miRNA-429 suppresses osteogenic differentiation of human adipose-derived mesenchymal stem cells under oxidative stress via targeting SCD-1.

Role of microRNA-429 (miRNA-429) in osteogenic differentiation of hADMSCs was elucidated to explore the potential mechanism. Serum level of miRNA-429 in osteoporosis patients and controls was determined by quantitative real-time polymerase chain reaction (qRT-PCR). After H2O2 induction in hADMSCs, cell viability and reactive oxygen species (ROS) level were determined by cell-counting kit (CCK-8) assay and flow cytometry, respectively. Alkaline phosphatase (ALP) activity in H2O2-induced hADMSCs was also detected. The binding condition between miRNA-429 and SCD-1 was verified by dual-luciferase reporter gene assay. Relative levels of osteogenesis-related genes influenced by SCD-1 and miRNA-429 were detected by qRT-PCR. Furthermore, regulatory effects of SCD-1 and miRNA-429 on ALP activity and calcification ability of hADMSCs were evaluated. miRNA-429 was significantly upregulated in serum of osteoporosis patients. During the process of osteogenesis differentiation, H2O2 induction gradually upregulated miRNA-429 in hADMSCs. Overexpression of miRNA-429 markedly reduced ALP activity. Subsequent dual-luciferase reporter gene assay verified that miRNA-429 could bind to SCD-1 and negatively regulated its protein level in hADMSCs. SCD-1 was obviously downregulated in the osteogenesis differentiation of hADMSCs under oxidative stress. Moreover, silencing of SCD-1 suppressed expression of osteogenesis-related gene, ALP activity and calcification ability. Notably, SCD-1 knockdown partially reversed the regulatory effect of miRNA-429 on the osteogenic differentiation of hADMSCs. miRNA-429 suppresses the osteogenic differentiation of hADMSCs under oxidative stress via downregulating SCD-1. Copyright: © Lan et al.

Introduction

The main pathogenic factors of osteoporosis include weakened osteogenic potential and enhanced osteoclastogenesis potential. The imbalance in primary bone remodeling eventually leads to bone destruction. Aging is considered to be a major reason for bone quality decline in osteoporosis. As the age increases, bone resorption gradually exceeds bone formation, resulting in bone mass reduction and bone micro-structural damage. In addition, oxidative stress is responsible for osteoporosis and aging. It damages intracellular components and accelerates the process of osteoporosis. Previous studies have indicated that antioxidants are of potential value in the prevention and treatment of osteoporosis. Therefore, it is crucial to explore the underlying mechanism of oxidative stress in preventing osteoporosis.

Mesenchymal stem cells (MSCs) are non-hematopoietic adult stem cells derived from mesoderm. MSCs can be isolated from various tissues and organs, such as trabecular bones (1), periosteums (2), synovial membranes (3), fats, skeletal muscles (4), perivascular cells (5), peripheral blood (6) and umbilical cord (7,8). Adipose-derived mesenchymal stem cells (ADMSCs) have certain advantages compared to MSCs derived from other tissues. It has been shown that ADMSCs are non-immunogenic, non-carcinogenic and available (9). ADMSCs, including adipocytes, chondrocytes, myocytes and genital cells, exhibit multi-directional differentiation (10–13). In this study, ADMSCs were selected for in vitro experiments.

MicroRNAs (miRNAs) are a class of evolutionarily conserved, non-coding RNAs, serving as key regulators in various biological processes. There are over 1,800 protein-encoding miRNAs in the human genome, and each is predicted to regulate several target genes. It is reported that >50% of human protein-coding genes can be regulated by miRNAs (14). The important roles of miRNAs in bone formation, as well as osteoblast differentiation and function have been identified. For example, miR-34b and miR-34c affect osteoblast differentiation by directly targeting osteoblast-associated factors, such as RUNX2, Satb2, Notch1 and Notch2 (15,16). Overexpression of miR-375 decreases activities of RUNX2, ALP, OC and IBSP, thus inhibiting osteogenic differentiation (17). As a member of the miR-200 family, miRNA-429 is located on chromosome 4 (18). Functionally, miRNA-429 is involved in the pathogenesis of AD. However, the exact function of miRNA-429 in osteoporosis has not been fully elucidated.

Stearoyl-CoA desaturase 1 (SCD-1) has an important role in the biosynthesis of monounsaturated fatty acids. SCD-1 is the rate-limiting enzyme in adipogenesis, which is highly expressed in liver and adipose tissues (19,20). Studies have shown that overexpression of SCD-1 can promote osteogenic differentiation of MSCs (21).

In this study, the function of miRNA-429 in regulating osteogenic differentiation of ADMSCs was specifically explored. The present study might provide a novel direction for the treatment of osteoporosis.

Patients and methods

Research subjects

Osteoporosis patients (n=30) and healthy controls (n=30) were enrolled from December 2016 to October 2018 in The First Affiliated Hospital of Jinan University (Guangzhou, China). Five milliliters of venous blood was harvested from each subject and let stand for 30 min. Subsequently, blood samples were centrifuged at 2,500 × g at 4°C for 10 min. The supernatant was collected, followed by centrifugation at 4°C, 12,000 × g for 15 min. The supernatant off the serum sample was subpacked in Eppendorf (EP) tubes and preserved at −80°C for later use. This experimental study was approved by the Medical Ethics Committee of The First Affiliated Hospital of Jinan University. Signed informed consents were obtained from the patients or the guardians.

Cell culture

hADMSCs (PCS-500-011) were provided by American Type Culture Collection (ATCC). All cells were cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) (both Gibco; Thermo Fisher Scientific, Inc.), 1% L-glutamine and 1% penicillin-streptomycin. Culture medium was replaced every three days.

For osteogenic differentiation, hADMSCs were cultured in DMEM containing 10% FBS, 10 nmol/l dexamethasone, 10 mmol/l β-glycerophosphate, 50 µg/ml ascorbic acid, 1% L-glucose and 1% penicillin-streptomycin for 14 days.

Cell-counting kit 8 (CCK-8) assay

Cells were first seeded into 96-well plates. Absorbance (A) at 450 nm was recorded at appointed time points using the CCK-8 kit (Dojindo Laboratories) for depicting the viability curve.

Cell transfection

hADMSCs were transfected with miRNA-429 mimics, miRNA-429 inhibitor or SCD-1 siRNA according to the instructions of Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Forty-eight hours after transfection, the cells were collected for subsequent experiments.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA in hADMSCs was extracted by TRIzol method (Invitrogen; Thermo Fisher Scientific, Inc.). RNA purity was measured by ultraviolet spectrophotometry, and RNA samples were stored at −80°C until use. Subsequently, extracted RNA was reverse-transcribed to complementary deoxyribose nucleic acids (cDNAs), and SYBR-Green method (Thermo Fisher Scientific, Inc.) was used for PCR detection. Primer sequences used in this study were as follows: miRNA-429, forward, 5′-UAAUACUGUCUGGUAAAACCGU-3′ and reverse, 5′-CAAGAUCGGAUCUACGGGUUUU-3′; SCD-1, forward, 5′-GGATGCTCGTGCCAGTG-3′ and reverse, 5′-ACTCAGTGCCAGGTTAGAAG-3′.

Western blot analysis

Total protein in cells was first extracted using radioimmunoprecipitation assay (RIPA) (Beyotime). Target proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk for 2 h, the membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies for 2 h. Immuno-reactive bands were exposed by electrochemiluminescence (ECL) and analyzed by Image Software (National Institutes of Health).

Determination of alkaline phosphatase (ALP) activity

hADMSCs were first lysed with cell lysis buffer [50 mM Tris-HCI (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.02% NaN3, 1 µg/ml aprotinin, 100 µg/ml MSF] on ice and incubated for 5 min. Then, cell lysis was centrifuged at 4°C, 750 × g at 10 min. The supernatant was collected for ALP activity (Abcam) determination at 450 nm.

Determination of reactive oxygen species (ROS) production

ROS production was determined based on the methods proposed by Tang et al (22). Briefly, hADMSCs were seeded into 6-well plates with 2×105 cells per well. Twenty-four hours later, the cells were induced with H2O2 and 20 µM 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min in the dark. Subsequently, DCFH-DA was removed and the cells were digested for preparing cell suspension. ROS level was determined at 488 nm of excitation wavelength and 525 nm of emission wavelength.

Alizarin red staining

hADMSCs were cultured in osteogenic medium containing 10 mol/l dexamethasone, 10 ng/ml β-glycerophosphate and 50 µg/ml vitamin C. After 21 days of incubation, the cells were washed with phosphate-buffered saline (PBS) twice, fixed in 4% paraformaldehyde for 10 min and stained with 2% alizarin red staining (pH 4.1) for 15 min. Calcified nodules were observed and captured using an inverted microscope.

Dual-luciferase reporter gene assay

hADMSCs were co-transfected with wild-type/mutant-type SCD-1 and miRNA-429 mimics/NC using Lipofectamine 2000. After 24 h, the cells were harvested. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega Corporation).

Statistical analysis

Statistical Product and Service Solutions (SPSS) 18.0 (SPSS Inc.) was used for all statistical analysis. Data were expressed as mean ± SD (standard deviation). t-test was used for analyzing inter-group differences. Comparison between groups was done using One-way ANOVA test followed by Post Hoc Test (Least Significant Difference). P<0.05 indicated significant difference.

Results

miRNA-429 is upregulated in osteoporosis patients and activated under oxidative stress

Serum level of miRNA-429 was significantly higher in osteoporosis patients relative to healthy controls (Fig. 1A). hADMSCs were then subjected to H2O2 induction at 0, 50, 100 and 200 µM for 24 h. CCK-8 assay revealed that cell viability only decreased by the induction of 200 µM H2O2 (Fig. 1B). ROS production was subsequently detected by flow cytometry. After 100 and 200 µM H2O2 induction for 24 h, ROS level was remarkably elevated in a dose-dependent manner (Fig. 1C). This indicated that 100 µM H2O2 induction simulated oxidative stress in vitro. Furthermore, miRNA-429 level was gradually upregulated by induction of 100 and 200 µM H2O2 in a dose-dependent manner (Fig. 1D). Before H2O2 induction, hADMSCs were pretreated with 1 mM NAC (an antioxidant commonly applied for suppressing ROS production). The results showed that upregulated level of miRNA-429 due to H2O2 induction was markedly reversed by NAC treatment (Fig. 1E). The above data demonstrated that miRNA-429 was upregulated in osteoporosis patients, and could be increased by oxidative stress stimulation.

Figure 1.

miR-429 is upregulated in osteoporosis patients and activated under oxidative stress. (A) Relative level of miR-429 in serum samples of osteoporosis patients (n=30) and healthy controls (n=30) determined by qRT-PCR. (B) Cell viability in hADMSCs induced with 0, 50, 100 and 200 µM H2O2. (C) ROS level in hADMSCs induced with 0, 50, 100 and 200 µM H2O2. (D) Relative level of miR-429 in hADMSCs induced with 0, 50, 100 and 200 µM H2O2. (E) Relative level of miR-429 in hADMSCs with no treatment, NAC induction, 100 µM H2O2 induction and 100 µM H2O2 + NAC induction. *P<0.05.

Knockdown of miRNA-429 accelerates osteogenic differentiation of hADMSCs

To evaluate the potential influence of miRNA-429 on osteogenic differentiation, hADMSCs induced with 100 mM H2O2 were cultured in osteogenesis medium for 0, 3, 7 and 14 days, respectively. miRNA-429 level was markedly elevated in H2O2-induced hADMSCs cultured in osteogenesis medium relative to those without H2O2 induction. Under oxidative stress, miRNA-429 level decreased obviously with the prolongation of osteogenic differentiation (Fig. 2A). During the process of osteogenic differentiation, ALP activity was significantly reduced by H2O2 induction. Moreover, ALP activity gradually decreased at 7 and 14 days of osteogenic differentiation in a time-dependent manner (Fig. 2B). Subsequently, miRNA-429 and mimics were constructed and transfected into hADMSCs. Transfection efficacy was evaluated by qRT-PCR (Fig. 2C). Under oxidative stress, miRNA-429 overexpression reduced ALP activity. Conversely, miRNA-429 knockdown enhanced its activity (Fig. 2D).

Figure 2.

Knockdown of miR-429 accelerated osteogenic differentiation of hADMSCs. (A) Relative level of miR-429 in hADMSCs cultured in osteogenic medium with or without 100 µM H2O2 induction for 0, 3, 7 and 14 days. (B) Relative ALP activity in hADMSCs cultured in osteogenic medium with or without 100 µM H2O2 induction for 0, 3, 7 and 14 days. (C) Relative level of miR-429 in hADMSCs cultured in osteogenic medium with 100 µM H2O2 induction and transfected with miR-429 mimics or inhibitor. (D) Relative ALP activity in hADMSCs cultured in osteogenic medium with 100 µM H2O2 induction and transfected with miR-429 mimics or inhibitor.*P<0.05.

SCD-1 is the target gene of miRNA-429

TargetScan was used to predict the potential target of miRNA-429. Binding sequences were identified in miRNA-429 and SCD-1 3′UTR (Fig. 3A). Dual-luciferase reporter gene assay was conducted to verify the binding relationship between miRNA-429 and SCD-1. Relative luciferase activity remarkably decreased in cells co-transfected with miRNA-429 mimics and wild-type SCD-1 plasmid. However, no significant changes in luciferase activity were observed in mutant-type group (Fig. 3B). The mRNA level of SCD-1 in hADMSCs was not influenced by miRNA-429 (Fig. 3C). However, the protein level of SCD-1 was downregulated in hADMSCs after miRNA-429 overexpression, whereas upregulated after silencing of miRNA-429 (Fig. 3D).

Figure 3.

SCD-1 is the target gene of miR-429. (A) Binding sequences between miR-429 and SCD-1 predicted by TargetScan. (B) Relative luciferase activity in hADMSCs co-transfected with miR-429 mimics/NC and wild-type/mutant-type SCD-1. (C) Relative level of SCD-1 in hADMSCs transfected with miR-429 mimics or inhibitor. (D) Protein level of SCD-1 in hADMSCs transfected with miR-429 mimics or inhibitor. *P<0.05.

miRNA-429 mediated osteogenic differentiation of hADMSCs via SCD-1

SCD-1 was downregulated in H2O2-induced hADMSCs cultured in osteogenesis medium relative to those without H2O2 induction (Fig. 4A). Transfection of SCD-1 siRNA significantly downregulated the mRNA levels of OC, RUNX2 and ALP. Expression of the above genes was upregulated after transfection of miRNA-429 inhibitor. Notably, upregulated levels of OC, RUNX2 and ALP due to miRNA-429 knockdown were partially downregulated after silencing of of SCD-1 (Fig. 4B). In H2O2-induced hADMSCs cultured in osteogenesis medium, increased ALP activity caused by miRNA-429 knockdown was partially reversed by co-transfection of SCD-1 siRNA (Fig. 4C). Identically, pronounced calcification in hADMSCs transfected with miRNA-429 inhibitor was reversed by silencing of SCD-1 (Fig. 4D). It was concluded that miRNA-429 inhibited osteogenic differentiation via downregulating SCD-1.

Figure 4.

miR-429 mediated osteogenic differentiation of hADMSCs via SCD-1. (A) Relative level of SCD-1 in hADMSCs cultured in osteogenic medium with or without 100 µM H2O2 induction for 0, 3, 7 and 14 days. hADMSCs cultured in osteogenic medium with100 µM H2O2 induction were transfected with scramble, SCD-1 siRNA, miR-429 inhibitor or SCD-1 siRNA+miR-429 inhibitor. (B) Relative levels of OC, RUNX2 and ALP; (C) ALP activity; (D) Calcification ability. *P<0.05.

Discussion

miRNAs are non-coding RNAs approximately 22 nucleotides in length. They exert biological functions by disrupting the stable structure of mRNA or inhibiting the translation of target genes (23). Many miRNAs have been reported to be involved in the process of osteogenesis (24–28). miRNAs can effectively regulate the expression of relevant transcription factors by mediating mRNA activities, which affects various cellular physiological processes at all times. RUNX2, OSX and other homologous domain proteins greatly influence the differentiation and maturation of osteogenic precursor cells. Moreover, the interaction between miRNAs and transcription factors coordinates bone formation (29). Therefore, searching for osteogenesis-related miRNAs with high specificity contributes in developing therapeutic strategies of bone fracture, osteoporosis, osteoarthritis, bone defect repair and joint function reconstruction. These miRNAs can also serve as biological hallmarks for improving clinical outcomes of affected patients.

The crucial function of miRNA-429 in diseases has been identified (18,30–32). Nevertheless, its potential role in osteoporosis is rarely reported. In this study, we first revealed that miRNA-429 was upregulated in osteoporosis patients, indicating its possible role in the progression of osteoporosis.

Increased cellular oxidative stress induces low turnover of osteopenia (33). Bone mass gradually decreases with downregulated levels of antioxidant enzymes (34). Studies have shown that free radicals and ROS affect osteoblast growth and function (35,36). In this study, hADMSCs were subjected to H2O2 induction (0, 50, 100 and 200 µM) to induce intracellular ROS production, which simulated oxidative stress in vitro. Our results showed that miRNA-429 was upregulated in hADMSCs under oxidative stress. Overexpression of miRNA-429 markedly decreased ALP activity during the osteogenic differentiation. It was concluded that miRNA-429 inhibits osteogenic differentiation of hADMSCs under oxidative stress.

Furthermore, we investigated the specific mechanism of miRNA-429 in inhibiting osteogenic differentiation of hADMSCs. Through TargetScan and dual-luciferase reporter gene assay, SCD-1 was predicted and verified as a direct target of miRNA-429. SCD-1 level was negatively regulated by miRNA-429 in hADMSCs. Silence of SCD-1 suppressed osteogenesis-related gene expression, ALP activity and calcification ability. Previous studies have demonstrated that lipid modification of Wnt is required for activation of Wnt pathway. SCD-1 has been shown to participate in Wnt biosynthesis and processing as well (37,38). The present study found that miRNA-429 knockdown induced β-catenin expression and its nuclear translocation, which were blocked by silencing of SCD-1. The above results suggest that miRNA-429 could regulate β-catenin activation by targeting SCD-1, thus activating Wnt pathway to inhibit osteogenic differentiation.

In conclusion, miRNA-429 is upregulated in osteoporosis patients and can be induced under oxidative stress. Furthermore, miRNA-429 suppresses osteogenic differentiation of hADMSCs via downregulating SCD-1.