenic differentiation.30,31 We wondered if miR-23b-3p activates the Wnt/b-catenin signaling pathway during the osteogenic differentiation of hMSCs. As revealed in Fig. 5A, upregulated miR-23b-3p reduced the protein levels of Wnt1 and bcatenin during osteogenic differentiation, while upregulation of MRC2 rescued such effects. The TOP and FOP luciferase reporter assay further revealed that upregulation of MRC2 counteracted the suppressive effects of miR-23b-3p upregulation on Wnt/b-catenin pathway in hMSCs (Fig. 5B). Moreover, effects of miR-23b-3p overexpression on the downstream targets of the Wnt/b-catenin signaling pathway including c-MYC, Cyclin D1 and Survivin were detected. c-MYC, Cyclin D1 and Survivin protein levels were found to be downregulated by miR-23b-3p overexpression, and such effects were rescued by MRC2 upregulation (Fig. 5C).
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Fig. 2. Suppression of miR-23b-3p facilitates hMSC osteogenic differentiation. (A) The expression of miR-23b-3p during osteogenic differentiation at day 0, day 10 and day 20 was assessed by RT-qPCR with U6 as an internal control. n ¼ 4. (B) MiR-23b-3p expression was decreased by infection of lenti-miR-23b-3p inhibitor in hMSCs, as revealed by RTqPCR with U6 as an internal control. n ¼ 4. (C) Alizarin Red S staining (scale bar: 100 mm) was used to evaluate the effect of lenti-miR-23b-3p inhibitor on matrix mineralization. n ¼ 4. (DeF) RT-qPCR was carried out to show RUNX2, Osterix and OCN expression in different groups using GAPDH as an internal control. n ¼ 4. (G) The protein levels of RUNX2, Osterix and OCN were determined by Western blot analysis during hMSC osteogenic differentiation. n ¼ 4. (H) ALP activity was quantified. n ¼ 4. ***p < 0.001.
ALP.38,39 It has been reported that miR-130a and miR-27b enhance osteogenesis by upregulating the expression of Runx2 and Osterix as well as increasing the ALP activity.40 MiR-10b facilitates osteogenesis by increasing the levels of ALP and Runx2.41 In the present study, we discovered that inhibition of miR-23b-3p significantly
decreased the levels of Runx2, Osterix and Ocn during hMSC osteogenic differentiation. Additionally, recent studies have indicated that miRNAs play significant roles in the self-renewal and differentiation of stem cells by targeting specific genes. For example, miR-199a-3p promotes 73
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Fig. 3. MRC2 is a direct target of miR-23b-3p. (A) The potential downstream targets of miR-23b-3p were predicted by starBase and shown by Venn diagram (CLIP data: high stringency (3); Degradome data: high stringency (3); overlap of 5 predicted programs: microT; miRanda; PITA; PicTar; TargetScan). (B) The overexpression efficiency of miR-23b3p was confirmed by RT-qPCR with U6 as an internal control. n ¼ 4. (C) RT-qPCR analysis with GAPDH as an internal control was conducted to measure the levels of candidate mRNAs in lenti-miR-23b-3p mimics infected cells. n ¼ 4. (D) The expression of MRC2 in OVX mice (n ¼ 8) and in sham mice (n ¼ 8) was detected by RT-qPCR with GAPDH as an internal control. (E) MRC2 expression during hMSC osteogenic differentiation was measured at indicated time points by RT-qPCR with GAPDH as an internal control. n ¼ 4. (FeG) Luciferase reporter and RIP assays were carried out to verify the binding relationship between miR-23b-3p and MRC2. n ¼ 4. (H) The protein levels of MRC2 were assessed during the osteogenic differentiation of lenti-miR-23b-3p mimics-infected hMSCs. n ¼ 4. ***p < 0.001.
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Fig. 4. MiR-23b-3p suppresses hMSC osteogenic differentiation by regulating MRC2. (A) The expression of MRC2 in lenti-sh-MRC2 infected hMSCs was evaluated by RT-qPCR with GAPDH as an internal control. n ¼ 4. (B) Alizarin Red S staining (scale bar: 100 mm) was utilized to assess matrix mineralization. n ¼ 4. (CeE) RT-PCR with GAPDH as an internal control was carried out to detect the expression of RUNX2, Osterix and OCN during osteogenic differentiation. n ¼ 4. (F) RUNX2, Osterix and OCN protein levels were assessed via Western blot analysis. n ¼ 4. (G) ALP activity was detected in infected cells. n ¼ 4. *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig. 5. MiR-23b-3p inhibits the Wnt/b-catenin pathway by downregulation of MRC2 during osteogenic differentiation. (A) Protein levels of Wnt1 and b-catenin were measured at day 0, day 10 and day 20 in hMSCs via Western blot analysis. n ¼ 4. (B) Effects of lenti-miR-23b-3p mimics and lenti-MRC2 on luciferase activity of TOP and FOP vectors in hMSCs on Day 20. n ¼ 4. (C) Protein levels of c-MYC, Cyclin D1 and Survivin were measured on day 20 in hMSCs via Western blot analysis. n ¼ 4. ***p < 0.001.
advanced osteosarcoma.46 Vinik Y revealed that binding of galectin-8 to receptor complexes (uPAR and MRC2) contributes to the secretion of RANKL by galectin-8-treated osteoblasts, which highlights a potential regulation of bone mass by animal lectins.47 Our results demonstrated that knockdown of MRC2 significantly counteracted the promoting effect of lenti-miR-23b-3p inhibitor on hMSC osteogenic differentiation. Wnt protein was first discovered in 1982.48 It is an essential activator of the Wnt signaling and participates in different developmental mechanisms, including cell differentiation, cell migration and cell proliferation.49e51 In recent years, increasing studies have indicated the essential role of the Wnt/b-catenin signaling pathway during stem cell differentiation.52e54 According to previous studies,
the MSC adipogenic differentiation by modulating the KDM6A/ WNT signaling.42 MiR-217 facilitates osteogenic differentiation of BMSCs by regulating DKK1 in steroid-induced osteonecrosis.43 MiR-133 suppresses hMSC osteogenic differentiation by targeting SLC39A1 in estrogen deficiency-induced osteoporosis.44 In the present study, we identified that miR-23b-3p directly targets the 30 UTR of MRC2. MiR-23b-3p suppressed the mRNA expression of MRC2, and further inhibited its protein expression. MRC2 is a constitutively recycling endocytic receptor belonging to the mannose receptor family, which has been found to be associated with collagen metabolism in murine osteoblasts.45 Sturge J has demonstrated that antibody targeting the MRC2 prevents osteolysis and bone destruction in a syngeneic model of 76
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the Wnt/b-catenin pathway involves in osteogenesis and hBMSCs osteogenic differentiation. For example, miR-346 positively regulates the osteogenic differentiation of hMSCs by activating the Wnt/ b-catenin signaling.55 MiR-139-5p inhibits the Wnt/b-Catenin pathway to regulate BMSC osteogenesis.56 In the present study, we found that miR-23b-3p inactivates the Wnt/b-catenin pathway by targeting MRC2 during the osteogenic differentiation of hMSCs. In conclusion, our results illustrated that the upregulated miR23b-3p inhibits hMSC osteogenic differentiation by targeting MRC2 to suppress the Wnt/b-catenin pathway. Our findings may offer a novel insight for PMOP treatment.
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Advanced glycation end products inhibit the osteogenic differentiation potential of adipose-derived stem cells by modulating Wnt/b-catenin signalling pathway via DNA methylation. Cell Prolif. 2020;53(6), e12834. 32. Finnegan EF, Pasquinelli AE. MicroRNA biogenesis: regulating the regulators. Crit Rev Biochem Mol Biol. 2013;48(1):51e68. 33. Kebschull M, Papapanou PN. Mini but mighty: microRNAs in the pathobiology of periodontal disease. Periodontol 2000. 2015;69(1):201e220. 34. Sala V, Bergerone S, Gatti S, et al. MicroRNAs in myocardial ischemia: identifying new targets and tools for treating heart disease. New Front miR-Med Cell Mol Life Sci: CMLS. 2014;71(8):1439e1452. 35. Bellavia D, Salamanna F, Raimondi L, et al. Deregulated miRNAs in osteoporosis: effects in bone metastasis. Cell Mol Life Sci: CMLS. 2019;76(19): 3723e3744. 36. Yavropoulou MP, Anastasilakis AD, Makras P, Tsalikakis DG, Grammatiki M, Yovos JG. Expression of microRNAs that regulate bone turnover in the serum of postmenopausal women with low bone mass and vertebral fractures. Eur J Endocrinol. 2017;176(2):169e176. 37. Yang Q, Zhou Y, Cai P, et al. Downregulation of microRNA-23b-3p alleviates IL1b-induced injury in chondrogenic CHON-001 cells. Drug Des Dev Ther. 2019;13:2503e2512. 38. Chen D, Gong Y, Xu L, Zhou M, Li J, Song J. Bidirectional regulation of osteogenic differentiation by the FOXO subfamily of Forkhead transcription factors in mammalian MSCs. Cell Prolif. 2019;52(2), e12540. 39. Ariffin SH, Manogaran T, Abidin IZ, Wahab RM, Senafi S. A perspective on stem cells as biological systems that produce differentiated osteoblasts and odontoblasts. Curr Stem Cell Res Ther. 2017;12(3):247e259. 40. Seenprachawong K, Tawornsawutruk T, Nantasenamat C, Nuchnoi P, Hongeng S, Supokawej A. miR-130a and miR-27b enhance osteogenesis in human bone marrow mesenchymal stem cells via specific down-regulation of peroxisome proliferator-activated receptor g. Front Genet. 2018;9:543. 41. Li H, Fan J, Fan L, et al. MiRNA-10b reciprocally stimulates osteogenesis and inhibits adipogenesis partly through the TGF-b/SMAD2 signaling pathway. Aging Dis. 2018;9(6):1058e1073. 42. Shuai Y, Yang R, Mu R, Yu Y, Rong L, Jin L. MiR-199a-3p mediates the adipogenic differentiation of bone marrow-derived mesenchymal stem cells by regulating KDM6A/WNT signaling. Life Sci. 2019;220:84e91. 43. Dai Z, Jin Y, Zheng J, et al. MiR-217 promotes cell proliferation and osteogenic differentiation of BMSCs by targeting DKK1 in steroid-associated osteonecrosis. Biomed Pharmacother Biomed Pharmacother. 2019;109:1112e1119. 44. Lv H, Sun Y, Zhang Y. MiR-133 is involved in estrogen deficiency-induced osteoporosis through modulating osteogenic differentiation of mesenchymal
Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper. Acknowledgement We thank all participators for their help. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jphs.2020.11.004. References 1. Kanis JA, Cooper C, Rizzoli R, Reginster JY. European guidance for the diagnosis and management of osteoporosis in postmenopausal women. Osteoporos Int: J Establ. Res Coop between Eur Found Osteoporos Nat Osteoporos Found USA. 2019;30(1):3e44. 2. Lindsay R. Pathogenesis of postmenopausal osteoporosis. Bailliere Clin Rheumatol. 1993;7(3):499e513. 3. Pacifici R. Estrogen, cytokines, and pathogenesis of postmenopausal osteoporosis. J Bone Miner Res: Off J Am Soc Bone Min Res. 1996;11(8):1043e1051. 4. Bonaccorsi G, Messina C, Cervellati C, et al. Fracture risk assessment in postmenopausal women with diabetes: comparison between DeFRA and FRAX tools. Gynecol Endocrinol: Off J Int Soc Gynecol Endocrinol. 2018;34(5):404e408. 5. Diab DL, Watts NB. Postmenopausal osteoporosis. Curr Opin Endocrinol Diabetes Obes. 2013;20(6):501e509. 6. Andersen TL, Abdelgawad ME, Kristensen HB, et al. Understanding coupling between bone resorption and formation: are reversal cells the missing link? Am J Pathol. 2013;183(1):235e246. 7. Lorentzon M. Treating osteoporosis to prevent fractures: current concepts and future developments. J Intern Med. 2019;285(4):381e394. 8. Farquhar C, Marjoribanks J, Lethaby A, Suckling JA, Lamberts Q. Long term hormone therapy for perimenopausal and postmenopausal women. Cochrane Database Syst Rev. 2009;(2), Cd004143. 9. Black DM, Rosen CJ. Postmenopausal osteoporosis. N Engl J Med. 2016;374(21): 2096e2097. 10. McCarus DC. Fracture prevention in postmenopausal osteoporosis: a review of treatment options. Obstet Gynecol Surv. 2006;61(1):39e50. 11. Lewiecki EM. Emerging drugs for postmenopausal osteoporosis. Expet Opin Emerg Drugs. 2009;14(1):129e144. 12. Delmas PD. Treatment of postmenopausal osteoporosis. Lancet (London, England). 2002;359(9322):2018e2026. 13. Rizzoli R, Sigaud A, Azria M, Herrmann FR. Nasal salmon calcitonin blunts bone microstructure alterations in healthy postmenopausal women. Osteoporos Int: J Establ. Res Coop between Eur Found Osteoporos Nat Osteoporos Found USA. 2015;26(1):383e393. 14. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science (New York, NY). 1999;284(5411):143e147. 15. Okolicsanyi RK, Camilleri ET, Oikari LE, et al. Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extended in vitro expansion. PloS One. 2015;10(9), e0137255. 16. Barry F, Boynton RE, Liu B, Murphy JM. Chondrogenic differentiation of mesenchymal stem cells from bone marrow: differentiation-dependent gene expression of matrix components. Exp Cell Res. 2001;268(2):189e200. 77
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Journal of Pharmacological Sciences 145 (2021) 69e78 51. Zhang J, Wen X, Ren XY, et al. YPEL3 suppresses epithelial-mesenchymal transition and metastasis of nasopharyngeal carcinoma cells through the Wnt/b-catenin signaling pathway. J Exp Clin Canc Res: CR (Clim Res). 2016;35(1):109. 52. Huang J, Chen C, Liang C, et al. Dysregulation of the Wnt signaling pathway and synovial stem cell dysfunction in osteoarthritis development. Stem Cell Dev. 2020;29(7):401e413. 53. Oh SH, Kim HN, Park HJ, Shin JY, Lee PH. Mesenchymal stem cells increase hippocampal neurogenesis and neuronal differentiation by enhancing the Wnt signaling pathway in an alzheimer's disease model. Cell Transplant. 2015;24(6): 1097e1109. 54. Zheng DH, Wang XX, Ma D, Zhang LN, Qiao QF, Zhang J. Erythropoietin enhances osteogenic differentiation of human periodontal ligament stem cells via Wnt/b-catenin signaling pathway. Drug Des Dev Ther. 2019;13:2543e2552. 55. Wang Q, Cai J, Cai XH, Chen L. miR-346 regulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting the Wnt/bcatenin pathway. PloS One. 2013;8(9), e72266. 56. Long H, Sun B, Cheng L, et al. miR-139-5p represses BMSC osteogenesis via targeting Wnt/b-catenin signaling pathway. DNA Cell Biol. 2017;36(8): 715e724.
stem cells. Med Sci Mon Int Med J Exp Clin Res: Int Med J Exp Clin Res. 2015;21: 1527e1534. Dong Y, Yang L, Luo W, et al. Mannose receptor C type 2 mediates 1,25(OH)(2) D(3)/vitamin D receptor-regulated collagen metabolism through collagen type 5, alpha 2 chain and matrix metalloproteinase 13 in murine MC3T3-E1 cells. Mol Cell Endocrinol. 2019;483:74e86. Sturge J. Endo180 at the cutting edge of bone cancer treatment and beyond. J Pathol. 2016;238(4):485e488. Vinik Y, Shatz-Azoulay H, Vivanti A, et al. The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice. eLife. 2015;4, e05914. €hle U, Ingham PW. Three Wnt genes expressed in a wide variety Blader P, Stra of tissues during development of the zebrafish, Danio rerio: developmental and evolutionary perspectives. Dev Gene Evol. 1996;206(1):3e13. Duchartre Y, Kim YM, Kahn M. The Wnt signaling pathway in cancer. Crit Rev Oncol-Hematol. 2016;99:141e149. Shen X, Pan B, Zhou H, et al. Differentiation of mesenchymal stem cells into cardiomyocytes is regulated by miRNA-1-2 via WNT signaling pathway. J Biomed Sci. 2017;24(1):29.
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MiR-23b-3p promotes postmenopausal osteoporosis by targeting MRC2 and regulating the Wnt/b-catenin signaling pathway Ran Li 1, Qing Ruan 1, Fei Yin, Kunchi Zhao* Department of Orthopedics, China-Japan Union Hospital of Jilin University, Changchun 130033, Jilin, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 September 2020 Received in revised form 26 October 2020 Accepted 9 November 2020 Available online 11 November 2020
Postmenopausal osteoporosis (PMOP) is one of the most common metabolic bone diseases in postmenopausal women. Increasing evidence has indicated that microRNAs (miRNAs) play vital regulatory roles during osteoporosis progression. This study aimed to investigate the potential function of miR-23b3p in the osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). PMOP was induced in mice by bilateral ovariectomy. X-ray absorptiometry was applied to detect BMD and BMC in PMOP mice. Luciferase reporter assay and RIP assay were utilized to investigate the relationship between miR-23b-3p and MRC2. We found the upregulation of miR-23b-3p in bone tissues of PMOP mice. Silencing of miR-23b-3p relieved PMOP in mice. Moreover, miR-23b-3p knockdown facilitated the osteogenic differentiation of hMSCs by increasing the expression of Runx2, OCN, Osterix and promoting ALP activity. Mechanistically, MRC2 is a downstream target gene of miR-23b-3p. MRC2 knockdown significantly rescued the promoting effect of lenti-miR-23b-3p inhibitor on osteogenic differentiation of hMSCs. Furthermore, miR-23b-3p targeted MRC2 to inhibit the Wnt/b-catenin pathway during the osteogenic differentiation of hMSCs. In summary, inhibition of miR-23b-3p alleviates PMOP by targeting MRC2 to inhibit the Wnt/b-catenin signaling, which may provide a novel molecular insight for osteoporosis therapy. © 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: miR-23b-3p MRC2 Wnt/b-catenin pathway Postmenopausal osteoporosis
1. Introduction Postmenopausal osteoporosis (PMOP) is a prevalent metabolic bone disease induced by ovarian failure and decreased estrogen.1e3 The abnormal bone loss and structural destruction increase the risk of bone fracture in postmenopausal women.4e6 Great efforts have been made to improve the treatment of PMOP.7,8 However, huge challenges including the side effects of drug and serious complications limit the effects of clinical therapy of PMOP. Low bone mass and degenerative bone tissues may increase the risk of fracture during the progression of PMOP.9,10 Nowadays, strategies for osteoporosis treatment including raloxifene, hormone replacement, calcium supplement mainly focus on repressing bone breakdown rather than stimulating the formation of new bone.11e13 Therefore,
* Corresponding author. China-Japan Union Hospital of Jilin University, No.126 Xiantai Street, Changchun, Jilin, China. E-mail address: [email protected] (K. Zhao). Peer review under responsibility of Japanese Pharmacological Society. 1 These authors contributed equally to this work.
to identify the underlying mechanism of new bone formation is needed to improve the clinical treatment of PMOP. Human mesenchymal stem cells (hMSCs) can be isolated from bone marrow and are potential to differentiate into multiple cell types.14e16 Under the appropriate stimuli, MSCs can undergo osteoblastic or adipocytic differentiation.17,18 Bone homeostasis depends on the balance between osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs). Disruption of the normal homeostasis may lead to the decline of bone mass and induce the development of OP.19,20 hMSCs are characterized by abundant sources, strong differentiation potential and easy access, and the mechanisms underlying hMSC osteoblastic differentiation have become a hot topic in bone-related research.21 MicroRNAs (miRNAs) are small non-coding RNAs with approximately 22 nucleotides in length. Accumulating evidence has indicated the importance of miRNAs in bone-related diseases. For example, miR-27b-3p plays a protective role in rheumatoid arthritis by repressing the apoptosis of chondrocytes.22 MiR-26a ameliorates the development of steroid-induced femoral head necrosis by reducing the apoptosis of osteocytes and increasing the
https://doi.org/10.1016/j.jphs.2020.11.004 1347-8613/© 2020 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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proliferation of osteoblasts.23 MiR-210 positively regulates VEGF expression and promotes osteoblast differentiation in PMOP.24 Moreover, miRNAs are closely associated with the differentiation of stem cells. For instance, miR-181a-3p targets BMP10 to negatively regulate the osteogenic differentiation of bone marrowderived MSCs.25 MiR-146a suppresses bone regeneration and the osteogenesis of adipose-derived MSCs.26 In addition, a recent study demonstrated that serum miR-23b-3p may serve as a potential biomarker for osteoporosis in postmenopausal women.27 However, the specific function of miR-23b-3p in the pathogenesis of PMOP remains to be further investigated. The recent study focused on the role and underlying regulatory mechanism of miR-23b-3p in osteogenic differentiation of hMSCs, which might shed a new insight into PMOP development.
was used for relative quantification. Relative sequences of primers are shown in Supplementary Table. 2.5. Hematoxylin and eosin (H&E) staining The morphology of the tibia bone tissues was evaluated by H&E staining using a light microscope. Briefly, tissues were mixed with 10% formaldehyde, followed by dehydrating, paraffin embedding and sectioned at 4 mm. H&E staining was performed after rehydration following the protocols from Beijing Solarbio Science & Technology (China). Sections were differentiated in hydrochloric acid ethanol, rinsed in water, recovered in ammonia water, and then stained with eosin. 2.6. Alizarin Red S staining
2. Materials and methods Cells were fixed with 95% cold ethanol for 20 min and air dried. 40 mmol/L of Alizarin Red S (SigmaeAldrich, USA) was dissolved in dH2O. Then, samples were stained by Alizarin Red S solution at 25 C for 30 min. Next, 10% (w/v) cetylpyridinium chloride (Sigma, USA) was prepared and used for destaining. Finally, Alizarin Red S in samples was analyzed by measuring absorbance at 560 nm.
2.1. Blood collection The peripheral blood from PMOP patients (n ¼ 30) and healthy volunteers (n ¼ 30) were collected in ChinaeJapan Union Hospital of Jilin University (Jilin, China). The participants had no bedridden history within six months before the blood collection. The written informed consents have been signed by all participants. This study was approved by the Ethical Review Committee of ChinaeJapan Union Hospital of Jilin University (Jilin, China).
2.7. Alkaline phosphatase (ALP) activity An ALP Detection Kit (Nanjing Jiancheng Bioengineering, Nanjing, China) was applied to evaluate ALP activity according to provided protocols. Before detection, cells were frozen and thawed to 25 C four times. Then, cell lysates were combined with the ALP substrate at 37 C for 30 min in 96-well plates. Finally, ALP activity was assessed by measuring absorbance at 405 nm via a microplate reader (Biorad, USA).
2.2. Cell culture and osteogenic differentiation The hMSCs (HUXMA-01001) were obtained from Cyagen Biosciences (China) and cultured in oriCellTM human Mesenchymal Stem Cell growth media (HUXMA-90011, Cyagen Biosciences) containing 10% fetal bovine serum (FBS; Gibco, USA) at the density of 5 104 cells/cm2. Afterwards, hMSCs were cultured in the humidified atmosphere with 5% CO2 at 37 C. Cells were detached by 0.25% trypsineEDTA (Gibco, USA) every 3 days and then passaged. The 3rd passage cells were used for subsequent experiments. When cells reached 70% confluency, they were treated with 50 mmol/L of ascorbic acid, 100 nM of dexamethasone and 10 mmol/L of b-glycerophosphate (SigmaeAldrich, St. Louis, USA) for 20 days to induce osteogenic differentiation. The media was changed every 3 days.
2.8. Western blot analysis Cells were lysed using RIPA buffer (Beyotime, Haimen, China) containing phenylmethylsulfonyl fluoride. A bicinchoninic acid protein assay kit (Beyotime, Haimen, China) was used to measure protein concentration. Equal amounts of proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to the PVDF membranes (Millipore, USA). Then, 5% skim milk was utilized to block membranes for 2 h at 25 C. Next, the membranes were incubated at 4 C overnight with the primary antibodies as described below (Abcam, Shanghai, China): Runx2 (ab192256), Osterix (ab209484), Ocn (ab13420), MRC2 (ab70132), Wnt 1 (ab15251), b-catenin (ab16051), c-Myc (ab32072), Cyclin D1 (ab40754), Survivin (ab76424), and b-actin (ab179467). Subsequently, blots were cultured at 25 C for 2 h with secondary antibodies. Finally, blots were visualized using an Enhanced Chemiluminescence Substrate kit (Millipore, USA). The ImageJ software was used for densitometry analysis of the band intensity.
2.3. Cell infection Lentivirus containing miR-23b-3p mimics (lenti-miR-23b-3p mimics), miR-23b-3p inhibitor (lenti-miR-23b-3p inhibitor), shMRC2 (lenti-sh-MRC2) and empty lentivirus vectors (lenti-mock) were purchased from Hanbio Biotechnology (Shanghai, China). hMSCs were infected with the lentivirus vectors according to a previous study.28 2.4. RNA extraction and reverse-transcription quantitative PCR (RTqPCR)
2.9. Luciferase reporter assay
TRIzol (Invitrogen, USA) was applied for total RNA extraction according to the provided protocols. Then, isolated RNAs were reverse transcribed into cDNA using the PrimeScript® RT Master Mix reagent kit (TaKaRa, Japan). To quantify miR-23b-3p expression, a stem-loop real-time PCR miRNA kit (Ribobio, China) was used. U6 served as an internal control for miR-23b-3p. For mRNA evaluation, GAPDH acted as an internal control. RT-qPCR reactions were performed using a SYBR Premix Ex Taq kit (TOYOBO, Japan) with a 7500 Real-Time PCR System (ABI, USA). The 2DDCt method
The wild type MRC2 30 untranslated region (UTR) containing the complementary binding site of miR-23b-3p (or the mutant 30 UTR) was inserted to the pmirGLO vectors to construct pmirGLO-MRC2Wt (or pmirGLO-MRC2-Mut) vectors. These vectors were then transfected into hMSCs infected with lenti-miR-23b-3p mimics or lenti-mock. For detection of the Wnt/b-catenin pathway, TOP flash or control FOP flash vectors were respectively transfected into Day 20 fully differentiated hMSCs infected with lenti-mock or lentimiR-216b-5p mimics. A luciferase Reporter Assay System 70
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3.2. MiR-23b-3p inhibition facilitates hMSC osteogenic differentiation
(Promega) was utilized to detect the relative luciferase activities 48 h later.
Based on the above investigations, we aimed to probe the potential function of miR-23b-3p in the osteogenic differentiation of hMSCs. As presented in Fig. 2A, miR-23b-3p expression was gradually decreased during hMSC osteogenic differentiation. RT-qPCR analysis suggested that miR-23b-3p expression was reduced by infection of lenti-miR-23b-3p inhibitor in hMSCs (Fig. 2B). Next, osteogenesis in lenti-miR-23b-3p inhibitor-infected hMSCs was evaluated on day 0, day 10, and day 20 after the addition of osteogenic medium. According to the results of Alizarin Red S staining, lenti-miR-23b-3p inhibitor-infected cells showed more intense staining consistent with increased mineralization on day 10 and day 20 (Fig. 2C). Subsequently, we detected the levels of osteogenic markers (Runx2, Osterix and Ocn) in infected hMSCs. The results demonstrated that inhibition of miR-23b-3p induced the upregulated expression of Runx2, Osterix and Ocn on both day 10 and day 20 (Fig. 2DeF). As shown in Fig. 2G, the same tendency was found in the protein expression of Runx2, Osterix and Ocn in hMSCs infected with lenti-miR-23b-3p inhibitor. Furthermore, we observed that lenti-miR-23b-3p inhibitor significantly increased ALP activity in hMSCs on day 10 and day 20 (Fig. 2H).
2.10. RNA immunoprecipitation (RIP) assay RIP assay was performed using an EZ-Magna RIPA Kit (Millipore, USA). The hMSCs lysate was cultured with RIPA buffer containing magnetic beads conjugated with Ago2 antibodies (Abcam, Shanghai, China). IgG was used as internal interference. Then, the immunoprecipitated RNAs were isolated using Proteinase K and the purified RNAs were assessed by RT-qPCR. 2.11. Ovariectomized osteoporosis model A total of 16 female mice (18e22 g, 5 weeks) were purchased from Vital River Co. Ltd. (Beijing, China). All procedures were approved by the ethics committee of ChinaeJapan Union Hospital of Jilin University. Mice were housed in pathogen-free laboratory animal room at 26 C with a 12 h light/dark cycle (relative humidity 50e60%). Mice could adapt to the environment for a week, and then bilateral ovariectomy was conducted in the mice to induce PMOP. Mice were randomly divided into OVX group and sham group. Mice in OVX group were anaesthetized by injecting 5% chloral hydrate (400 mg/kg animal body weight) into abdominal cavity and extirpated both sides of the ovaries. Mice in sham group received the same operation without bilateral ovariectomy. Four weeks later, OVX mice were injected with lenti-mock or lenti-miR-23b-3p mimics via caudal vein on days 1e3 of the first and fourth week, respectively. Six weeks after the first injection, the left proximal femurs were used for the detection of bone mineral density (BMD), bone mineral content (BMC), thickness of trabeculae (Tb.Th), number of trabeculae (Tb.N), and separating degree of trabeculae (Tb.Sp). BMD and BMC were measured by dual-energy X-ray absorptiometry with a PIXImus II densitometer (Skyscan, Antwerp, Belgium). Tb.Th, Tb.N and Tb.Sp were measured by a microcomputed tomography (mCT) system (Skyscan).
3.3. MRC2 is a direct target of miR-23b-3p Subsequently, downstream targets of miR-23b-3p were searched. Eight potential target genes were screened using starBase tool,29 as shown by the Venn diagram in Fig. 3A. As presented by RT-qPCR, miR-23b-3p expression was increased in hMSCs infected with lenti-miR-23b-3p mimics (Fig. 3B). Then, we assessed the levels of 8 candidate mRNAs in infected hMSCs and discovered that, compared to other 7 targets, MRC2 expression exhibited the significant downregulation by lenti-miR-23b-3p mimics (Fig. 3C). Therefore, we aimed to perform subsequent experiments with MRC2. As revealed in Fig. 3D, expression of MRC2 was higher in OVX group, than in sham group of the mice. Fig. 3E indicated that the expression of MRC2 was gradually increased during the osteogenic differentiation of hMSCs. As indicated in Fig. 3F, the 30 UTR of MRC2 contains a site complementary to the seed region of miR23b-3p, which is predicted from the starBase tool. In addition, lenti-miR-23b-3p mimics significantly reduced the luciferase activity of pmirGLO-MRC2-Wt vectors, while no evident change was found on mutant reporters. RIP assay revealed that both miR-23b3p and MRC2 were enriched in Ago2-conjugated beads rather than in IgG-conjugated beads (Fig. 3G). Furthermore, the protein level of MRC2 was significantly reduced by miR-23b-3p upregulation during hMSC osteogenic differentiation (Fig. 3H).
2.12. Statistical analysis Data are expressed as the mean ± standard deviation and analyzed by SPSS (v16.0; SPSS, USA). Comparisons between different groups were performed via the unpaired Student's t test or ANOVA, with p < 0.05 as the significant threshold. 3. Results 3.1. MiR-23b-3p inhibition alleviates the development of PMOP in mice To figure out the potential effect of miR-23b-3p on PMOP, RTqPCR analysis was conducted to measure miR-23b-3p expression in the peripheral blood of healthy individuals and PMOP patients. As shown in Fig. 1A, miR-23b-3p was significantly upregulated in peripheral blood of PMOP patients. Next, we performed OVX experiments on mice. H&E staining was carried out to determine the pathological changes of tibia bone tissues in mice. As revealed in Fig. 1B, tibia bone tissues exerted loosely arranged bone trabeculae structure in OVX group compared with sham group. Moreover, miR-23b-3p was highly expressed in tibia bone tissues of OVX mice than in sham group (Fig. 1C). As presented in Fig. 1DeH, OVX mice exhibited lower BMD, BMC, Tb.Th and Tb.N, and higher Tb.Sp than sham mice. However, the injection of lenti-miR-23b-3p inhibitor rescued these effects.
3.4. MiR-23b-3p suppresses hMSC osteogenic differentiation by regulating MRC2 We next explored whether miR-23b-3p participates in hMSC osteogenic differentiation via regulating MRC2. First, MRC2 was knocked down in hMSCs by infection of lenti-sh-MRC2 (Fig. 4A). As illustrated by Alizarin Red S staining in Fig. 4B, MRC2 knockdown counteracted the promotive effects of miR-23b-3p inhibition on matrix mineralization. Compared with the lenti-miR-23b-3p inhibitor group, MRC2 knockdown significantly decreased the mRNA and protein levels of Runx2, Osterix and Ocn during hMSC osteogenic differentiation (Fig. 4CeF). In addition, inhibited miR-23b-3pmediated increase in ALP activity on both day 10 and day 20 were partially recovered by MRC2 knockdown (Fig. 4G). 71
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Fig. 1. MiR-23b-3p inhibition alleviates the development of PMOP. (A) The levels of miR-23b-3p in blood samples from PMOP patients (n ¼ 30) and healthy controls (n ¼ 30) were measured by RT-qPCR. (B) H&E staining (scale bar: 50 mm) was performed to reveal the pathological changes of tibia bone tissues in mice. (C) RT-qPCR analysis was conducted to evaluate the expression of miR-23b-3p in tibia bone tissues (n ¼ 8) with U6 as an internal control. (DeH) Bone mineral density (BMD), bone mineral content (BMC), thickness of trabeculae (Tb.Th), number of trabeculae (Tb.N) and separating degree of trabeculae (Tb.Sp) were detected in mice (n ¼ 8). *p < 0.05, **p < 0.01, ***p < 0.001.
3.5. MiR-23b-3p inhibits the Wnt/b-catenin pathway during osteogenic differentiation of hMSCs via targeting MRC2
4. Discussion MiRNAs are a class of highly conserved single chain small noncoding RNAs, which can combine with the 30 -untranslated region of target mRNA sequences.32 It has been reported that miRNAs exert crucial functions in the occurrence and development of many diseases, including PMOP.33e36 MiR-23b-3p has been found to participate in the progression of degenerative joint disease.37 Upregulated serum level of miR-23b-3p has been considered to associate with osteoporosis in postmenopausal women.27 Consistent with the previous study, we revealed that miR-23b-3p expression was upregulated in the peripheral blood of PMOP patients compared with healthy individuals. Moreover, our findings indicated that miR-23b-3p was highly expressed in PMOP mice bone tissues and miR-23b-3p inhibition alleviated PMOP development. MSCs osteogenic differentiation is closely correlated with several osteogenic factors, including Runx2, Osterix, OCN and
The Wnt/b-catenin singling pathway has been found to play crucial roles during osteogenic differentiation.30,31 We wondered if miR-23b-3p activates the Wnt/b-catenin signaling pathway during the osteogenic differentiation of hMSCs. As revealed in Fig. 5A, upregulated miR-23b-3p reduced the protein levels of Wnt1 and bcatenin during osteogenic differentiation, while upregulation of MRC2 rescued such effects. The TOP and FOP luciferase reporter assay further revealed that upregulation of MRC2 counteracted the suppressive effects of miR-23b-3p upregulation on Wnt/b-catenin pathway in hMSCs (Fig. 5B). Moreover, effects of miR-23b-3p overexpression on the downstream targets of the Wnt/b-catenin signaling pathway including c-MYC, Cyclin D1 and Survivin were detected. c-MYC, Cyclin D1 and Survivin protein levels were found to be downregulated by miR-23b-3p overexpression, and such effects were rescued by MRC2 upregulation (Fig. 5C).
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Fig. 2. Suppression of miR-23b-3p facilitates hMSC osteogenic differentiation. (A) The expression of miR-23b-3p during osteogenic differentiation at day 0, day 10 and day 20 was assessed by RT-qPCR with U6 as an internal control. n ¼ 4. (B) MiR-23b-3p expression was decreased by infection of lenti-miR-23b-3p inhibitor in hMSCs, as revealed by RTqPCR with U6 as an internal control. n ¼ 4. (C) Alizarin Red S staining (scale bar: 100 mm) was used to evaluate the effect of lenti-miR-23b-3p inhibitor on matrix mineralization. n ¼ 4. (DeF) RT-qPCR was carried out to show RUNX2, Osterix and OCN expression in different groups using GAPDH as an internal control. n ¼ 4. (G) The protein levels of RUNX2, Osterix and OCN were determined by Western blot analysis during hMSC osteogenic differentiation. n ¼ 4. (H) ALP activity was quantified. n ¼ 4. ***p < 0.001.
ALP.38,39 It has been reported that miR-130a and miR-27b enhance osteogenesis by upregulating the expression of Runx2 and Osterix as well as increasing the ALP activity.40 MiR-10b facilitates osteogenesis by increasing the levels of ALP and Runx2.41 In the present study, we discovered that inhibition of miR-23b-3p significantly
decreased the levels of Runx2, Osterix and Ocn during hMSC osteogenic differentiation. Additionally, recent studies have indicated that miRNAs play significant roles in the self-renewal and differentiation of stem cells by targeting specific genes. For example, miR-199a-3p promotes 73
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Fig. 3. MRC2 is a direct target of miR-23b-3p. (A) The potential downstream targets of miR-23b-3p were predicted by starBase and shown by Venn diagram (CLIP data: high stringency (3); Degradome data: high stringency (3); overlap of 5 predicted programs: microT; miRanda; PITA; PicTar; TargetScan). (B) The overexpression efficiency of miR-23b3p was confirmed by RT-qPCR with U6 as an internal control. n ¼ 4. (C) RT-qPCR analysis with GAPDH as an internal control was conducted to measure the levels of candidate mRNAs in lenti-miR-23b-3p mimics infected cells. n ¼ 4. (D) The expression of MRC2 in OVX mice (n ¼ 8) and in sham mice (n ¼ 8) was detected by RT-qPCR with GAPDH as an internal control. (E) MRC2 expression during hMSC osteogenic differentiation was measured at indicated time points by RT-qPCR with GAPDH as an internal control. n ¼ 4. (FeG) Luciferase reporter and RIP assays were carried out to verify the binding relationship between miR-23b-3p and MRC2. n ¼ 4. (H) The protein levels of MRC2 were assessed during the osteogenic differentiation of lenti-miR-23b-3p mimics-infected hMSCs. n ¼ 4. ***p < 0.001.
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Fig. 4. MiR-23b-3p suppresses hMSC osteogenic differentiation by regulating MRC2. (A) The expression of MRC2 in lenti-sh-MRC2 infected hMSCs was evaluated by RT-qPCR with GAPDH as an internal control. n ¼ 4. (B) Alizarin Red S staining (scale bar: 100 mm) was utilized to assess matrix mineralization. n ¼ 4. (CeE) RT-PCR with GAPDH as an internal control was carried out to detect the expression of RUNX2, Osterix and OCN during osteogenic differentiation. n ¼ 4. (F) RUNX2, Osterix and OCN protein levels were assessed via Western blot analysis. n ¼ 4. (G) ALP activity was detected in infected cells. n ¼ 4. *p < 0.05, **p < 0.01, ***p < 0.001.
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Fig. 5. MiR-23b-3p inhibits the Wnt/b-catenin pathway by downregulation of MRC2 during osteogenic differentiation. (A) Protein levels of Wnt1 and b-catenin were measured at day 0, day 10 and day 20 in hMSCs via Western blot analysis. n ¼ 4. (B) Effects of lenti-miR-23b-3p mimics and lenti-MRC2 on luciferase activity of TOP and FOP vectors in hMSCs on Day 20. n ¼ 4. (C) Protein levels of c-MYC, Cyclin D1 and Survivin were measured on day 20 in hMSCs via Western blot analysis. n ¼ 4. ***p < 0.001.
advanced osteosarcoma.46 Vinik Y revealed that binding of galectin-8 to receptor complexes (uPAR and MRC2) contributes to the secretion of RANKL by galectin-8-treated osteoblasts, which highlights a potential regulation of bone mass by animal lectins.47 Our results demonstrated that knockdown of MRC2 significantly counteracted the promoting effect of lenti-miR-23b-3p inhibitor on hMSC osteogenic differentiation. Wnt protein was first discovered in 1982.48 It is an essential activator of the Wnt signaling and participates in different developmental mechanisms, including cell differentiation, cell migration and cell proliferation.49e51 In recent years, increasing studies have indicated the essential role of the Wnt/b-catenin signaling pathway during stem cell differentiation.52e54 According to previous studies,
the MSC adipogenic differentiation by modulating the KDM6A/ WNT signaling.42 MiR-217 facilitates osteogenic differentiation of BMSCs by regulating DKK1 in steroid-induced osteonecrosis.43 MiR-133 suppresses hMSC osteogenic differentiation by targeting SLC39A1 in estrogen deficiency-induced osteoporosis.44 In the present study, we identified that miR-23b-3p directly targets the 30 UTR of MRC2. MiR-23b-3p suppressed the mRNA expression of MRC2, and further inhibited its protein expression. MRC2 is a constitutively recycling endocytic receptor belonging to the mannose receptor family, which has been found to be associated with collagen metabolism in murine osteoblasts.45 Sturge J has demonstrated that antibody targeting the MRC2 prevents osteolysis and bone destruction in a syngeneic model of 76
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the Wnt/b-catenin pathway involves in osteogenesis and hBMSCs osteogenic differentiation. For example, miR-346 positively regulates the osteogenic differentiation of hMSCs by activating the Wnt/ b-catenin signaling.55 MiR-139-5p inhibits the Wnt/b-Catenin pathway to regulate BMSC osteogenesis.56 In the present study, we found that miR-23b-3p inactivates the Wnt/b-catenin pathway by targeting MRC2 during the osteogenic differentiation of hMSCs. In conclusion, our results illustrated that the upregulated miR23b-3p inhibits hMSC osteogenic differentiation by targeting MRC2 to suppress the Wnt/b-catenin pathway. Our findings may offer a novel insight for PMOP treatment.
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