miR-539 mediates osteoblast mineralization by regulating Distal-less genes 2 in MC3T3-E1 cell line

Jianguo Han; Li Su; Chunyang Zhang; Rongcai Jiang

doi:10.1515/biol-2017-0034

Article Open Access

miR-539 mediates osteoblast mineralization by regulating Distal-less genes 2 in MC3T3-E1 cell line

Jianguo Han , Li Su , Chunyang Zhang and Rongcai Jiang

Published/Copyright: October 23, 2017

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From the journal Open Life Sciences Volume 12 Issue 1

Abstract

microRNAs (miRNAs) play an important role in osteoblast differentiation. However, the mechanisms of miRNAs regulating osteoblast mineralization still needs to be further cleared. Distal-less genes 2 (Dlx2) plays an important role in osteoblast differentiation. We have found that miR-539 was significantly downregulated and Dlx2 was found to be inversely correlated with miR-539 in MC3T3-E1 cell line during osteoblast mineralization. The overexpression of miR-539 significantly decreased the expression level of Dlx2 and suppressed the osteogenic marker gene expression level, alkaline phosphatase activity and matrix mineralization. Our study showed that miR-539 was a negative regulator in osteoblast mineralization and that the targeting of Dlx2 gene partly contributes to this inhibitory effect exerted by miR-539.

Keywords: miR-539; Distal less genes 2; osteoblast mineralization

1 Introduction

MicroRNAs (miRNAs), endogenous noncoding 18- to 25-nucleotide RNAs, regulate gene expression posttranscriptionally [1]. They inhibit protein translation and promote the degradation of the target mRNAs by binding to the 3’ untranslated regions (UTR) of specific mRNAs [2]. miRNAs have been shown to be key factors involved in the control of almost every aspect of cell activity, including growth, differentiation, proliferation, metabolism, apoptosis, tumorigenesis, mobile genetic element stability and drug-induced tissue adaptation [3-5]. An expanding body of evidence has showed that the miRNAs control osteoblasts-mediated bone formation and osteoclasts related bone remodeling [6].

Some researchers have showed that microRNAs play important roles in the regulation of bone cellular processes such as development, differentiation, proliferation and apoptosis [7]. There are some reports about the functions of miR-539. miR-539 can be a factor for sensing cellular biotin levels through targeting holocarboxylase synthetase (HCS), can be a suppressor for O-GlcNAcase expression in the failing heart, can be a regulator for mitochondrial activity and apoptosis through targeting PHB2 and a novel regulator of migration and invasion in human thyroid cancer cells by targeting CARMA1 [8-11]. In our exploration, experiment with skull repair using Next Generation Sequencing (NGS) (unpublished data), we found that miR-539 may play a role in mineralization of osteoblast cells.

Matrix mineralization is an important stage in bone formation, which is a tightly regulated process. Bone-specific genes, such as Dlx2, are involved in the regulation of osteoblast mineralization. This study was carried out to detect the roles of miR-539 in osteoprecursor cell mineralization using MC3T3-E1 cell line that is induced with ascorbic acid and β-glycerophosphate.

2 Materials and methods

2.1 Cell culture

MC3T3-E1 cells were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and grown in a-minimum essential medium (a-MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 1% penicillin (100 U/ml, GIBCO), streptomycin (100 ug/ml streptomycin; GIBCO, USA), in a humidified atmosphere of 5% CO₂ and 95% air at 37°C.

2.2 Mineralization induced by β-glycerophosphate and ascorbic acid

MC3T3-E1 cells were divide into 2 groups: control group (a-MEM ) and mineralization group (a-MEM with 50 mg/L ascorbic acid and 10 mM β-glycerophosphate). The culture medium was changed every 3 days.

2.3 miRNA-539 construct

The amplified precursor miRNA-539 and scrambled control were amplified and cloned into pLenti6/V5 plasmid as described by Muthusamy et al. [9].

2.4 Lentivirus preparation and transfection

miR-539 lentivirus and scrambled control lentiviruses were prepared by Hanbio Biocompany (WUHAN, CHINA).

2.5 Measuring mRNA levels by real-time PCR

Total RNA was prepared from cells using the RNeasy Mini Kit (Qiagen, USA). Total RNA (1 μg) was reverse transcribed with Superscript II reverse transcriptase (Invitrogen, USA). One microliter of the resulting cDNAs was subjected to amplification in a total volume of 25 μl containing Real-time mix (Kaiji, China) and a pair of specific primers (0.2 μmol/l each). Primers for mouse miR-539, DLX2, U6 and β-actin were designed using the Primer Premier 5.0 software, and sequences were obtained from GenBank.

2.6 Western blot

Cell protein was extracted with RIPA buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined with the bicinchoninic acid (BCA) method. Cell lysate (40μg) was separated by SDS/PAGE (12%) at 30 mA for 2.5 h and then blotted onto a nitrocellulose membrane. The membrane was then incubated for 1h in blocking buffer (Tris-buffered saline containing 10% skimmed milk powder) at room temperature. Next, the membrane was incubated for 16 h at 4 °C with a rabbit anti-DLX2 polyclonal antibody (novus biologicals) and a mouse anti-GAPDH polyclonal antibody (Sigma, St. Louis, Mo.), followed by incubation with secondary antibodies for 1h at room temperature. After each antibody incubation, membranes were washed three times with Tris-buffered saline containing 0.05% tween20. Protein signals were detected by an electrochemiluminescence detection system (Pierce Biotechnology, Rockford, Ill., USA), in which membranes were exposed to the detection solution for 5 min.

2.7 Luciferase reporter Assay

The luciferase reporter assay containing 3’UTR of mouse Dlx2 was constructed by our lab as described by Qadir et al. [12]. MC3T3-E1 cells which transfected with lentiviral pre-miR-539 or scrambled control were transfected with the reporter plasmids at 19 days. Firefly luciferase activity of each sample was measured 48 h after reporter plasmids transfection and normalized to Renilla luciferase activity.

2.8 Alkaline phosphatase activity (Alp) and Matrix mineralization

ALP staining was used to determine osteogenic differentiation and Alizarin red S staining was used to observe matrix mineralization. MC3T3-E1 cells were transfected with miR-539 lentivirus and scrambled control lentiviruses and then cultured in mineralization induced medium for 21 days. ALP staining and Alizarin red S staining were performed as described by Qadir et al. [12].

Ethical approval

The conducted research is not related to either human or animals use

2.9 Statistical analysis

The optical density (OD) of bands produced by Western Blot and PCR experiments were obtained with a Gel-Doc system and analyzed with SigmaGel (Jandel Scientific, San Rafael, Calif., USA). All western blot data were normalized to β-actin and are presented as relative abundances. The threshold cycle (CT) value for target genes were normalized to those of β-actin reference genes and were calculated as ΔCT = CT_target −CT_β-actin. Relative mRNA concentrations for each target gene were expressed as F = 2^ΔCT. Data were presented as 1 in scrambled control groups and other data were expressed as mean ±SD (standard deviation). Statistical analysis was performed with an analysis of variance (ANOVA) model for intra-group comparisons and the Tukey HSD test for comparisons made between different groups. All statistical analyses were conducted with SPSS Version 10.0 (SPSS Inc., Chicago, Ill., USA), and only those results with p<0.05 were considered to be statistically significant.

3 Results

3.1 miRNA-539 was down-regulated during osteoblast differentiation and mineralization

To investigate the role of miR-539 in osteoblast mineralization, the expression of miR-539 was measured in control group and mineralization group by qRT-PCR at 3d, 5d, 7d, 14d and 21d. miR-539 level did not change in the control group (p>0.05). miR-539 level were significantly down regulated in the mineralization group at 14d and 21day compared with the control group and with the expression level at 3d, 5d and 7d (p>0.05) (Figure 1).

Figure 1

miR-539 RNA level in MC3T3-E1 cells which cultured with mineralization medium or not for3,5,7,14 and 21 days. Y-axis indicates ratio value of miR-539 RNA abundance to U6 RNA abundance. Gene mRNA abundance was got by real-time PCR. p < 0.05, vs. the same day in control group; #p < 0.05, vs. 3 days in mineralization group, n=6).

3.2 DLX2 was up-regulated during osteoblast differentiation and mineralization

To identify the role of DLX2 in osteoblast mineralization, mRNA and protein expressions of DLX2 were measured in the control group and the mineralization group by qRT-PCR and Western blot at 3d, 5d, 7d, 14d and 21d. mRNA and protein expressions of DLX2 were not changed in the control group. DLX2 mRNA and protein levels were significantly up-regulated in the mineralization group at 14d and 21day compared with the control group and with expression at 3d, 5d and 7d (Figure 2A and 2C). A negative correlation was found between the upregulated DLX2 protein and downregulated miR-539 (r= −0.607, P<0.05) (Figure 2D).

Figure 2

Dlx2 level In MC3T3-E1 cells which cultured with mineralization medium or not for3,5,7,14 and 21 days. A: Real-time PCR analysis of Dlx2 mRNA expression, GAPDH was used as control. Y-axis indicates ratio value of Dlx2 mRNA abundance to GAPDH mRNA abundance; B: Typical result of Dlx2 Western blot; C: semi-quantitative analysis indicated the protein changes in levels of Dlx2. Y-axis indicates ratio value of Dlx2 protein abundance to GAPDH protein abundance; D: correlation analysis of miR-539 RNA and Dlx2 mRNA. p < 0.05, vs. the same day In control group; #p < 0.05, vs. 3 days In mineralization group.

3.3 miR-539 overexpression inhibited the expression of DLX2 and osteoblast mineralization

The effects of miR-539 on DLX2 and cell osteoblast mineralization was determined by overexpressing experiment. MC3T3-E1 cells were transfected with lentiviral pre-miR-539 or scrambled control and cultured with mineralization medium (a-MEM with 50 mg/L ascorbic acid and 10mM β-glycerophosphate) for 21 days. Real-time PCR was used to evaluate the level of miR-539 and DLX2 mRNA and we found miR-539 was increased and DLX2 mRNA was decreased in lentiviral pre-miR 539 transfection cells (p<0.05) (Fig. 3A and 3B). Western blot was used to evaluate the level of DLX2 protein and we found that DLX2 protein was decreased in lentiviral pre-miR-539 transfection cells (p<0.05) (Fig. 3C and 3D). ALP staining and Alizarin Red S staining were used to evaluate mineralized matrix formation. We found miR-539 was over-expressed than the control and it can reduced the osteogenic differentiation and mineralized nodules of osteoblasts (Fig. 3E, 3F and 3G). These results indicated that overexpression of miR-539 inhibited the expression of DLX2 and the mineralization of osteoblasts.

Figure 3

Overexpression of a miR-539 inhibited the expression of Dlx2, reduced the osteogenic differentiation and mineralized nodules. A: Real-time PCR analysis of miR-539 RNA expression; B: Real-time PCR analysis of Dlx2 mRNA expression; C: Typical result of Western blot showed Dlx2; D:semi-quantitative analysis indicated the protein changes in levels of Dlx2; E:ALP activity; GF: The Alizarin red staining analysis of mineralized nodules (G: scrambled control; F: miR-539). (p < 0.05, vs. scrambled control, n=6).

3.4 Dlx2 may be a direct target of miR-539

Dlx2 was predicted as a potential target of miR-539 by MiRanda (http://www.microrna.org/microrna/searchMirnas.do).The 3′-UTR of DLX2 mRNA contained a complementary site for the seed region of miR-539 (Figure 4A). Compared to luciferase activity in scrambled control, the luciferase activity of the reporter was significantly suppressed in miR-539 overexpression cells (Figure 4B). This result indicated that miR-539 may suppress gene expression through miR-539 binding sequence at the 3′-UTR of DLX2.

Figure 4

Dlx2 may be a direct target of miR-539. (A: The putative miR-539 binding sequence in the 3′-UTR of Dlx2 mRNA. B: Suppressed luciferase activity of 3′-UTR of Dlx2 mRNA by miR-539 mimic. (p < 0.05, vs. scrambled control, n=6).

4 Discussion

miRNAs are key regulators of numerous cellular processes including osteoblast differentiation, proliferation, and mineralization [13]. This study showed that miR-539 played a role in osteoblast mineralization and exerted its roles through regulating the mineralization factor Dlx2.

Dlx are divergent homeobox genes of the Hox gene family, including Dlx2, Dlx3 and Dlx5, that play roles in the regulation of maxillofacial bone development [14-16]. Newborn gene knockout Dlx2-/-mice were found to die after birth with structural abnormalities observed in the bones originating from the first branchial arch maxillary process, bone dysplasia in long bones [17,18]. Bone morphogenetic protein-2 (BMP-2)-induced Dlx3 expression is regulated by p38/Smad5 signaling pathway in osteoblasts [16]. Dlx5 is a target gene of the BMP signaling pathway and acts as an important regulator of both osteogenesis and dorsoventral patterning of embryonic axis [15]. These observations indicated that Dlx may be crucial in regulating craniofacial bone development and differentiation.

Some osteogenic differentiation-related miRNAs, such as miR-26a, -133, -135, miR-141 and -200a have been reported to be involved in the differentiation using different cell models [19-21]. MiR-141 and -200a are involved in the osteogenic differentiation by targeting Dlx5 using MC3T3E1 cells [19]. miR-124 is a negative regulator in osteogenic differentiation and in vivo bone formation by targeting of Dlx5, Dlx3, and Dlx2 genes [12]. Regulating osteoblast mineralization through miRNAs may be an important mechanism of bone formation by targeting Dlx.

Dlx2 is a key molecular regulator in osteogenic differentiation [14,22-24]. There is a putative miR-539 binding sequence in the 3′-UTR of Dlx2 mRNA. In the present study, we observed that miR-539 was downregulated and Dlx2 was upregulated during osteoblast mineralization. Osteoblasts were transfected with the pre-miR-539 or scrambled control for 21 days. Overexpression miR-539 induced downregulation of Dlx2 mRNA protein and induced decrease of ALP activity, which is widely accepted as a potential osteoblast differentiation marker. Moreover, our results showed that Dlx2 acted as a target gene for miR-539 using a sensor luciferase reporter assay. Taken together, we demonstrate for the first time that miR-539 involved in pre-osteoblast differentiation by targeting Dlx2.

The mineralization of bone in vitro is a very complex process, including organization of organic and inorganic matter [25]. In the present study, mineralization in the culture of MC3T3-E1 bone cells induced by ascorbic acid and β-glycerophosphate was used as in vivo model and we found that miR-539 mediated MC3T3-E1 mineralization. Nanomaterials can affect MC3T3-E1 mineralization process by regulating the expression of miRNA [26]. This suggests that, the miR-539 should be a crucial mediator in the mineralization of MC3T3-E1 bone cells which are treated by ascorbic acid and β-glycerophosphate and roles of miR-539 in mineralization of bone in vitro without extraneous ascorbic acid and β-glycerophosphate need to be further defined.

In conclusion, we here provide evidence that miR-539 plays an important role in osteoblast mineralization by regulating DLX2. Therefore, this study also provides new insights into the roles and regulatory mechanisms of miRNAs in osteoblast mineralization.

Acknowledgments

This project was supported by the National Natural Science Foundation of China (No. 81360164) and the Natural Science Foundation of Inner Mongolia(No.2014MS0806).

Conflict of interest: Authors state no conflict of interest.

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Received: 2017-5-24

Accepted: 2017-8-8

Published Online: 2017-10-23

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