Increased microRNA-93-5p inhibits osteogenic differentiation by targeting bone morphogenetic protein-2
Increased microRNA-93-5p inhibits osteogenic differentiation by targeting bone morphogenetic protein-2
Ying Zhang 0 1
Qiu-Shi Wei 1
Wei-Bin Ding 1
Lei-Lei Zhang 0 1
Hui-Chao Wang 0 1
Ying- Jie Zhu 0 1
Wei He 1
Yu-Na Chai 1
You-Wen Liu 0 1
0 Medical Centre of Hip, Luoyang Orthopaedic-Traumatological Hospital (Orthopaedic Hospital of Henan Province) , Luoyang , China , 2 The First Affiliated Hospital, Guangzhou University of Chinese Medicine , Guangzhou, China, 3 Guangzhou Ginkgo Biotechnology Co. , LTD. , Guangzhou , China , 4 The First Affiliated Hospital of Zhengzhou University , Zhengzhou , China
1 Editor: Jung-Eun Kim, Kyungpook National University School of Medicine , REPUBLIC OF KOREA
Background and purpose Trauma-induced osteonecrosis of the femoral head (TIONFH) is a major complication of femoral neck fractures. Degeneration and necrosis of subchondral bone can cause collapse, which results in hip joint dysfunction in patients. The destruction of bone metabolism homeostasis is an important factor for osteonecrosis. MicroRNAs (miRNAs) have an important role in regulating osteogenic differentiation, but the mechanisms underlying abnormal bone metabolism of TIONFH are poorly understood. In this study, we screened specific miRNAs in TIONFH by microarray and further explored the mechanism of osteogenic differentiation.
Blood samples from patients with TIONFH and patients without necrosis after trauma were
compared by microarray, and bone collapse of necrotic bone tissue was evaluated by
micro-CT and immunohistochemistry. To confirm the relationship between miRNA and
osteogenic differentiation, we conducted cell culture experiments. We found that many miRNAs
were significantly different, including miR-93-5p; the increase in this miRNA was verified by
Q-PCR. Comparison of the tissue samples showed that miR-93-5p expression increased,
and alkaline phosphatase (ALP) and osteopontin (OPN) levels decreased, suggesting
miR93-5p may be involved in osteogenic differentiation. Further bioinformatics analysis
indicated that miR-93-5p can target bone morphogenetic protein 2 (BMP-2). A luciferase gene
reporter assay was performed to confirm these findings. By simulating and/or inhibiting
miR93-5p expression in human bone marrow mesenchymal stem cells, we confirmed that
osteogenic differentiation-related indictors, including BMP-2, Osterix, Runt-related transcription
factor, ALP and OPN, were decreased by miR-93-5p.
experimental material expense to this study. The
commercial company Guangzhou Ginkgo
Biotechnology Co., LTD did not provide funding to
this study, only its employee Wei-Bin Ding
contributed to analyzing the data.
Competing interests: The commercial company
Guangzhou Ginkgo Biotechnology Co., LTD and its
employee just provided some help to this study,
and this does not alter our adherence to PLOS ONE
policies on sharing data and materials.
Our study showed that increased miR-93-5p in TIONFH patients inhibited osteogenic differentiation, which may be associated with BMP-2 reduction. Therefore, miR-93-5p may be a potential target for prevention of TIONFH.
TIONFH is an important type of femoral head necrosis caused by femoral neck fractures, hip
dislocation and other hip trauma. Patients who develop osteonecrosis eventually suffer bone
collapse, and many patients require hip replacement surgery. The facilitation of fracture
healing and the preservation of the femoral head for prevention or delay of total hip arthroplasty to
avoid osteonecrosis are currently recognised as the goals of TIONFH treatment. Bone
metabolism homeostasis is pivotal for fracture healing. The dynamic balance of bone resorption and
bone formation primarily involves osteoclasts and osteoblasts. Promoting osteogenic
differentiation is an important strategy to enhance bone repair.
MicroRNAs (miRNAs), a class of small, conserved non-coding RNA molecules (averaging
19±22 nucleotides), have important regulatory functions in osteogenic differentiation,
including adipogenesis, osteogenesis and cartilage development [
]. Osteogenic cells derived
from bone marrow mesenchymal stem cells (BMSCs) show a relative decrease in number
during femoral head necrosis, and this change in activity directly contributes to osteonecrosis.
Human BMSCs (hBMSCs), which have the capacity of self-renewal and differentiation, can
differentiate to form a variety of tissues, including bone, cartilage, adipose and endothelium
], which is useful in tissue regeneration. These properties suggest that hBMSCs are
potential treatments for various diseases and are critical in regulating bone differentiation.
Endogenous miRNAs play a regulatory role in hBMSC osteogenic differentiation. Therefore, we
selected hBMSCs for in vitro experiments.
Bone morphogenetic protein (BMP), an endogenous mediator, is necessary for fracture
]. It belongs to the acidic glycoprotein family, is primarily synthesised and secreted
from osteoblasts and is widely present in the bone matrix. There are currently more than 20
members in the BMP family, a class of relatively strong osteogenic factors, which are capable
of stimulating bone mesenchymal progenitor cells to differentiate into mature osteoblasts.
BMP-2 is the most important extracellular signalling molecule, promoting bone formation
and inducing bone cell differentiation [
], and regulates the gene expression of a variety of
transcription factors, such as Osterix and Runx-2, critical osteogenic transcription factors.
Runx-2 is expressed in premature osteoblasts, osteoblasts, premature chondrocytes and early
hypertrophic chondrocytes. BMP-2 induces Runx-2 expression at both the transcriptional and
posttranscriptional levels [
]. Osterix is expressed in all developmental osteoblasts and
osteocytes and is positively regulated by BMP-2 during osteogenic differentiation. Loss of BMP-2
results in severely impaired osteogenesis [
]. However, the role of BMP-2 in osteogenic
differentiation has yet to be confirmed, and its complementary miRNA as well as the related
mechanism remains to be further elucidated.
In this study, we aimed to identify specific miRNAs involved in TIONFH and the
mechanism of osteogenic differentiation to provide a foundation for diagnosing and treating
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Materials and methods
Clinical sample data
The inclusion and exclusion criteria of the samples were as follows: from January to April in
2015, patients with femoral neck fractures in the Henan Orthopaedic Hospital Hip Disease
Treatment Study Centre were treated by manual reduction and percutaneous cannulated
screw fixation (without gender limitations). Among these patients, 10 met the diagnostic
criteria of ONFH (osteonecrosis of the femoral head) [
], and another 10 patients lacked ONFH
after treatment. None of the patients had received drug treatment in the last six months, and
they lacked other joint diseases, such as gout, rheumatoid arthritis and others. In addition,
patients with any systemic inflammation, autoimmune disease, or chronic malignant disease
were not included in this study.
All patients underwent evaluation for the following: age, sex, body mass index (BMI), pulse,
respiration, body temperature, blood pressure, Harris hip score [
], visual analogue score
(VAS score) [
], bilateral hips joint X-ray of orthotopic and frog-leg position, and bilateral
hip MRI examination. Bone tissues of the TIONFH patients were evaluated with micro-CT
and histological examination [haematoxylin-eosin (HE) staining].
The Harris score was used as hip function scoring system, which includes Pain、Limp、
Support、Distance Walked、Sitting、Enter public transportation、Stairs、Put on Shoes
and Socks、Absence of Deformity (All yes = 4; Less than 4 = 0)、Range of Motion (indicates
normal) and Range of Motion Scale.
VAS score was used as pain scoring system. Draw a 10 cm line on the paper with number
0±10, which indicates varying degrees of pain. Let the patient mark the line according to his
feeling, indicating the extent of the pain.
The experimental programme was fully in accordance with the relevant provisions of the
1964 Helsinki Declaration. This statement was approved by the Henan Provincial Hospital
orthopaedic clinical trials ethics committee of Luoyang Orthopaedic Hospital. The study team
obtained the written consent from each clinical subject and then implemented the
Screening of differentially expressed genes
Elbow venous blood samples were collected from subjects under fasting conditions and stored
at -80ÊC. Samples were stored until RNA extraction to avoid freezing and thawing. Blood
samples were used for microarray detection, and miRNA differences were identified.
Normal bone tissue and necrotic bone tissue from five osteonecrosis patients were assessed.
Immunohistochemistry was used to detect protein expression of BMP-2, ALP and OPN. The
femurs were dissected and fixed in 4% paraformaldehyde for 48 h, followed by decalcification
in 8% EDTA (pH 7.4) at 4ÊC. The decalcified specimens were processed for paraffin
embedding, and serial 5-mm sections were prepared. The anti-BMP-2 mAb (Santa Cruz) was used
at a dilution of 1:300. ALP (Abcam) and OPN (Santa Cruz), antibodies were diluted 1:200.
Mouse IgG, at the same concentrations as the corresponding primary antibodies, was used to
replace the primary antibodies in the negative control experiments. All immunohistochemistry
experiments were performed using the DAB kit according to the manufacturer's instructions,
and images were obtained with an Olympus cx31 system software.
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Target gene prediction
Online software (http://www.microrna.org/microrna/home.do) was used to predict potential
target genes of miR-93-5p.
Cell isolation, culture and identification
The hBMSCs used in this study were isolated from the bone marrow of donors with trauma
who had provided informed consent. These hBMSCs were harvested and cultured as
previously described [
]. hBMSCs were isolated by Ficoll density-gradient centrifugation .
A PBS mixture was allowed to stand for 10 min, and the supernatant was slowly transferred to
a 15 ml centrifuge tube containing an equal amount of Ficoll-Paque (GE Healthcare, USA).
After 35 min of centrifugation (2,000 rpm), mononuclear cells were collected from the
interface (the cloud-like cell layer) and resuspended in 8 ml culture medium. Cell lines were
cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated foetal
bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin in 5% CO2 in a humidified
incubator at 37ÊC. All experiments using hBMSCs were performed at passages 2±5. For
osteogenic differentiation, cells were cultured using osteogenic induction solution containing
0.1 μmol/L dexamethasone, 50 mg/L ascorbic acid, and 10 mmol/L β-glycerophosphate [
hBMSCs were analysed for the expression of CD29, CD34, CD44, CD45, CD29, CD34,
CD44, and CD45 by flow cytometry. Approximately 5.0×104 second-passage hBMSCs were
stained with 20 μl FITC-conjugated antibody (BD, USA) for 30 min at 4ÊC. IgG1 was used as a
control. Cells were analysed for fluorescence within 6 h using BD Accuri C6 Flow Cytometry.
Oligonucleotides and plasmids
The miR-93-5p mimic/inhibitor, negative control mimics (NC) and primers were obtained
from RiboBio Biotechnology (Guangzhou, China). A 580 bp region of BMP-2 3' untranslated
region (UTR), containing the potential miR-93-5p binding site cloned into the luciferase assay
plasmid PSICHECK2.0, was obtained from Ginbio (Guangzhou, China) to demonstrate a
direct interaction between miR-93-5p and BMP-2. All primers were synthesised by Sangon
Biotech (Shanghai, China) (Table 1).
Luciferase reporter assays
To construct a luciferase reporter for wild-type (WT) BMP-2 3'UTR, we synthesised this UTR
sequence and sub-cloned it into the NotI and XhoI sites in the apsiCHECK-2 vector
(Promega). A mutant BMP-2 3'UTR was generated by site-directed mutagenesis using Phusion™
High-Fidelity DNA Polymerase (Thermo Scientific) according to the manufacturer's protocol.
All constructs were confirmed by sequence analysis. For transfection, 5 × 104 293T cells were
plated in complete medium on a 24-well plate. The next day, the cells were transfected with 50
nM of miR-93-5p mimics or NC mimics and inhibitor using Lipo3000 (Thermo Scientific)
according to the manufacturer's protocol. Three groups of cells (miR-93-5p NC group,
miR93-5p mimic group and miR-93-5p inhibitor group) were harvested 72 h after transfection in
cell lysis buffer and then assayed for luciferase activity using the Dual-Luciferase Reporter
Assay System (Beyotime, Shanghai) and a luminometer according to the manufacturer's
protocol. Transfection of each construct was performed in triplicate for each assay, and the ratios
of Renilla luciferase activity to firefly luciferase activity were averaged for each experiment. For
protocol see: dx.doi.org/10.17504/protocols.io.irbcd2n.
MTT assay for cell proliferation
For the MTT assay, 5×103 cells per well were seeded with complete growth medium on a
96-well plate. The cells were divided into three groups: miR-93-5p NC group, miR-93-5p
mimic group and miR-93-5p inhibitor group. Treated cells were assessed for 1 to 3 days via the
previously described MTT assay. The data were measured using a microtiter plate reader with
a 570 nm filter (Thermo Scientific).
Q-PCR analysis 1: miR-93-5p. Q-PCR for miR-93-5p was performed according to
standard protocols using a Bio-Rad CFX96 Touch™ Deep Well Q-PCR Detection System. The
expression of miR-93-5p was evaluated using a mirVana™ qRT-PCR miRNA Detection Kit
(RiboBio Biotechnology, Guangzhou). The primers were designed and synthesised by RiboBio
Biotechnology (Guangzhou). U6 was used as an internal control.
Total RNA was isolated from the four groups of cells (as above) after 0, 3, and 7 days in
differentiation medium with TRIzol (TaKaRa, Dalian, China), and cDNA was synthesised using
a special miR-93-5p primer. The quantity and quality of isolated RNA was determined using a
2000 Spectrophotometer (Thermo Scientific).
Q-PCR analysis 2: BMP-2, OPN, Runx-2, ALP and Osterix. After 7 days in
differentiation medium with osteogenic induction, two cell groups (miR-93-5p NC, and miR-93-5p
mimic groups) were harvested, and RNA was reverse-transcribed into cDNA using oligT
primers and M-MLV reverse transcriptase (TaKaRa, Dalian, China). For Q-PCR of transcripts,
cDNA was mixed with Power SYBR Green PCR Mastermix (Applied Biosystems, Foster City,
CA) and analysed on a Bio-Rad Real-Time PCR system. The primers are shown in the
Supplementary materials and include primers for BMP-2, OPN, Runx-2, ALP and Osterix. β-actin
was used as an internal control (Table 1). Quantification of the fold change in gene expression
was determined with the ΔΔCt method.
Western blotting analysis of BMP-2, Runx-2, OPN, and ALP
After 7 days in differentiation medium with osteogenic induction, two cell groups (miR-93-5p
NC group and miR-93-5p mimic group) were seeded on 60 mm plastic dishes (WHB) and
cultured for 7 days in osteogenic differentiation medium. Total protein was isolated using RIPA
buffer. Proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane for
1.5 h at 4ÊC. Membranes were blocked with 5% milk in TBST for 2 h at room temperature and
incubated with primary antibodies against BMP-2 (1:200, Santa Cruz, USA), OPN (1:200,
Santa Cruz, USA), Runx-2 (1:200; Santa Cruz, USA), ALP (1:100, Boster, China), Osterix
(1:200, Santa Cruz, USA), or GAPDH (1:3000, Tianjin Sungene, China) at 4ÊC overnight.
Membranes were incubated with HRP-conjugated secondary antibody (1:2000, Boster, China)
for 1 h at room temperature, followed by scanning with X-ray film. The integrated intensity
for each detected band was then determined with ImageJ, v.1.46.
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Alkaline phosphate (ALP) assay
Three cell groups (miR-93-5p NC and miR-93-5p mimic groups) were seeded in 24-well plates
(Costar) for the ALP activity assay. Cells were harvested and resuspended in 250 μl culture
supernatants, which was followed by cell rupturing with an ultrasound breaker. After
centrifugation, the ALP activities in the cell supernatants were quantified using an ALP Detection Kit
(Nanjing Jiancheng Biotech Institute, China) and a spectrophotometer at a wave length of 520
nm. Each value was normalised to the protein concentration.
Alizarin red (AR) staining
Two cell groups (miR-93-5p NC and miR-93-5p mimic groups) were seeded on 96-well plastic
dishes (Costar) to examine mineralisation. Cells were cultured in differentiation medium for
21 days and then washed twice with PBS and fixed with 4% paraformaldehyde at room
temperature for 10 min. The dishes were then washed three times with distilled water and incubated
with 0.1% AR (Sigma, USA) at 37ÊC for 30 min. Cells were thoroughly washed with distilled
water, and images were captured using a scanner.
SPSS 19.0 statistical software (SPSS Inc., Chicago, IL) was used for statistical analysis. All data
are presented as the mean ± standard deviation. Data between the two groups were analysed
using t tests, and multiple comparisons between groups were assessed by one-way ANOVA,
assuming double-sided independent variance. P values < 0.05 were considered statistically
Clinical data of 20 patients are shown in Table 2. There were no significant differences for the
two groups of patients in age, blood pressure and other vital signs; Harris scores of the
TIONFH group were lower than those without necrosis (p = 0.008), and VAS scores were
higher than those without necrosis (p = 0.009). These results indicated that hip function in the
necrosis group was significantly limited. MRI test results are shown as follows. In patients
without necrosis, there appeared hollow spike-like images without a significant ªdouble lineº
sign (Fig 1 A). Necrosis patients had a clear ªdouble lineº sign and displayed high signals on
fat-suppressed images, which is a typical feature of TIONFH (Fig 1 B). X-ray analysis showed
Fig 1. MRI and X-ray examination of TIONFH patients and the patients without necrosis. (A) MRI of
patients without necrosis showed hollow spike-like images without a significant ªdouble lineº. (B) Necrosis
patients showed a clear ªdouble lineº sign and displayed high signals on fat-suppressed images. (C) X-ray of
patients without necrosis showed a smooth surface without collapse. (D) Necrosis patients showed femoral
head collapse, density changes, cystic changes, or hollow or pierced femoral head surface.
that patients without necrosis had a smooth surface without collapse and necrosis patients
showed femoral head collapse or density changes, cystic changes, or a hollow or pierced
femoral head surface, (Fig 1C and 1D). These findings are typical, demonstrating that the bone
structure of TIONFH patients showed significant changes.
Histological structure changes of necrotic bone tissue
The micro-CT results showed that compared to the normal area, the necrotic area showed
structural relaxation (Fig 2A and 2B). Three-dimensional reconstruction showed trabecular
bone collapse deformation compared to the normal area (Fig 2C and 2D). Further pathology
staining demonstrated a necrotic area with significant bone necrosis and bone marrow tissue
necrosis between the trabecular and simultaneous granulation tissue (Fig 2E and 2F). This
evidence suggests that TIONFH patients had significant bone structure changes and osteoblast
death, which is an important potential cause of bone collapse and hip dysfunction.
miRNA array of peripheral blood and Q-PCR confirmation
We analysed TIONFH patients (n = 10) and patients without necrosis after femoral neck
fracture (n = 10) by microarray and found 297 differentially expressed miRNAs in peripheral
blood. These differences included 35 up-regulated miRNAs, such as miR-93-5p, miR-7i-5p,
miR-320a, miR-25-3p, and miR-16-2-3p (fold > 2.0, P < 0.05). A partially differentially
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Fig 2. TIONFH patient bone collapse and osteoblast death. (A) Micro-CT of a normal area. (B) Micro-CT of a necrotic area,
which appeared to indicate trabecular bone disorder, shows structural relaxation. (C) Three-dimensional reconstruction of normal
areas. (D) Three-dimensional reconstruction of a necrotic area showing trabecular bone collapse deformation. (E) HE staining of
normal areas. (F) HE staining of a necrotic area shows significant bone necrosis and bone marrow tissue necrosis between the
trabecular and simultaneous granulation tissue.
expressed miRNA cluster diagram is shown (Fig 3A), and miR-93-5p, with a fold change more
than 3, was further examined.
Q-PCR was used to further confirm the miR-93-5p expression and showed that miR-93-5p
was significantly increased in the peripheral blood of TIONFH patients (Fig 3B), consistent
with the microarray analysis. At the same time, Q-PCR analysis of bone tissue showed that the
expression of miR-93-5p in necrotic areas was significantly higher than that in normal areas
Whether increased miR-93-5p in TIONFH patients is involved in bone metabolism
abnormalities caused by bone necrosis remains to be elucidated. To assess this hypothesis, we
performed further experiments.
Association of miR-93-5p and osteoblast differentiation
Immunohistochemistry of necrotic bone tissue samples from TIONFH patients demonstrated
that ALP and OPN expression was decreased (Fig 4A), indicating that patients with TIONFH
had a low osteoblast differentiation ability.
Patients with bone necrosis tend to exhibit decreased cell differentiation. Is this process
directly related to abnormal expression of miR-93-5p? To address this issue, we further verified
the impact of miR-93-5p on osteoblast differentiation in cell experiments.
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Fig 3. miRNA array and miR-93-5p expression. (A) Cluster analysis of partially differentially expressed
miRNAs from TIONFH patients and controls. (B) miR-93-5p expression level in plasma was significantly
increased as shown by Q-PCR. (C) miR-93-5p expression increased in bone tissue as shown by Q-PCR.
Cell culture and identification experiments were performed. Flow cytometry revealed that
the cultured cells were negative for HLA-DR, CD45, CD34, CD14, and CD11b and positive for
CD166, CD105, CD73, CD44, and CD29. These cells are characterised by their surface marker
]; they possessed the stem cell characteristics of hBMSCs and could thus be used for
miR-93-5p expression during hBMSCs differentiation was also assessed. Q-PCR results
showed that the miR-93-5p expression was decreased in the NC group when they were
cultured in differentiation medium after 3 and 7 d, but the miR-93-5p mimic group showed
higher rates of expression compared to the other groups, indicating successful plasmid
transfection (Fig 4B)
The results of MTT assays indicated that the miR-93-5p mimic significantly promoted
hBMSC cell proliferation in basic medium after 48 h, which was more pronounced after 72 h
(p<0.05) (Fig 4C).
To further validate the effect of miR-93-5p on hBMSC osteogenic differentiation, we used
an ALP assay kit to test ALP activity of hBMSCs that were induced for 7 d with an osteogenic
agent. The results showed that ALP activity in the mimic group was significantly inhibited (Fig
4D). Meanwhile, AR staining of osteogenic agent-induced cells was performed after 21 d. The
formation of calcium nodules was significantly reduced in the mimic group. (Fig 4E). Q-PCR
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Fig 4. Association of miR-93-5p and osteoblast differentiation. (A) ALP and OPN expression in TIONFH
patients with necrotic bone tissue samples by immunohistochemistry. Brown represents positive signals. (B)
Real time Q-PCR detected the miR-93-5p expression level during differentiation at 0, 3, and 7 d. (C) Effects of
miR-93-5p on hBMSCs] proliferation after 24, 48 and 72 h by MTT assays. (D) ALP enzyme activity was
detected with a reagent kit. (E) AR staining to assess mineralisation. Two cell groups were seeded on 96-well
plastic dishes (Costar). Cells were cultured in differentiation medium for 21 d, and images were captured
using a scanner. (F) mRNA expression level of ALP, OPN, Runx-2 and Osterix using real time PCR analysis.
(G) Western blotting was performed to detect protein levels of ALP, OPN, Runx-2 and Osterix. (* P < 0.05 and
** P < 0.01).
and Western blotting were used to detect the gene and protein changes of bone
formationrelated indicators, including ALP, OPN, Runx-2 and Osterix, after osteogenic agent induction
for 7 d. The results showed that the gene and protein levels of ALP, OPN, Runx-2 and Osterix
were significantly suppressed in the mimic group (Fig 4F and 4G). These data indicated that
miR-93-5p can suppress hBMSC osteogenic differentiation in vitro and suggest the important
role of miR-93-5p in osteogenic differentiation.
Identification of BMP-2 as a miR-29 target
Online software was used to screen potential miR-93-5p targets. Based on the complementarity
with the miR-93-5p seed sequence, we focused BMP-2 (Fig 5A). The BMP2 30-UTR with either
the predicted WT miR-93-5p binding site or a mutant binding site was inserted into the
psiCHECK2 vector (Fig 5A). WT-BMP2 significantly repressed the expression of Renilla
luciferase without affecting the MUT-BMP2 (Fig 5B). These results demonstrated that miR-93-5p
can bind to the 30-UTR of the BMP-2.
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Fig 5. Identification of BMP-2 as a target of miR-93-5p. (A) miR-93-5p targets the 3 'UTR of BMP-2.
BMP2-3'UTR WT or MUT was cloned into psiCHECK2.0 and co-transfected with the miR-93-5p mimic. miR-93-5p
mimics bind specifically to BMP2-3'UTR WT. (B) Effect of miR-93-5p on BMP-2 RNA 3'UTR region by the
luciferase gene reporter assay. (* P < 0.05 and ** P < 0.01).
Luciferase gene reporter experiments showed that in the WT group, luciferase activity was
significantly reduced due to binding to the miR-93-5p mimic, while the miR-93-5p mimic did
not affect the luciferase activity of the MUT group (Fig 5C). These results demonstrate that
miR-93-5p inhibits the expression of BMP-2 by binding to a specific region of the BMP-2
miR-93-5p regulation of BMP-2 expression
Immunohistochemical analysis of samples showed that BMP-2 was significantly reduced, as
shown in Fig 6A. In vivo, we further validated the gene and protein levels of BMP-2. BMP-2
expression was significantly reduced in the Inducer + miR-93-5p mimic group (Fig 6B and
6C), which is consistent with the luciferase reporter gene results. The above results confirmed
that low expression of the osteogenic protein BMP-2 was associated with high miR-93-5p
expression in TIONFH patients and decreased osteoblast differentiation.
This study reveals a major mechanism of TIONFH patient bone collapse. TIONFH is a femoral
head blood circulation disorder with various causes that can result in in bone cell and bone
marrow component death and then structural changes in the femoral head. Early necrosis
occurs in the outer range of the femoral head and is characterised by increased local bone
mineral density with unclear trabecular bone and a flat area showing collapse in the avascular
necrosis area. Avoiding femoral head necrosis and facilitating fracture healing is pivotal for treatment
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Fig 6. miR-93-5p regulation of BMP-2 expression. (A) BMP-2 immunohistochemical analysis of normal
bone tissue from patients with necrotic areas and the area of necrosis. (B) BMP-2 protein expression in
hBMSCs by Western blotting analysis. (C) BMP-2 mRNA expression level in hBMSCs by real time Q-PCR
analysis. (* P < 0.05 and ** P < 0.01).
of this condition. Therefore, exploring osteoblast differentiation and proliferation as well as
promoting osteogenic potential of the femoral head in patients is crucial for the prevention and
treatment of this condition. The BMP-2 gene has been genetically linked to osteoporosis and
]. Abnormal BMP-2 levels cause congenital anomalies and diseases involving
the mesenchymal cells, which differentiate into muscle, fat, cartilage, and bone [
]. The 3'UTR
of BMP-2 genes from mammals to fish are extraordinarily conserved, which indicates that the
BMP-2 3'UTR is under stringent selective pressure, and the conserved region is a strong
posttranscriptional regulator of BMP-2 expression [
]. In this study, we demonstrated that
miR93-5p posttranscriptionally regulates BMP-2 by binding to the 3'UTR. Our study provides the
first evidence that TIONFH patients have high miR-93-5p expression in the peripheral blood
and that TIONFH inhibits osteoblast differentiation by reducing BMP-2 expression.
miR-93-5p, as a member of the microRNA gene cluster miR-106b~25 (miR-106b, miR-93,
and miR-25), is expressed in primary stem cells [
] and normal tissue [
] as well as in
pathological contexts, such as tumour development [
], ageing [
], bone formation [
cardiovascular disease [
]. This miRNA promotes cell proliferation in osteosarcoma [
bladder cancer [
], and neural stem cells [
]. However, miR-93-5p was also shown to inhibit
cell expansion in human colon cancer [
]. The coexistence of these seemingly contradictory
effects suggests that the function of this miRNA is related to the specific tissue or
microenvironment involved [
In recent years, the roles of small RNAs in orthopaedic diseases have been investigated and
analysed. miRNAs are important for osteogenic differentiation. miR-204 and miR140-5p were
shown to regulate the proliferation of BMSCs, and miR140-5p was associated with the
expression of BMP-2 [
]. However, miRNAs involved in TIONFH have not been unidentified.
We screening genes between TIONFH patients and patients without necrosis and
demonstrated that miR-93-5p was increased in the subjects, which was verified by Q-PCR. These
results provide potential new diagnostic strategies for TIONFH.
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Notably, while screening miRNA, we chose postoperative necrosis patients after trauma
without necrosis rather than healthy individuals as controls. Therefore, interference from
hormonal necrosis or other factors were ruled out.
To further explore the mechanism of miR-93-5p in TIONFH, we analysed its target gene,
BMP-2, after prediction by bioinformatics methods and luciferase reporter gene analysis. In
addition, in situ expression of BMP-2 in TIONFH patients was tested by immunochemistry.
BMP-2 is an important protein in osteogenic differentiation, and miR-93-5p is a negative
regulator of BMP-2. miR-93-5p and the BMP-2 RNA 3'UTR region binding were not completely
paired, and thus, the function of hBMSCs expressing BMP-2 was inhibited. This blocks
bonerelated transcription of other genes in the cells, suppressing the formation of osteoblasts. In
vivo, BMP-2 protein expression was low in tissues of femoral head necrosis patients and had
an inverse correlation with the miR-93-5p level. This is consistent with the in vitro results.
Therefore, miR-93-5p likely inhibits osteoblast differentiation via BMP-2. These observations
contribute to our understanding of BMP-2-associated miRNAs and also elucidate the
mechanism of TIONFH-mediated inhibition of osteogenic differentiation.
ALP is an important indicator of early differentiation in osteogenic differentiation. OPN is
involved in osteoblast adhesion and mineralisation [
]. In patient tissue samples, we found
that ALP and OPN were significantly reduced. Furthermore, we performed in vitro studies.
The regulation of miRNAs was time-dependent and based on the time required for osteoblast
differentiation and mineralisation (7 d and 21 d, respectively). At the corresponding time, the
formation of ALP and calcium nodules and the expression of osteogenic genes in hBMSCs all
decreased. In vitro and in vivo experiments indicated that osteogenic differentiation inhibition
was associated with miR-93-5p. Combined with clinical results showing that BMP-2 was
decreased, these findings indicated that TIONFH bone lesions occur in patients with hip
collapse phenomenon. The level of Runx-2 determines the degree of skeletal maturation and the
rate of conversion [
], and Osterix is expressed in all developing osteocytes and osteoblasts;
these proteins are downstream molecules of BMP-2 signalling [
]. Thus, based on
multiple lines of evidence, miR-93-5p inhibits osteogenic differentiation by BMP-2.
Interestingly, we found that miR-93-5p promoted cell proliferation of hBMSCs during in
vitro experiments. Other evidence, such as the significantly reduced ALP and the formation of
calcium nodules as well as the expression of BMP-2, Runx-2 and Osterix, demonstrated the
inhibition of bone cell differentiation in response to miR-93-5p. However, this is a complex and
interesting issue. These results prompted us to assess whether hBMSCs have multi-lineage
differentiation potential [
] and whether miR-93-5p induces hBMSCs toward other types of
differentiation. These questions should be further investigated and will promote enhanced
understanding and novel treatment approaches. Currently, although the function of miR-93-5p
in promoting hBMSC proliferation and suppressing osteogenic differentiation is unclear,
specific suppression of miR-93-5p in hBMSC osteogenic differentiation has been demonstrated.
Based on comprehensive experimental results in vitro and in vivo, miR-93-5p was shown to
be significantly increased in TIONFH patients, and its osteogenic suppression function was
related to target genes, such as BMP-2, which may be one of the mechanisms of bone necrosis
collapse. Our findings improve our understanding of the osteonecrosis mechanisms. However,
increased miR-93-5p will have broad pleiotropic effects. As with all miRNAs, miR-93-5p has
many targets, and BMP-2 is just one target. It is unclear whether the effects of miR-93-5p on
osteogenic differentiation depend solely/primarily on targeting BMP-2 or whether other
targets are involved as well. In this study, we only assessed the effects of miR-93-5p in TIONFH,
and more detailed examinations are required. These studies provide a new approach for
diagnosing and treating TIONFH as well as other diseases associated with bone necrosis, such as
steroid femoral head necrosis.
13 / 16
S1 Figs. Original uncropped and unadjusted Western blotting of ALP, OPN, Runx-2,
Osterix, BMP-2, GAPDH.
S1 File. Editorial certification from AJE.
S1 Data. Microarray data on GEO. The related data were submitted to GEO as required, and
the following link has been created to allow review of record GSE89587 while it remains in
private status: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=clefscoqbpgxdid&;acc=
Data curation: Ying Zhang, Wei-Bin Ding, Lei-Lei Zhang, Hui-Chao Wang.
Formal analysis: Ying Zhang.
Funding acquisition: You-Wen Liu.
Methodology: Ying Zhang, Qiu-Shi Wei, Hui-Chao Wang, Ying-Jie Zhu, Yu-Na Chai,
Project administration: Ying Zhang, Qiu-Shi Wei, Wei He, Yu-Na Chai, You-Wen Liu.
Software: Wei-Bin Ding.
Supervision: Ying Zhang.
Writing ± original draft: Ying Zhang, Lei-Lei Zhang, Hui-Chao Wang, Ying-Jie Zhu, Yu-Na
Chai, You-Wen Liu.
Writing ± review & editing: Ying Zhang, Wei-Bin Ding, Lei-Lei Zhang, Hui-Chao Wang,
Wei He, Yu-Na Chai, You-Wen Liu.
14 / 16
15 / 16
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