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Abstract. – OBJECTIVE: It was the aim of this study to investigate whether miR-339 may affect osteogenic differentiation of bone marrow-de-rived mesenchymal stem cells (BMSCs) by tar-geting DLX5, thereby alleviating osteoporosis.

MATERIALS AND METHODS: BMSCs were isolated from the bone marrow of mice. The ex-pression levels of miR-339 and DLX5 during the process of osteogenesis was detected by quan-titative Real Time-Polymerase Chain Reaction (qRT-PCR). Meanwhile, the expression of down-stream osteogenesis-associated proteins, such as runt-related transcription factor 2 (RUNX2) and osteopontin (OPN), were also detected after overexpression or inhibition of miR-339. The al-kaline phosphatase (ALP) activity was measured in cells by ALP activity assay kit. Alizarin red staining was performed to reveal the cell min-eralization ability. The luciferase reporter gene assay was used to identify the targeted pairings of miR-339 and DLX5 genes. In addition, the ex-pression of DLX5 was detected by Western blot analysis after overexpression or knockdown of miR-339. Rescue test was applied to evaluate whether miR-339 could affect the differentiation of BMSCs by inhibiting the expression of DLX5.

RESULTS: QRT-PCR showed that miR-339 ex-pression gradually decreased while the expres-sion of DLX5 increased during the induction cul-ture of BMSCs. After overexpression of miR-339 in BMSCs, the expression levels of ALP, RUNX2, and OPN were reduced. Besides, ALP activi-ty assay showed a decreased cell ALP activity. RUNX2 protein expression was also decreased. In addition, Alizarin red staining detected a sig-nificant increase in cell mineralization, where-as silencing miR-339 resulted in an opposite re-sult. These results indicated that miR-339 could regulate the osteogenic differentiation of BM-SCs. Subsequently, we predicted using bioinfor-matics software that miR-339 might target DLX5, and validated this hypothesis by luciferase re-porter assay. Finally, Western blot and ALP ac-tivity assay revealed that DLX5 could reverse the inhibitory effect of overexpression of miR-339 on osteogenic differentiation of BMSCs.

CONCLUSIONS: Down-regulation of miR-339 can promote osteogenic differentiation of BM-SCs by targeting DLX5, thereby relieving oste-oporosis.

Key Words:BMSCs, miR-339, DLX5, Osteoporosis.

Introduction

Osteoporosis is a type of systemic metabolic bone disease characterized by reduced bone mass and abnormal bone structure, leading to increa-sed bone fragility and prone to fracture1. Osteo-porosis and its resulting fractures, especially hip fractures, are one of the leading causes of disabi-lity and death clinically. The root cause of osteo-porosis is the imbalance between bone resorption and bone formation. The research on osteoclast differentiation has been extensive and deep, and several anti-osteoporosis drugs that inhibit bone resorption have been applied, such as bisphospho-nates. However, in the long-term application pro-cess, it has been found2 that osteoclast inhibitors may inhibit the process of bone remodeling, lea-ding to many adverse reactions such as jaw ne-crosis. Therefore, how to promote bone formation has received more attention in recent years.

Mesenchymal stem cells (MSCs) are stem cel-ls derived from the mesoderm and have multiple differentiation potentials. They exist in many tissues such as liver, skin, and placenta3. Among them, bone marrow-derived MSCs (BMSCs) are the most widely studied. BMSCs are precursor stem cells of osteoblasts and adipocytes, the pro-liferation, and differentiation of which play a key role in bone balance4. Studies have found that the imbalance of BMSCs is an important mechanism in the pathogenesis of osteoporosis. Therefore, it is one of the important therapeutic directions to

European Review for Medical and Pharmacological Sciences 2019; 23: 29-36

J. ZHOU, H. NIE, P. LIU, Z. WANG, B. YAO, L. YANG

Department of Orthopedic, East Campus of Sichuan Provincial People’s Hospital, Chengdu, China.

Corresponding Author: Ji Zhou, MM; e-mail: doctorzhouji@163.com

Down-regulation of miR-339 promotes differentiation of BMSCs and alleviates osteoporosis by targeting DLX5

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promote the differentiation of BMSCs and correct bone imbalance or alleviate osteoporosis.

MicroRNA (miRNA) is a small non-coding RNA with 21 to 25 nucleotides in length. By complementary binding to the 3’UTR of the tar-get gene mRNA, miRNA can degrade or prevent the translation of the target mRNA and thus par-ticipate in the regulation of gene expression du-ring cell differentiation, cell proliferation, and disease progression. Bioinformatics analysis also showed5,6 that each miRNA regulates hundreds of genes, which also reflects that miRNAs are invol-ved in almost every biological process. Many stu-dies5,7-9 have shown that miRNAs can participate in the development of a variety of diseases, such as tumors, inflammation, and neurodevelopment. Our experimental results showed that the expres-sion level of miR-339 gradually decreased during the differentiation of human BMSCs into osteo-blasts; however, its specific role and mechanism still remain elusive. In this study, target genes of miR-339 were predicted by online software such as Targetscan and miRanda, and DLX5 was found to be a potential target gene. Some studies10-12 have shown that DLX5 plays an important part in mammalian osteogenic differentiation. The-refore, it is hypothesized that miR-339 may exert its biological function through targeted inhibition of DLX5. This study discusses the role of miR-339 in promoting the differentiation of BMSCs and thus relieving osteoporosis through targeting DLX5 gene.

Materials and Methods

Cell Culture and TransfectionThe SPF mice were sacrificed, and their fe-

murs and iliac bones were taken with the bone marrow washed repeatedly. The bone marrow was collected in a 15 mL centrifuge tube. The cell suspension was filtered, seeded into a six-well plate at 2.5×106/mL, and cultured at 37°C in a 5% CO2 incubator. After about ten days of culture, cells were digested by trypsin and seeded at 5×105/mL as the Passage 1 BMSCs. Cells were passaged to 6-well plates and grown to a densi-ty of 60-80%. Transfection was performed using LipofectamineTM 2000 (Invitrogen, Carlsbad, CA, USA) according to standard protocols. After 24 to 48 hours of transfection, further operations were performed according to the purpose of the experiment. The mice were purchased from the Animal Model Center of Nanjing University. All

operations have been approved by the Sichuan Provincial People’s Hospital Animal Ethics Com-mittee. miR-NC, miR-339 mimics, inhibitor and overexpression plasmids were synthesized by Gene Pharma (Shanghai, China).

Osteogenic DifferentiationWhen BMSCs reached 70%-80% confluence,

the osteogenic induction mediumα-MEM contai-ning 10 μmol/L dexamethasone, 50 μmol/L VitC, 10 mmol/L β-sodium glycerophosphate was ad-ded and was changed every 3 days.

RNA Extraction and Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR)

BMSCs were collected and total RNA was iso-lated by TRIzol (Invitrogen, Carlsbad, CA, USA). After reverse transcription completed, the qRT-PCR was performed using SybrGreen (MedChem Express, Monmouth Junction, NJ, USA), with U6 used as an internal reference. Primers are listed below:

Mir-339 F: 5’-CTAGCTGAGAAGGGGCCA-CAGGC-3’, R: 5’-ACGCGTCACACTGCATCA-GAAGACC-3’;

DLX5 F: 5’-TTCCAAGCTCCGTTCCAGAC-3’, R: 5’-GAATCGGTAGCTGAAGACTCG-3’;

Alkaline phosphatase (ALP) F: 5 ‘-CA-TCGCCTACCAGCTCATG-3’, R: 5’-CTC-GTCACTCTCATACTCCACA-3’;

Runt-related transcription factor 2 (RUNX2) F: 5’-TGGTTACTGTCATGGCGGGTA-3’, R: 5’-TCTCAGATCGTTGAACCTTGCTA-3’;

Osteopontin (OPN) F: 5’-CAGTGATTT-GCTTTTGCCTCCT-3’R: 5’-CAGCATCTGG-GTATTTGTTGTAA-3’,

GAPDH (F: 5’-AGCCACATCGCTCAGA-CAC-3’, R: 5’-GCCCAATACGACCAAATCC-3’),

U6 (F: 5’-CTCGCTTCGGCAGCAGCACATA-TA-3’, R: 5’-AAATATGGAACGCTTCACGA-3’).

Western BlotThe total proteins of transfected cells were

extracted by radioimmunoprecipitation assay (RIPA) lysate (Beyotime, Shanghai, China). The total protein amount was determined by the bicin-choninic acid (BCA) method (Pierce, Rockford, IL, USA). 15 μL of protein was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After blocked in 4% bovine serum albumin (BSA) for 1 h, the mem-brane was incubated with the primary antibodies (diluted 1:1000) overnight at 4°C. Afterwards,

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the membrane was incubated with the correspon-ding secondary antibody for 1 h, and the protein bands were detected by gel imaging system using enhanced chemiluminescence (ECL; Thermo Fi-sher Scientific, Waltham, MA, USA).

Alkaline Phosphatase (ALP) Activity AssayAfter osteogenic induction for 7 days, cells

were fixed in 4% paraformaldehyde for 30 minu-tes, rinsed 3 times with phosphate buffered saline (PBS) and stained using the ALP activity assay kit. Generally, cells were incubated with the incu-bation liquid for 15 min at 37°C and then washed for 2 min. After further stained with the hema-toxylin for 5 min, the cells were observed and photographed by an optical microscope.

Alizarin Red (ARS) StainingAfter transfection, the cells were cultured for

24 hours; then, they were washed 2 times with PBS and fixed in 70% ethanol for 60 minutes. After that, the samples were covered with alizarin red staining solution and incubated at 37°C for 60 minutes in the dark and washed 5 times with PBS (pH 7.2) without calcium and magnesium. Then, the cells were observed and photographed by an optical microscope.

Luciferase Reporter Gene AssayThe PCR amplification product of the 3’UTR

fragment of DLX5 gene was cloned into the XbaI restriction site of pGL3 plasmid to construct a wild-type pGL3-WT-DLX5 reporter gene plas-mid. A mutant pGL3-MUT-DLX5 eukaryotic ex-pression vector was constructed by mutating the miR-339 binding site on the DLX5 3’ UTR ac-

cording to the GeneTailorTM Site-Directed Muta-genesis System kit instructions. Human BMSCs were transfected with the following four groups of plasmids: wild-type DLX5 plasmid and miR-NA mimic control, wild-type DLX5 plasmid and miR-339 mimic, mutant DLX5 plasmid and miR-NA mimic control, mutant DLX5 plasmid and miR-339 mimic. After 24 hours of transfection, cells were collected, and the luciferase activity of each group of cells was detected using the Du-al-Luciferase Reporter System.

Statistical AnalysisStatistical Product and Service Solutions

(SPSS) 22.0 statistical software (IBM, Armonk, NY, USA) was used for data analysis. Measured data were expressed as mean±standard deviation. A two-sample t-test was used for comparison be-tween groups. p<0.05 was considered statistically significant.

Results

Dynamic Expression of MiR-339 and DLX5 During Osteogenic Differentiation

qRT-PCR was used to observe the dynamic expression of miR-339 and DLX5 during the osteogenic differentiation of BMSCs at 1, 3, 5, and 7 days, respectively. Results indicated that miR-339 expression in cultured BMSCs gradually decreased over time (Figure 1A); meanwhile, the expression of DLX5 gradual-ly increased (Figure1B), which suggested that miR-339 and DLX5 were involved in osteogen-ic differentiation.

Figure 1. Dynamic expression of miR-339 and DLX5 during osteogenic differentiation. BMSCs were cultured in osteogenic medium for different days. qRT-PCR showed that the expression of A, miR-339 gradually decreased. B, DLX5 expression gradually increased.

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Knockdown of MiR-339 Promotes Osteogenic Differentiation of BMSCs

To further investigate the role of miR-339 in osteogenic differentiation, we overexpressed and knockdowned miR-339 in BMSCs before osteogenic differentiation. After osteogenic cul-ture for 7 days, the qRT-PCR analysis showed that ALP, RUNX2, OPN expression levels were decreased in the miR-339 overexpression group while the opposite result was observed in the miR-339 knockdown group (Figure 2A). We then examined the effect of miR-339 on ALP activi-ty. Results showed that the ALP activity in the miR-339 overexpressed cells was markedly de-creased, whereas significantly increased in cells of miR-339 silencing group (Figure 2B). Besides, Western blot analysis revealed a marked reduced

RUNX2 protein expression after overexpression of miR-339, while the result was corrected by si-lencing miR-339 (Figure 2C). In addition, after overexpression of miR-339, alizarin red staining detected a marked reduction of mineralization ability of cells, and vice versa (Figure 2D). These results demonstrated that silencing miR-339 could promote osteogenic differentiation of BMSCs

MiR-339 Can Target DLX5 and Regulate Its Expression

To verify the binding site between the miRNA and the target gene, we used Targetscan and mi-Randa online software to predict the target gene of miR-339 through bioinformatics analysis and predicted that the target gene of miR-339 might be DLX5. Based on the results of bioinformatics

Figure 2. Silencing miR-339 promotes osteogenic differentiation of BMSCs. A, After overexpression and knockdown of miR-339, BMSCs were cultured in osteogenic medium for 7 days. Expression levels of ALP, RUNX2, and OPN were detected by qRT-PCR. B, After overexpression and knockdown of miR-339, BMSCs were cultured in osteogenic medium for 7 days. The ALP activity assay kit was used to detect the ALP activity. C, After overexpression and knockdown of miR-339, BMSCs were cultured in osteogenic medium for 7 days. The expression of RUNX2 protein in the cells was detected by Western blot. D, After overexpression and knockdown of miR-339, BMSCs were cultured in osteogenic medium for 14 days. Alizarin red staining was applied to detect the osteogenic differentiation ability.

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analysis, we used the luciferase reporter gene to confirm the binding of miR-339 to DLX5. Re-sults showed that miR-339 overexpression sig-nificantly reduced the luciferase intensity of the wild-type reporter gene (Figure 3A, 3B). To test whether miR-339 could directly regulate DLX5 expression, we used qRT-PCR and Western blot to detect mRNA and protein expression of DLX5 after overexpression and silencing of miR-339, re-spectively. As shown in the figure, the target gene DLX5 mRNA and protein levels decreased after overexpression of miR-339, while knockdown of miR-339 elevated the mRNA and protein expres-sion of target genes (Figure 3C, 3D). These results suggested that miR-339 could target DLX5 and regulate its expression.

MiR-339 Regulates Osteogenic Differentiation of BMSCs Through DLX5

To further investigate whether miR-339 func-tioned through DLX5, we overexpressed DLX5 in miR-339 overexpressing groups, and then exam-ine the expression levels of osteogenesis-related genes, ALP activity, and RUNX2 protein expres-sion. The results showed that after overexpression of miR-339, the expression of ALP, RUNX2, and OPN were markedly decreased, and the cell ALP

activity was also significantly reduced. Howev-er, overexpression of DLX5 partially reversed the inhibitory effect of miR-339 (Figure 4A, 4B). Similarly, Western blot results also showed that overexpression of miR-339 suppressed RUNX2 protein expression, while simultaneous overex-pression of DLX5 increased that (Figure 4C). These results indicated that miR-339 could regu-late osteogenic differentiation of BMSCs through DLX5.

Discussion

Bone is a continuous dynamic equilibrium tis-sue, which mainly includes two kinds of cells, os-teoblasts, and osteoclasts. The former promotes bone formation and the latter promotes bone re-sorption. The two types of cells in the normal bone tissue are balanced. Once this balance is broken, various types of bone metabolic diseases are initiated3,13. Osteoporosis and other bone me-tabolism-related diseases have become an urgent problem in China. An effective therapeutic mea-sure must be found as soon as possible to treat these diseases. Enhancing bone regeneration is the key to the treatment of osteoporosis.

Figure 3. miR-339 targets DLX5. A, The predicted binding site of miR-339 and DLX5. B, The luciferase reporter assay was applied to detect the binding of miR-339 to DLX5. C, qRT-PCR was performed to detect that the expression of DLX5 after over-expression of miR-339 and knockdown of miR-339

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However, the role and mechanism of miR-339 in the differentiation process of bone mesenchymal stem cells remain elusive.

In this work, we first examined the expression levels of miR-339 and DLX5 in BMSCs cultured in vitro. It was found that miR-339 gradually de-creased with induction time. We speculated that miR-339 may play a key role in the differentia-tion of BMSCs. Therefore, we constructed miR-339 low-expressed human BMSCs and induced their differentiation in vitro. By ARS staining, detection of alkaline phosphatase activity, and expression of osteogenic differentiation mark-ers including Runx2 and OPON, we found that miR-339 indeed promoted osteogenic differen-tiation of human BMSCs. Subsequently, bioin-formatics were used to predict the downstream gene regulation of miR-339 and DLX5 was found as the target gene. DLX5, a member of the DLX

MiRNAs, a class of small endogenous non-cod-ing RNAs, have been shown to be widely involved in the regulation of gene expression during life activities. Some miRNAs can promote osteogen-ic differentiation of BMSCs, while others inhibit that14-16. Mir-31 regulates osteogenic differenti-ation by targeting Runx2 and Satb2 formation regulatory loops17. MiR-204 affects precursor by regulating Runx2 expression18. Besides, miR-205 regulates BMSC osteogenic differentiation by influencing SATB2/Runx2 and ERK/MAPK pathways19. MiR-339 is a recently investigated miRNA; however, its research has been main-ly focused on the field of oncology. Many stud-ies12,20-23 have reported that its abnormal expres-sion is related to the occurrence, metastasis, and prognosis of colorectal cancer, gastric cancer, etc. In ovarian tumor cell lines, miR-339-5p has also been shown to inhibit migration and invasion24.

Figure 4. miR-339 regulates osteogenic differentiation of BMSCs through DLX5. After overexpression of miR-339 and si-multaneous over-expression of DLX5, BMSCs were cultured in osteogenic medium for 7 days. A, qRT-PCR was performed to detectthe expression of osteogenic related genes ALP, RUNX2, OPN after DLX5 overexpression. B, The ALP activity assay kit was used to detect the ALP activity afterDLX5 overexpression. C, Western blot was used to detect the expression of RUNX2 after overexpression of DLX5.

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family, is located on human chromosome 7. The encoded protein can participate in the bone de-velopment and fracture healing by activating a series of promoters such as ALPL, SP7, and MYC25. We found by luciferase assay that DLX5 was indeed a direct target gene of miR-339. Also, mRNA and protein expression levels of DLX5 in human BMSCs after miR-339 overexpression were examined. As a result, it was confirmed that overexpression of miR-339 can significant-ly reduce mRNA and protein expression levels of DLX5, suggesting that miR-339 had a direct effect on DLX5. Finally, we used an experiment to investigate whether miR-339 can promote the differentiation of human BMSCs into osteoblasts and exerts biological effects by targeting DLX5. DLX5 was overexpressed into human BMSCs which was pre-treated with miR-339 overexpres-sion. DLX5 was found to reverse the decrease in ALP/RNUX2 expression as well as the reduction of ALP activity caused by miR-339 overexpres-sion during BMSC osteoblast differentiation. Therefore, we concluded that low expression of miR-339 promoted osteogenic differentiation of human BMSCs by targeting DLX5.

Conclusions

During the process of osteogenic differenti-ation of human BMSCs, we found that miR-339 can play an important part in the differentiation of human BMSCs, which provides a new clue and theoretical basis for the prevention and treatment of osteoporosis and bone metabolism-related dis-eases.

Conflict of InterestThe Authors declare that they have no conflict of interest.

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