Biochimie xxx (2014) 1e9

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Research paper

MicroRNA-125b regulates osteogenic differentiation of mesenchymal stem cells by targeting Cbfb in vitro Ke Huang a,1, Jingshu Fu a,1, Wei Zhou a, Wei Li a, Shiwu Dong b, Shengpeng Yu a, Zongkai Hu a, Huaqing Wang a, Zhao Xie a, * a National & Regional United Engineering Laboratory of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing, China b Department of Biomedical Materials Science, School of Biomedical Engineering, Third Military Medical University, Chongqing 400038, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 August 2013 Accepted 10 February 2014 Available online xxx

Differentiation of mesenchymal stem cells (MSCs) into a specific lineage is firmly and precisely regulated via crucial transcription factors and signaling cascades, but the accurate mechanisms still need to be revealed. MicroRNAs (miRNA) negativity regulates the target mRNA protein synthesis to regulate various kinds of biological processes. In the present study we investigate miRNAs mediated regulatory mechanisms of osteoblastic differentiation in C3H10T1/2 cells and we identified that the level of miR-125b expression was obviously decreased compared with undifferentiated ones during differentiation process. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-125b targets a putative binding site in the 30 -UTR of Cbfb gene, a key transcription factor for osteogenesis. We observed over and interferential expression of miR-125b down-regulate for Cbfb protein in C3H10T1/2 cells and the over-expression decrease the mRNA levels of three osteoblastic marker genes, alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OPN) by BMP-2-induced, whereas, anti-miR-125b increased the expression of these marker genes and hence up-regulated mRNA levels of Cbfb. It is concluded from the result that miR-125b is a key regulatory factor of osteoblastic differentiation by directly targeting Cbfb and indirectly acting on Runx2 at an early stage osteoblastic differentiation. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: MiR-125b C3H10T1/2 cell Cbfb Runx2

1. Introduction Bone marrow mesenchymal stem cells (MSCs) exist in the bone marrow and many mature cells [1], with a great potential of selfrenewal [2] and differentiation to give cell lineages including osteoblasts, chondrocytes, adipocytes [3]. Multiple levels of regulation and control mechanisms are involved in osteoblastic differentiation from the MSCs and some studies have proven that a variety of hormones and paracrine and/or autocrine cell factor participated in this process [4,5]. A cofactor Cbfb, is essential for transcription factor Runx2 in osteoblastic differentiation and could not directly attach with DNA due to their own seedless binding sites. But Cbfb could enhance the ability of Runx2 and DNA binding with DNA and promote osteogenesis in vitro [6,7], while the upstream regulation process remains to be fully elucidated. Thus, it can further deepen

* Corresponding author. Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Gaotanyan No. 30, 400038 Chongqing, China. Tel.: þ86 23 68765785. E-mail address: [email protected] (Z. Xie). 1 These authors contributed equal to this work.

our understanding of skeletal development and related disease of the molecular mechanisms via test upstream regulatory processes. MicroRNAs (miRNAs) are endogenous, non-coding and 21e24 nucleotide small RNAs, which are important regulators for gene expression and for protein expression on the post-transcription level. It can accurately regulate gene expression in real time through the translation inhibition and direct degradation of specific target genes of mRNA [8,9]. It has recently found that miRNAs could regulate some basic biological processes such as cell proliferation, differentiation, apoptosis and stress reaction [10,11]. Recent studies suggested that miRNAs promoted the directional differentiation of stem cells through down-regulated of certain genes to maintain stem cell in undifferentiated state and activate specific genes of stem cell lineage [12]. Many little miRNAs like miR-26a, miR-138 and miR-204 are involved in the differentiation of osteoblasts from the tissue stem cells and a strong research is a dire need for the screening of specificity of miRNAs, and analysis of the relationship between miRNAs and osteoblastic differentiation is less. MicroRNA-125b (miR-125b) is one of the early discovered miRNAs that plays a key role in the cellular functions of many cells especially in the control of proliferation and differentiation [13e15].

http://dx.doi.org/10.1016/j.biochi.2014.02.005 0300-9084/Ó 2014 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: K. Huang, et al., MicroRNA-125b regulates osteogenic differentiation of mesenchymal stem cells by targeting Cbfb in vitro, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.02.005

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K. Huang et al. / Biochimie xxx (2014) 1e9

Such as, miR-125b suppressed the tumor genes and regulated cell proliferation in human breast cancer, thyroid anaplastic cancer and nerve cell proliferation and differentiation [16e18]. Studies proved that miR-125b expression gradually decreased during BMP-4induced osteoblastic differentiation of ST2 cells regulate breast cancer and nerve cell proliferation and differentiation [19]. Lee and co-workers [15] have proved that miR-125b is an important factor for regulating stem cell directional differentiation by using gene knock-out technology and found a crucial role in regulating stem cell directional differentiation but the whole mechanism still needs to be defined. In our study, we evaluated that miR-125b expression gradually decreased via miRNA microarray technology testing analysis miRNA expression spectrum in osteoblastic differentiation of murine mesenchymal stem cells. Hence, let us have more confidence in the previous report. We explained that miR-125b plays a key role in inhibiting osteoblastic differentiation of muridae MSCs lines C3H10T1/2 cells. MiR-125b had a total of 562 potential target genes. To further affirm which function plays a key role in osteoblastic differentiation of MSCs, we demonstrated that miR-125b could act as the core members of the family with factor Cbfb through the dual-luciferase reporter gene detection, and confirmed Dong [20] and Iorio’s description [16]. Significantly, over-expression and interferential expression of miR-125b respectively inhibited and promoted osteoblastic differentiation by down-regulation and up-regulation mRNA level of Cbfb.

2.3. Osteoblastic differentiation assay Before osteoblastic differentiation, C3H10T1/2 cells readied for miRNA microarray technology analysis, were transfected with pre-miR-125b, anti-miR-125b and with their negative controls. MSCs made ready for miR-125b expression analysis were directly induced to osteoblastic differentiation. After transfection and incubation for 24 h, high density micromass cultures were treated as previously described [23]. The cells were trypsinized by 0.25% trypsin, modulated at a density of 107 cells/mL and placed into the center of each well on a 6-well plate (Shengyou Biotechnology). After incubation for 2 d at 37  C and 5% CO2, wells were mixed density for 100 ng/ml of BMP-2 (Shanghai PrimeGene). The BMP-2 was replaced every 2 d. 2.4. Alkaline phosphatase measurement

2. Materials and methods

The activity of alkaline phosphatase (ALP) was measured by using a commercially available Alkaline phosphatase assay kit (Nanjing Jiancheng Bioengineering Institute, China) [24]. Cells were washed twice and then lysed on ice for 30 min in lysis buffer composed of Mammalian Protein Extraction Reagent and Protease Inhibitor Cocktail (Cwbiotech, China). In the following step, lysates were collected and centrifuged at 14,000 rmp/min for 8 min, and the supernatants were transferred into a new tube for ALP activity detection. Supernatants were also analyzed for protein content using a BCA Assay kit (Beyotime, China), and ALP activity was normalized for total protein concentration. The absorbance at 490 nm and 570 nm, respectively for ALP and BCA analysis, were detected with Multifunctional Microplate Reader (Bio-rad, USA).

2.1. Cell culture

2.5. Alizarin red staining

Isolation of mouse MSCs from medulla ossium is done by the method previously described [21,22]. In brief, murine femur cavum ossis was rinsed by the culture medium under aseptic conditions, which consisted of low glucose DMEM (L-DMEM, Hyclone), 10% fetal bovine serum (FBS, Hyclone), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma) in humidified air containing 5% CO2, at 37  C. The culture medium was replaced every 3 d until the cells reached 90% confluence. Cells were passaged by 0.25% trypsin (Hyclone) for 2 min at room temperature. The fourth generation cells were used for the subsequent experiments. The C57BL/6 mice (six to eight weeks of age) were obtained from the Institute of Animal, the Third Military Medical University (Chongqing, China). The Southwest institutional Animal Care and Use Committee at the Third Military Medical University approved all animal protocols. Mouse embryonic mesenchymal stem cell lineage C3H10T1/2 cells and HEK293 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12, Hyclone) containing 10% fetal bovine serum (FBS, Hyclone), 100 units/ml penicillin and 100 mg/ml streptomycin (Sigma) in humidified air containing 5% CO2, at 37  C. The culture medium was replaced every 2e3 d.

The calcium (Ca) deposits were detected using Alizarin Red staining [25]. Cells were washed twice with phosphate-buffered saline (PBS) following fixation with paraformaldehyde for 30 min at 4  C and washed by double distilled water (ddH2O) for three times. Then, the fixed cells were incubated in 0.1% Alizarin redeTriseHCl (pH 8.3) staining solution at 37  C for 30 min. In last the cells were washed by with distilled water dried and mounted for further analysis.

2.2. Transfection assay To testify the functional relevance of miR-125b, pre-miR-125b (a final concentration of 50 nM), anti-miR-125b (a final concentration of 150 nM) or their negative controls were respectively transfected into C3H10T1/2 cells in 6-well plates (105 cell per well) with Lipofectamine 2000 transfection agent (Invitrogen) following the manufacturer’s instructions. After incubation for 24 h, the transfected cells were trypsinized and suffered to the osteoblastic differentiation assay. After indicated time points, the cells were reaped for mRNA and protein analysis.

2.6. Quantitative RT-PCR analysis To demonstrate the expression pattern of miR-125b, which appeared in the microarray results, qRT-PCR was performed. U6 served as an internal control. Gross RNA was used to generate cDNA by All-in OneÔ miRNA qRT-PCR Detection Kit (GeneCopoeia) according to manufacturer’s instructions. The RT-PCR primers are as follows e 50 -AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTVN30 . The primers of real-time PCR are as follows e U6 forward: 50 GCTTCGGCAGCACATATACTAAAAT-30 , U6 reverse primer used Universal adaptor primer; miR-125b forward: 50 -TCCCTGAGACCCTAACTTGTGA-30 , miR-125b reverse primer used Universal adaptor primer. To determine the expression levels of Cbfb, ALP, OCN, OPN, PPAR-g and Runx2, total RNA was performed RT-PCR using the Rever Tra Acea-First Strand cDNA Synthesis Kit (Toyobo) followed by real-time quantitative PCR with SYBR Green. GAPDH acted as an internal control. The cycle parameters for the RT reaction were 95  C for 5 min, 95  C 10 s, 72  C 20 s and 58  C for 15 s. Next, a reaction mixture (Promega) containing the SYBR Green and the appropriate primers was added to a 0.2 ml MicroAmp (ABI), together with 1 ml of cDNA template, for a final reaction volume of 20 ml. The amplification parameters were 94  C for 5 min, followed by 40 cycles of 94  C for 30 s, 57  C for 30 s, 72  C for 30 s. The primers of real-time PCR are as follows e GAPDH forward: 50 -GGAAGGTGAAGGTCGGAGT-30 , GAPDH reverse:

Please cite this article in press as: K. Huang, et al., MicroRNA-125b regulates osteogenic differentiation of mesenchymal stem cells by targeting Cbfb in vitro, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.02.005

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Table 1 Synthesized oligonucleotides sequences for generation of luciferase reporter constructs.

50 -CCTGGAAGATGGTGATGGG-30 ; ALP forward: 50 -GGACAGGACACACACACACA-30 , ALP reverse: 50 -CAAACAGGAGAGCCACTTCA-30 ; OCN forward: 50 -GGCTTAAAGACCGCCTACAG-30 , OCN reverse: 50 -GAGAGGACAGGGAGGATCAA-30 ; OPN forward: 50 -GATGATGATGACGATGGAGACC-30 , OPN reverse: 50 -CGACTATAGGGACGATTGGAC-30 ; Cbfb forward: 50 -TTCGTTAAGTGGAGCACAGC-30 , Cbfb reverse: 50 AGTGGGTGACGTCTTCACTG-30 ; Runx2 forward: 50 -CCACCTCTGACTTCTGCCTC-30 , Runx2 reverse: 50 -ATGAAATGCTTGGGAACTGC30 ; PPAR-g forward: 50 -GCTGTGGGGATGTCTCACAATGC-30 , PPAR-g reverse: 50 -CAGGGGGGTGATATGTTTGAA-30 . All real-time quantitative PCR reactions were performed in the PCR System 7500 (ABI). Data were analyzed using the 2-ddCt method. 2.7. Western-blot analysis Total cell extracts were harvested in lysis buffer containing 50 mM Tris-base, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS,1 mM PMSF and 0.2% Aprotinin (Sigma). In the next procedure we detected the protein concentration, the equal protein samples were mixed with 2 sample buffer (Beyotime) and boiled. Proteins were resolved by 10% SDS-PAGE gel and transferred on PVDF membrane (Millipore) by using the semi-dry transfer method. After blocking in 5% nonfat dried milk in TTBS for 2 h, the blots were incubated with anti-Cbfb (1:1000, Santa Cruz Biotech) or anti-bactin antibody (1:100, Santa Cruz Biotech) at 4  C overnight. b-actin acted as an internal control. After washing by TTBS, the blots were incubated with a horse-radish peroxidase-conjugated secondary antibody (1:10,000, Santa Cruz Biotech) at room temperature for 1 h. The ECL chemiluminescence kit was from Pierce (Rockford, IL, USA). 2.8. Generation of luciferase reporter constructs According to prediction binding site sequence of Cbfb 30 -UTR were accordingly synthesized miR-125b fully complementary sequence (Perfect Target, PT), the wild type binding site sequence (microRNA recognition element, MRE) and removing the seeds with the mutation forms combined with sequence (MRE e mutation, mut). To increase efficiency, we separately used pMIR-2 PT, pMIR-2 MRE, and pMIR-2 mut (Table 1). The positive-sense and antisense strand of synthesis 2 PT, 2 MRE and 2 mut were annealed into double chain. Application of classical DNA cloning method inserted pMIR-REPORT Firefly Luciferase reporter vector HindIII/SpeI enzyme site, and to find the correctness of the insertion sequence, we used DNA sequencing method for this purpose. 2.9. Dual-luciferase reporter gene analysis Using Lipofectamine 2000Ô (Invitrogen) we built three luciferase reporter gene carrier (pMIR-2 PT, pMIR-2 MRE, pMIR-2

mut) and NaCan plasmid phRL-TK (Ambion) according to the 5:1 transfecting into 293T cells. Meanwhile, we transfected miR-125b mimic, anti-miR-125b and negative control (Ambion). After incubation for 48 h, application of double luciferase reporter gene system (Promega) detected luciferase activity. Measurements of luminescence were performed on the luminometer (Glomax20/20, Promega). The independent experiment was performed for 3 times to take the averages, and analyzed the results. 2.10. Statistical analysis Data is expressed as the mean  SD. Statistical comparisons were made between two groups with the t-test and between multiple groups with one-way ANOVA. A value of p < 0.05 was considered significant unless otherwise described. 3. Results 3.1. MiR-125b is down regulated during BMP-2-induced murine MSCs osteoblastic differentiation In the present previous study, miRNA microarray technology was used to detect miRNAs expression patterns of three distinct phases during osteoblastic differentiation of murine mesenchymal stem cells C3H10T1/2 and osteoblastic induction at 7 d and 14 d after BMP-2 treatment (Fig. 1A). The expression of miR-125b was markedly decreased during osteoblastic differentiation. Subsequently, we accomplished bioinformatic analyses to predict the target genes of miR-125b as Pictar [26] and Targetscan [27]. Noticeably, we found the Cbfb was regulated by miR-125b and was the potential target gene. Because miRNA microarray technology merely preliminary screened. According to the main role of Cbfb in the process of MSCs differentiation into osteoblasts lineage, we hypothesized that Cbfb may be inhibited by miR-125b, which prevents MSCs differentiating into osteoblasts. Furthermore, down-regulation of miR-125b may play a positive effect in osteoblastic differentiation. Thus, tried to confirm the expression profile of miR-125b via qRT-PCR test in this study and the results validated that miR-125b gradually reduced in BMP-2-induced MSCs (Fig. 1B). These results are in accordance with finding of Yosuke Mizuno et al. [19]. 3.2. MiR-125b targets Cbfb by binding 30 -UTR of Cbfb mRNA MiRNAs suppress mRNA expression through binding at the MREs (MiRNA Recognition Elements) sites located in 30 -UTR of target mRNA [28]. The Cbfb 30 -UTR includes one hypothesis miR125b seed site which is bound with incomplete complementation (Table 2). We utilized the dual-luciferase reporter gene test to detect interaction of miR-125b and potential target sites. In start,

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Fig. 1. (A) Differential expression of miR-125b during osteoblastic differentiation of C3H10T1/2 cells. Down-regulated expression of miR-125b in C3H10T1/2 induced by BMP-2 treatment was detected by miRNA microarray at different stage of osteoblastic differentiation (0 d, 7 d, and 14 d). (B) Relative expression of endogenous miR-125b and measurement of cell proliferation during osteoblastic differentiation. After plating of C3H10T1/2 cells, BMP-2 was added at a final concentration of 100 ng/ml. Cells were harvested after 1, 3 and 5 days after BMP-2 addition, and endogenous expression level of miR-125b was measured. White bar and black bar indicate samples treated with or without BMP-2 (control), respectively. An asterisk indicates the t-test result is significant (p < 0.05).

according to the predicted binding site sequence of Cbfb 30 -UTR were respectively synthesized the negatively controlled of miR125b fully complementary sequence (Perfect Target, PT), the wild type binding site sequence of experimental group (microRNA recognition element, MRE) and removed the seeds with the mutation forms combined with sequence for mutation group (MRE e mutation, mut). To increase the efficiency, we respectively used of pMIR-2 PT, pMIR-2 MRE, and pMIR-2 mut. The increased sequence copy number carrier construction method has been effectively confirmed [29,30]. We found that luciferase activity significantly decreased in the 293T cells when transfected with pre-miR-125b, upon co-transfection the pMIR-2 PT into 293T cells with pre-miR-125b or miRNA negative control was achieved. The results suggested that pre-miR-125b could inhibit its expression with combining Cbfb 30 -UTR binding site, and confirmed that it was effective for dual-luciferase reporter gene experimental system. Then we co-transfected the pMIR-2 MRE into 293T cells with pre-miR-125b and found significant reduction in luciferase activity. But we found that luciferase activity had been transformed without any difference after co-transfection of the pMIR2 mut into 293T cells with pre-miR-125b (Fig. 2A). Furthermore, it was found that anti-miR-125b may prevent the inhibitory function and even increases luciferase activity after co-transfection the pMIR-2 MRE into 293T cells with pre-miR-125b and antimiR-125b (Fig. 2A). However, anti-miR-125b preventive affection was unobvious when we change the transfer vector to pMIR-2 mut (Fig. 2B). Taken together, the data suggested that miR-125b could target Cbfb by binding complementation within the Cbfb mRNA 30 -UTR. 3.3. MiR-125b inhibits Cbfb and Runx2 expression at an early stage of osteoblastic differentiation Cbfb and Runx2 were important transcription factors during MSCs differentiation into osteoblasts [7]. The diverse locus of complementary between miRNA and its target mRNA presumably determined that miRNA suppresses target mRNA through two unique pathways. MiRNA represses mRNA translation bearing

imperfect complementary target sequences and degrades mRNA bearing perfect complementary target sequences [31e33]. To demonstrate whether miR-125b regulated the expression of Cbfb, we transfected into C3H10T1/2 cells with pre-miR-125 for 1 d and 7 d time intervals but not at 14 d by BMP-2 treatment, qRT-PCR assay suggested that miR-125b showed over-expression, western-blot tests prompted that level of Cbfb protein has evidently reduced (Fig. 3A). These results indicated that miR-125b might act on Cbfb gene and inhibits its protein expression. Moreover, we transfected anti-miR-125b into C3H10T1/2 cells in order to decrease endogenous expression of miR-125b. qRT-PCR and western-blot assay showed that level of Cbfb protein (Fig. 3A) and mRNA (Fig. 3B) somewhat increased. To further explore the function of miR-125b in the process of osteoblastic differentiation of mesenchymal stem cells, we detected expression of the essential transcription factor Runx2. qRT-PCR and western-blot assay showed that miR-125b may be a negativity control expression of Runx2 (Fig. 3A, C). Consequently, these data illustrated that miR-125b could regulate the expression of Cbfb and Runx2 at early stage of osteoblastic differentiation. 3.4. MiR-125b inhibits early osteoblastic differentiation To explore whether miR-125b has an effect on osteoblastic differentiation, we transfected both pre-miR-125b and anti-miR-125b into C3H10T1/2 cells by BMP-2 induced. After induction of osteoblastic differentiation for 1 d, 7 d and 14 d, qRT-PCR analysis that transfects pre-miR-125b of C3H10T1/2 cells revealed a significant reduction in mRNA expression levels of osteoblastic marker genes including ALP, OCN and OPN during early stage. Using the same method detected a transcript of miR-125b interferential expression, and mRNA expression levels of osteoblastic marker genes evidently increased at early stage (Fig. 4AeC). But adipocyte marker genes containing PPAR-g un-obviously changed both transfected premiR-125b and anti-miR-125b (Fig. 4D). However, the same effect did not last for 14 d time period treatment. These data suggested that miR-125b plays a key negative regulator of early osteoblastic differentiation.

Table 2 The 50 end of miR-125b contains a sequence complementary to the specific miRNA binding site within the 30 -UTR of Cbfb mRNA.

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Fig. 2. MiR-125b directly targets Cbfb. (A) miR-125b mimic and its negative control were co-transfected with the specific pMIR-REPORT construct containing a consensus miR-125bbinding site (pMIR-2 PT) into 293T cells. MiR-125b mimic or its negative control was co-transfected with pMIR-2 MRE into 293T cells. pMIR-2 PT acts as a positive control. (B) Anti-miR-125b or its negative control was co-transfected with either miR-125b mimic together with pMIR-2 MRE or miR-125b mimic together with pMIR-2 mut into 293T cells. pMIR-2 PT acts as a positive control. All cells (A, B) were harvested at 48 h after transfection, and then luciferase activities were measured and normalized to the phRL-TK activities. Three independent transfection experiments were done and data was represented as mean  SD. * p < 0.05, when compared with control.

Fig. 3. Mir-125b represses the expression of Cbfb and Runx2 at protein level in early stage of osteoblastic differentiation. (A) C3H10T1/2 cells induced by BMP-2 were transfected with pre-miR-125b, anti-miR-125b or their control individually. At 1 d, 7 d and 14 d, the cells were harvested for measurement of Cbfb and Runx2 protein expression using Western blot. b-actin acts as an internal control. Quantitation of the Cbfb and Runx2 protein level was performed using Quantity One software. The result is shown in the below panels. (B) (C) C3H10T1/2 cells were transfected with pre-miR-125b, anti-miR-125b or their control, and then mRNA level of Cbfb and Runx2 were measured using qRT-PCR at 1 d, 7 d and 14 d. GAPDH acts as an internal control in qRT-PCR analysis. The relative expression level of Cbfb and Runx2 mRNA in cells transfected with control oligonucleotide was set to one, as control. Three independent experiments were done and an asterisk indicates the t-test result is significant (p < 0.05).

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Fig. 4. MiR-125b inhibits early osteoblastic differentiation. (A) ALP expressing after differentiation 1 d, 7 d and 14 d. (B) OCN expressing after differentiation 1 d, 7 d and 14 d. (C) OPN expressing after differentiation 1 d, 7 d and 14 d. (D) PPAR-g expressing after differentiation 1 d, 7 d and 14 d.

3.5. Alkaline phosphatase measurement and alizarin red staining ALP activity and calcium nodus formation on day 14 were measured to estimate the effect of miR-125b on cell differentiation. Fig. 5 showed that pre-miR-125b can significantly inhibit ALP activity (p < 0.05), which is a marker for osteoblastic cells differentiation. Additionally, the formation of calcium nodus is the most important event in bone formation, and alizarin red staining results in the present study indicated that inhibit miR-125b expression with anti-miR-125b can promote calcium nodus formation in the two types of cells (Fig. 6). Together, these data clearly demonstrated

that reduced miR-125b expression may be the reason for the enhanced osteoblast differentiation and the formation of mineral nodule. 4. Discussion In the present study, we ascertained that miR-125b played a function of part in negative regulation in C3H10T1/2 cells line that differentiated into osteoblasts, and complementarily combined at Cbfb gene 30 -UTR, and observably decreased the level of Cbfb protein. We confirmed that miR-125b was an endogenous attenuator

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Fig. 5. ALP activity assay. (A) C3H10T1/2 cells and (B) C57BL/6 mice MSCs were transfected with pre-miR-125b and anti-miR-125b respectively, and then treated with BMP-2 (100 ng/ml). Control cells were only treated with BMP-2. Cells were cultured for 14 days, and then ALP activity was determined in cell lysates and normalized to protein content. * p < 0.05.

of Cbfb expression and inhibited osteoblastic differentiation. But the regulation of miR-125b for MSCs differentiation into adipocyte had unobvious influence. Our results further demonstrated that miR-125b modulates MSCs differentiation into osteoblast at an early stage. The regulation effect was achieved by directly regulating expression of Cbfb and by indirectly regulating expression of Runx2. Our microarray and qRT-PCR data proved that the expression of miR-125b gradually decreased in murine MSCs while differentiation into osteoblasts by BMP-2-induction when compared with control group. This differential expression of miR-125b has been reported by Mizuno Y et al. [19] who used microarray detection in ST2 cells with BMP-4-induction. Therefore, combining our microarray and qRT-PCR data, miR-125b seemed to be involved in regulating osteoblastic differentiation of MSCs. In previous reports, miR-125b is not only involved in mediating osteogenesis [19] and hematopoietic activity [34e37], but also is up regulated in gastric cancer [38]. Core binding factor has three a subunits includes Cbfa1, Cbfa2 and Cbfa3, which partaken in regulation of osteogenesis [39,40], hematopoietic stem cell differentiation [41,42] and gastric epithelial cell proliferation [43], respectively. So we hypothesized that between miR-125b and family of core binding factors may be the regulatory effect. The

bioinformatic analyses showed a number of target genes of miR125b which had not binding site for core binding factor a subunit, but it may conservatively unite with Cbfb in regulating osteogenesis. Dual luciferase reporter gene analysis revealed that exogenic miR-125b and anti-miR-125b inhibitor might regulate luciferase activity through complementarily combing at Cbfb gene 30 -UTR. Furthermore the Cbfb 30 -UTR contained a target site for miR-125b complementary sequence. Moreover, big and small inhibitors were emerged in dose-dependent forms. This data suggested that miR-125b regulates the expression of Cbfb through binding complementarily in Cbfb 30 -UTR, and prompted us to explore whether miR-125b regulated early during osteogenesis through targeted Cbfb. Our results showed that miR-125b inhibits osteoblastic differentiation of MSCs C3H10T1/2 cells through the over expression of pre-miR-125b, and acted on Cbfb at post-transcription level to reduce osteoblastic marker gene mRNA expression such as ALP, OCN, and OPN via down-regulation of Cbfb and Runx2 levels. Thereby, this data revealed that Cbfb have a key role in osteogenesis. Conversely, in start there was a remarkable enhancing of ALP, OCN and OPN mRNAs expression but later it decreased with the time and proved that miR-125b may take indirect role in Runx2 by direct action on Cbfb, which is in accordance with the findings of Toshihisa Komori

Fig. 6. Alizarin red staining. C3H10T1/2 cells and C57BL/6 mice MSCs were transfected with pre-miR-125b and anti-miR-125b respectively, and then treated with BMP-2 (100 ng/ ml). Cells were cultured for 14 days, and then stained for calcium nodus by using alizarin red.

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[44]. In experiment with over-expression and interferential expression of miR-125b, we found that differential expression of Cbfb is not only at post-transcription level but also in mRNA formation level. This result suggested that miR-125b completed translation with imperfect complementation combined at Cbfb 30 UTR to inhibit expression of Cbfb. Moreover, the expression level of PPAR-g mRNA was not influenced with miR-125b in osteoblastic differentiation. This data showed that miR-125b have the crucial role in osteoblastic differentiation by BMP-2-induction. In addition, we also conducted the ALP enzyme activity measurement and alizarin red staining finding that pre-miR-125b treatment repressed ALP activity and formation of mineral nodule. Treatment of antimiR-125b had the opposite effects. Overall, miR-125b was a primary inhibitor in osteoblastic differentiation by directly targeting Cbfb in C3H10T1/2 cells with BMP-2-induction. This led us to find miR125b-Cbfb-Runx1/Runx3 pathway in hemopathy and gastric cancers. However, we could not find a definite proof that miR-125b would have any effect on the terminal differentiation of osteoblastic by long-time effecting (for 14 d). On the basis of the present results, we only demonstrated that miR-125b could regulate osteogenesis at an early stage of differentiation. MicroRNAs are small molecular regulators of gene expression and play a key role in self-renewal, differentiation and functions of stem cells [45,46]. For instance, miR-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation [47], while miR-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9 [22]. Our studies identified that transfected miR-125b and anti-miR-125b separately inhibited and promoted the osteoblastic differentiation of C3H10T1/2 cells via down-regulating and up-regulation expression level of Cbfb in vitro. Recent studies have proved that miR-125b have a pivotal role in potential targeted therapy in human diseases, such as, miR-125b inhibited ovarian cancer cell proliferation via effecting on BCL3 in ovarian cancer [48], repress the development of bladder cancer by targeting on E2F3 in bladder cancer cells [49], suppress keratinocyte proliferation and promoted their differentiation through targeting of FGFR2 in psoriasis [50]. In addition, miR-31 plays a negative role in osteogenic differentiation, and knockdown of miR-31 could enhance the potential of adipose tissue-derived stem cells in repairing critical sized bone defects [51,52]. Study has also demonstrated that miR-125b regulates osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting Smad4 [53]. Our results confirm that miR-125b could suppress osteoblastic differentiation of MSCs through acting on Cbfb. Osteogenic differentiation genes targeted by miR-125b still need to be explored. Consequently, these results may provide the knowledge essential for target therapies of osteoporosis and bone defect. It is concluded during the present study, that expression of miR-125b was decreased during BMP-2-induced osteoblastic differentiation of murine MSCs. Furthermore, miR-125b could inhibit osteoblastic differentiation via directly suppressing Cbfb expression. These conclusions provided a theoretical and experimental foundation for studying stem cell osteogenesis directional differentiation. Acknowledgments This work was supported by a grant from the National Natural Science Foundation of China (30973065), the National Basic Research Program (2011CB964701), and the National Key Technology Research and Development Program of China (2012BAI42G01), and General Program of the National Natural Science Foundation (81271980), and the Funding of State Key Laboratory of Trauma, Burns and Combined Injury (SKLKF201313).

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Please cite this article in press as: K. Huang, et al., MicroRNA-125b regulates osteogenic differentiation of mesenchymal stem cells by targeting Cbfb in vitro, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.02.005