Cellular and Molecular Life Sciences (2023) 80:75
https://doi.org/10.1007/s00018-023-04719-6
Cellular and Molecular Life Sciences
ORIGINAL ARTICLE
MiR‑422a promotes adipogenesis via MeCP2 downregulation
in human bone marrow mesenchymal stem cells
Angelica Giuliani1 · Jacopo Sabbatinelli1,2 · Stefano Amatori3 · Laura Graciotti1,4 · Andrea Silvestrini1 ·
Giulia Matacchione1 · Deborah Ramini5 · Emanuela Mensà1 · Francesco Prattichizzo6 · Lucia Babini1 ·
Domenico Mattiucci7 · Elena Marinelli Busilacchi7 · Maria Giulia Bacalini8 · Emma Espinosa9 · Fabrizia Lattanzio10 ·
Antonio Domenico Procopio1,5 · Fabiola Olivieri1,5 · Antonella Poloni7 · Mirco Fanelli3 · Maria Rita Rippo1
Received: 14 April 2022 / Revised: 16 December 2022 / Accepted: 22 January 2023 / Published online: 27 February 2023
© The Author(s) 2023
Abstract
Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly
focused on the central nervous system because alterations of its expression are associated with neurological disorders such
as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2
in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and
in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation
nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also
up-regulated in hBMSC-derived osteoblasts, suggesting a specific role of the latter in the adipogenic process. Experimental
modulation of intracellular levels of miR-422a and miR-483-5p affected MeCP2 expression through direct interaction with
its 3′ UTR elements, and the adipogenic process. Accordingly, the knockdown of MeCP2 in hBMSCs through MeCP2targeting shRNA lentiviral vectors increased the levels of adipogenesis-related genes. Finally, since adipocytes released a
higher amount of miR-422a in culture medium compared to hBMSCs we analyzed the levels of circulating miR-422a in
patients with osteoporosis—a condition characterized by increased marrow adiposity—demonstrating that its levels are
negatively correlated with T- and Z-scores. Overall, our findings suggest that miR-422a has a role in hBMSC adipogenesis
by downregulating MeCP2 and its circulating levels are associated with bone mass loss in primary osteoporosis.
Keywords Methyl CpG binding protein 2 · Mesenchymal stromal cells · Adipogenesis · Osteogenesis · MicroRNA ·
Osteoporosis
* Angelica Giuliani
angelica.giuliani@staff.univpm.it
5
Clinic of Laboratory and Precision Medicine, IRCCS INRCA
, Ancona, Italy
* Maria Rita Rippo
m.r.rippo@univpm.it
6
IRCCS MultiMedica, Milan, Italy
7
Section of Hematology, Department of Clinical
and Molecular Sciences, Università Politecnica delle Marche,
Ancona, Italy
8
IRCCS Istituto delle Scienze Neurologiche di Bologna,
Laboratorio Brain Aging, Bologna, Italy
9
Geriatrics, Santa Croce Hospital, Azienda Ospedaliera
Ospedali Riuniti Marche Nord, Fano, Italy
10
Scientific Direction, IRCCS INRCA, Ancona, Italy
1
Department of Clinical and Molecular Sciences, Università
Politecnica delle Marche, Via Tronto 10/A, Ancona, Italy
2
SOD Medicina di Laboratorio, Azienda Ospedaliero
Universitaria delle Marche, Ancona, Italy
3
Department of Biomolecular Sciences, Molecular Pathology
Laboratory “PaoLa”, University of Urbino Carlo Bo, Fano,
PU, Italy
4
Department of Biomedical Sciences and Public Health,
Università Politecnica delle Marche, Ancona, Italy
13
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Introduction
The methyl-CpG binding protein 2 (MeCP2) is known
principally for its ability to inhibit the transcription complex assembly on DNA by binding methylated CpG islands
across the genome [1]. Beyond its role in transcriptional
repression, more recent studies revealed that MeCP2
may play a complex multifunctional role, coordinating
also transcriptional activation, chromatin architecture,
and RNA splicing, depending on the molecular context
[2–4]. MeCP2 expression is ubiquitous throughout the
body, although it is particularly abundant and studied in
brain cells. Indeed, in females X-linked mutations of the
MECP2 gene cause Rett syndrome (RTT), a neurodevelopmental disorder characterized by loss of acquired motor
and language skills, autistic features, and unusual stereotyped movements [5, 6]. However, the variety of phenotypes identified in RTT patients and MeCP2 mutant mouse
models points to important roles for MeCP2 in peripheral
systems, including altered lipid metabolism, unbalanced
adipose tissue endocrine activity [7, 8], and decreased
bone mineral density, among others [9, 10].
In bone marrow (BM), mesenchymal stromal cells
(BMSCs), the precursors of adipocytes and osteoblasts, are
exposed to a plethora of stimuli that determine the balance
between adipogenesis and osteogenesis which in turn are
competing and reciprocal [11, 12]. The differentiation of
MSCs, in fact, is a two-step process: lineage commitment
(from BMSCs to lineage-specific progenitors) and maturation (from progenitors to specific cell types). Studying
the mechanisms that regulate bone marrow adipogenesis
is important because the marrow adipose tissue (MAT)
is not only a passive space-filler. Indeed, MAT actively
participates in a broad spectrum of physiological functions—e.g. energy homeostasis, immunity, hematopoiesis,
coagulation, and regulation of blood pressure—through
the release of several molecular mediators [13], including
adiponectin, of which BM adipocytes are among the major
contributors [14]. Variations in BM adipocyte mass have
been reported in primary osteoporosis and other systemic
conditions like aging, type 2 diabetes, obesity, myelodysplastic syndrome, cancer therapy, and anorexia nervosa,
suggesting that an abnormal differentiation of BMSCs
could contribute to pathogenic skeletal manifestations
associated with such diseases [15].
Adipogenesis is a finely tuned multi-step process requiring the sequential activation of numerous transcription factors driving the typical physiological and morphological
changes observed in the progenitor cells, i.e. cell cycle
arrest, metabolic reprogramming, and lipid accumulation
[16]. The expression of peroxisome proliferator-activated
receptor gamma (PPARγ) is critical to promote fat cell
13
differentiation, and survival of adult adipocytes, inducing the expression of genes involved in insulin sensitivity,
lipogenesis, and lipolysis [17–19].
Among the small non-coding RNAs, microRNAs (miRNAs, miRs) represent an additional mechanism for controlling adipogenic gene expression [20]. Given their unique
ability to simultaneously regulate multiple protein targets
and processes, it has been suggested that miRNAs may play
a leading role in BMSC differentiation. Their involvement
in adipogenesis has been investigated through experimental
and bioinformatic, target-based approaches [21–23]; some
identified miRNAs would appear to be involved in lineage
commitment while others in maturation. The latter, in some
cases, would involve switches that suppress the osteogenic
process by activating the adipogenic one [24, 25]. Interestingly, several studies in the nervous system, cancer, and
smooth muscle cell differentiation suggest that miRNAs can
directly regulate the expression of MeCP2 [26–32].
The role of MeCP2 in the BMSC differentiation process
is still unknown but given the relevance of fine control of the
transcriptional and epigenetic processes in BMSCs differentiation, it is conceivable that MeCP2 and miRNAs regulating
its expression could affect their fate.
Therefore, the aim of our study was to identify miRNAs
able to modulate the adipogenic process and their role in
the MeCP2-mediated modulation of adipogenesis. For this
purpose, we (i) evaluated MeCP2 expression in hBMSCs
undergoing differentiation; (ii) screened for miRNAs differentially regulated during hBMSC differentiation in vitro;
(ii) validated miRNAs selectively upregulated in adipocytes
compared to their precursors; (iii) tested their role in enhancing adipogenesis, and (iv) assessed their ability to modulate
MeCP2 expression in hBMSCs. Finally, the levels of circulating miRNAs involved in hBMSC differentiation have been
assessed in a cohort of elderly subjects with primary type II
osteoporosis, to investigate their association with bone mass
loss due to the expansion of the MAT compartment.
Results
MeCP2 is downregulated in adipose tissue
and during adipogenesis of hBMSC in vitro
To determine the role of MeCP2 during adipogenesis,
MeCP2 expression was analyzed in human bone marrow
MSCs induced to differentiate into adipocytes and osteoblast. As shown in Fig. 1A MeCP2 protein expression was
downregulated in BMSC-derived adipocytes compared to
undifferentiated cells, whereas an opposite modulation was
observed in osteoblasts. Furthermore, immunohistochemical
(IHC) staining performed in human bone marrow sections
obtained from healthy donors revealed a high expression of
Page 3 of 16 75
MiR‑422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal…
A
B
hBMSCs OS
b
a
b
AD
75 kDa
MeCP2
43 kDa
β-actin
a
MeCP2/B-actin ratio (a.u.)
2.5
1
***
2.0
**
c
***
c
1.5
1.0
0.5
0.0
2
50 μm
Ad
a
de
di
ff
s
AT
ip
oc
yt
e
s
D
C
AD
100 μm
AD
-M
SC
hBMSCs OS
75 kDa
MeCP2
58 kDa
PPARγ
43 kDa
α-tubulin
a
20
10
SC
oc
-M
AT
ip
Ad
de
di
ff
0
50 m
s
2
30
yt
e
100 m
*
*
40
s
b
50
AD
b
MeCP2/α-tubulin ratio (a.u.)
1
Fig. 1 MeCP2 expression in adipocytes and adipogenic process. A
Western blot and densitometric analysis of MeCP2 in human bone
marrow mesenchymal stromal cells (hBMSCs) and hBMSC-derived
adipocytes (AD) and osteoblasts (OS) after 14 days of pro-differentiating treatment. Data were normalized to β-actin. B Immunohistochemical detection of MeCP2 reactivity in the human femoral
bone marrow. 1, Numerous MeCP2-positive nuclei are present. a,
Periosteal positive cells (arrows). b, Hematopoietic positive cells.
2, Negative human BM adipose tissue; c, positive hematopoietic
cells (arrows) in close contact with adipocyte membrane. Images
were taken at 200× and 400× magnification. C Immunohistochemical detection of MeCP2 reactivity in rat. 1, Femoral bone marrow,
MeCP2 positive hematopoietic cells in close contact with adipocyte
membrane are present (a, arrows), no staining was observed in adipocyte nuclei. 2, Inguinal white adipose tissue (AT), low reactivity
in adipocytes was confirmed, whereas interstitial and blood cells
were positive (b, arrows). Images were taken at 200× and 400× magnification. D Western blot and densitometric analysis of MeCP2 and
PPARγ expression in human adipocytes, mesenchymal stromal cells
(AT-MSCs) and dedifferentiated adipocytes (AD dediff) obtained
from subcutaneous adipose tissue of the same donor. Western blot
image is relative to 1 out of 3 different analyzed donors. Data were
normalized to α-Tubulin. Data are mean ± SD of three independent
experiments. *t-test p < 0.05; **t-test p < 0.01; ***t-test p < 0.001
MeCP2 in the nuclei of periosteal cells (Fig. 1B(a)) and of
several hematopoietic cells (Fig. 1B(b)); on the contrary,
MeCP2 expression was not found in adipocytes (Fig. 1B(c)).
The same results are confirmed in the bone marrow and adipose tissue (AT) samples derived from rats (Fig. 1C). To
strengthen this observation, MeCP2 expression was analyzed
in human adipocytes and MSCs, isolated from subcutaneous
fat of the same donors, in addition to dedifferentiated adipocytes obtained as previously described [33, 34]. Here, we
show that MeCP2 expression is significantly lower in adipocytes compared to MSCs and dedifferentiated cells. PPARγ
was used as a control of the differentiation state (Fig. 1D).
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Page 4 of 16
MiR‑422a and miR‑483‑5p are upregulated
during adipogenesis and affect MeCP2 expression
in hBMSCs and hBMSC‑derived adipocytes
MeCP2
43 kDa
β-actin
MeCP2 mRNA relative expression (a.u.)
75 kDa
MeCP2/B-actin ratio (a.u.)
1.5
*
1.0
0.5
0.0
EV
sh-MeCP2
*
1.5
1.0
0.5
0.0
EV
sh-MeCP2
C
6
*
*
4
2
P2
C
N
BM
R
U
O
N
X2
0
PP
AR
AD G
IP
O
Q
LE
PT
FA
BP
4
PL
IN
1
To unravel the role of MeCP2 modulation in adipogenesis, we reduced its expression in hBMSCs by infecting
these cells with a pool of three MeCP2-targeting shRNAs
(shMeCP2) or control empty-lentiviral vectors (EV). MeCP2
expression was significantly decreased by about 50% both
at protein and mRNA levels in undifferentiated shMeCP2BMSCs compared to EV-BMSCs (Fig. 2A, B). MeCP2
silencing, although showing a not complete abrogation of
the protein level, was sufficient to significantly increase
PPARγ and PLIN1 mRNA expression in undifferentiated
shMeCP2-BMSCs compared to EV-BMSCs (Fig. 2C) and
to up-regulate the late adipogenic markers adiponectin and
leptin, tested at different time points, once induced to differentiate into adipocytes using complete adipogenic medium
(Fig. 2D). Notably, no significant modulation of the osteogenesis-related markers RUNX2, OCN, and BMP2 was
observed in shMeCP2-BMSCs (Fig. 2C). Figure 2D shows
mRNA levels of genes related to adipogenesis in shMeCP2BMSCs undergoing adipogenic differentiation. Notably,
mRNA of ADIPOQ and LEPT were upregulated after 3 days
while LEPT, FABP4, and PLIN1 after 14 days of differentiation in shMeCP2-BMSCs compared to hBMSCs induced
to differentiate into adipocytes with empty vector (ADEV). No significant difference between shMeCP2-BMSCs
and AD-EV was observed for PPARγ mRNA expression
(Fig. 2D).
B
A
sh-MeCP2 infected hBMSC
Fold change vs EV
MeCP2 downregulation promotes adipogenic
program in hBMSCs
D
5
sh-MeCP2 infected
adipocyte-differentiated hBMSC
Fold change vs AD-EV
75
*
3 days of AD
14 days of AD
4
*
*
3
*
*
2
1
0
The silencing of MeCP2 demonstrates its role in adipogenesis, therefore, we deeply explored the mechanisms
underlying MeCP2 protein expression during hBMSC differentiation. We analyze the MeCP2 mRNA levels and the
methylation status of CpGs among a genomic region covering the MeCP2 gene in undifferentiated-BMSCs and cells
cultured for 14 days in pro-adipogenic or -osteogenic media.
The results showed that MeCP2 modulation in differentiated
hBMSCs does not depend on the transcription nor on the
methylation status of its gene (Fig. 3A, B).
These observations prompted the hypothesis that the
decline in MeCP2 expression during adipogenesis could
be related to post-transcriptional mechanisms, i.e. the
interaction between miRNAs and MeCP2 mRNA that may
inhibit the protein translation. To identify a set of miRNAs
potentially involved in BMSC adipogenesis, the differential
miRNA expression profiles of hBMSC-derived adipocytes
(obtained cultivating hBMSCs in adipogenic medium (AD)
for 14 days) and undifferentiated hBMSCs were analyzed
13
PPARγ
ADIPOQ
LEPT
FABP4
PLIN1
Fig. 2 MeCP2 partial silencing induces adipogenesis. MeCP2 expression in undifferentiated hBMSCs infected with shRNA-containing
(sh-MeCP2) or empty lentiviral vectors (EV) was analyzed by A
western blot and densitometric analysis, (data were normalized to
β-actin) and B by RT-PCR. C Adipogenesis (left)- and osteogenesis
(right)-related mRNA fold change in hBMSCs infected with shRNAcontaining lentiviral vectors vs cells infected with empty vector (EV).
D Adipogenesis-related mRNA fold change in hBMSCs induced to
differentiate for 3 and 14 days with shRNA-containing vectors vs
hBMSCs induced to differentiate for 3 and 14 days with empty vector (AD-EV). Data are mean ± SD of three independent experiments.
*t-test p < 0.05
by performing a Taqman miRNA qRT-PCR-array. PPARγ
and ADIPOQ RT-PCR analysis and fat-soluble staining
Oil Red images were used to verify hBMSC differentiation
(Supplementary Fig. 1A). Out of 384 tested miRNAs, 15
miRNAs were significantly upregulated (fold change ≥ 5.0),
MiR‑422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal…
while 14 were downregulated (fold change ≤ −5.0) in AD
compared to hBMSCs (Fig. 3C). Complete profiling results
have been deposited in NCBI’s Gene Expression Omnibus
(GEO) (https://www.ncbi.nlm.nih.gov/geo) with accession
reference GSE189508.
In the attempt to identify miRNAs involved in adipogenesis with a possible role in the post-transcriptional modulation of MeCP2, we first proceeded with qPCR validation of
the five most upregulated miRNAs (fold change (log2) > 4),
miR-98, -139-3p, -422a, -330-5p, and -483-5p. The analysis confirmed the results of the microarray with miR-422a
showing the highest expression among all miRNAs tested
(Fig. 3D). However, miR-139-3p validation yielded high Ct
values (> 30) in all conditions tested, suggesting its negligible expression (data not shown).
To identify miRNAs endowed with a pro-differentiating
effect and those specific for adipogenesis, we also assessed
their expression in osteoblasts obtained by culturing hBMSCs for 14 days in an osteogenic medium (OS) (Fig. 3D).
All miRNAs were significantly up-regulated in OS, except
for miR-422a which did not show a significant modulation
in OS compared to hBMSCs. Osteogenesis was assessed
by mRNA analysis of Osteocalcin, BMP2 and Runx2, and
Alizarin Red S staining (Supplementary Fig. 1B).
To further confirm these results, the expression of the
four miRNAs was tested in human BMSCs and AD isolated
from the bone marrow of the same donors. Consistently,
the expression of all validated miRNAs was significantly
upregulated in isolated AD compared to BMSCs (Fig. 3E).
Overall, these results suggest that miR-98, miR-422a,
miR-330-5p and miR-483-5p may have a role in hBMSC
differentiation, with miR-422a being more specific for
adipogenesis.
To test whether miR-98, miR-422a, miR-330-5p, and
miR-483-5p may affect hBMSC differentiation by interacting with MeCP2, we performed a search into the ENCORI
miRNA–mRNA interaction database, which collects information from five different prediction tools, observing that
MeCP2 mRNA is a predicted target of miR-422a (PITA
score = −16.08, interaction supported by 2 Ago CLIP-seq
experiments) and miR-483-5p (PITA score = −19.01, Pancancer score = 6, interaction supported by 2 Ago CLIP-seq
experiments), which has been previously demonstrated to
downregulate MeCP2 expression by binding the 3′ UTR
of its mRNA [32]. Given the upregulation of the former in
differentiated hBMSCs and the specific expression of the
latter in hBMSC-differentiated into adipocytes, we focused
the subsequent experiments on the role of miR-422a and
miR-483-5p.
To investigate whether these two miRNAs are responsible for adipocyte MeCP2 down-regulation, hBMSCs
and hBMSC-differentiated adipocytes were transfected
for 72 h with specific miRNA mimics or inhibitors,
Page 5 of 16 75
respectively. Figure 3F shows that both miRNA mimics strongly reduced MeCP2 expression in undifferentiated cells. In line with these results, miR-422a and miR483-5p inhibitors induced upregulation of MeCP2 protein
in adipocytes (Fig. 3G). On the other hand, miR-422a and
miR-483-5p expression were not significantly modulated
by MeCP2 silencing (Supplementary Fig. 2). We also performed a luciferase reporter assay, which confirmed that
miR-422a binds at least one of the two 3′ untranslated
regions of MeCP2 (Fig. 3H).
MiR‑422a and miR‑483‑5p promote adipogenesis
in hBMSCs
To investigate the role of miR-422a in adipogenesis, we
analyzed the expression of several specific molecular markers of adipogenesis in hBMSCs induced to differentiate for
14 days in the adipogenic medium in which miR-422a mimic
was added instead of indomethacin, i.e. the component of
the adipogenic cocktail exerting the greatest impact on the
expression of key regulator genes of adipogenesis and lipid
accumulation [35]. We also tested the effect of miR-483-5p,
which we observed to be significantly up-regulated both in
adipogenesis and osteogenesis (Fig. 3B) and whose role in
promoting adipogenesis has already been demonstrated by
previous reports [36]. We found that supplementation with
miR-422a mimic induced a significant expression of the
transcripts of all the genes tested (PPARγ, GLUT4, FATP1,
FATP4, ACSL1, LEP, ADIPOQ, and PLIN1), in some
cases (PPARγ, GLUT4, FATP1, FATP4, ACSL1) with an
efficiency comparable to indomethacin (CTR +) (Fig. 4A).
MiR-483-5p mimic promoted the upregulation of ACSL1,
PLIN1, and ADIPOQ but less efficiently than miR-422a.
qPCR analysis of intracellular miR-422a and -483-5p confirmed the efficiency of the transfection (Supplementary
Fig. 3).
To strengthen these data, we further analyzed the effect
of miR-422a or miR-483-5p inhibition during hBMSC differentiation by adding their specific antagomiRs to the complete adipogenic medium Notably, after 14 days of culture,
the expression of PPARγ, PLIN1, LEP, and ADIPOQ was
significantly reduced in miR422a-antagomiR transfected
cells compared to adipogenic medium alone (CTR +). MiR483-5p inhibition yielded similar results only for LEP and
ADIPOQ (Fig. 4B).
The effect of both miR-422a and miR-483-5p on adipogenesis was further investigated by Oil Red O staining of
hBMSC transfected with miRNA mimics or inhibitors in
the same conditions described earlier (Fig. 4C). Lipid content is increased by miR-422a as well as by miR-483-5p
mimic transfection compared to the adipogenic medium
without indomethacin (CTR-). Accordingly, miR-422a or
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miR-483-5p inhibition by antagomirs significantly reduced
lipid droplet formation induced by complete adipogenic
medium (CTR +).
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A. Giuliani et al.
Since adiponectin is an essential factor secreted by
mature adipocytes and miR-422a and miR-483-5p mimics
alone were able to induce adiponectin (ADIPOQ) mRNA
MiR‑422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal…
◂Fig. 3 miRNA expression in adipogenesis. A MeCP2 mRNA relative
expression in arbitrary units (a.u.). Data were normalized to IPO8.
B DNA methylation profile of the genomic region encompassing
MeCP2 (chrX:153282264–153368188, hg19 assembly). The position of microarray probes along the chromosome is reported in the
x-axis, while the y-axis reports DNA methylation levels expressed
as beta values, ranging from 0 to 1. C Bar chart reporting the (log2)
fold changes of the miRNAs showing a differential expression in
adipocytes (AD) compared to hBMSCs. The figure showed miRNAs with absolute (foldchange) ≥ 5. In green and in red are shown
miRNA upregulated and downregulated in adipocytes, respectively.
D miRNA relative expression validated through Real-time PCR. E
Fold-change relative expression of miRNA of adipocytes compared
with MSCs from the same donors. F Western blot and densitometric
analysis of MeCP2 in hBMSCs transfected with miRVANA miRNA
mimic negative control #1 (CTR) and in hBMSCs transfected with
miR-422a and miR-483-5p miRNA mimics. Data were normalized to
β-actin. G Western blot and densitometric analysis of MeCP2 in adipocytes transfected with miRNA inhibitor negative control #1 (ADCTR) and in adipocytes transfected with miR-422a and miR-483-5p
inhibitors. Data were normalized to β-actin. H Luciferase reporter
assay. HEK293 cells were infected with either negative control (NC)
or miR-422a mimic, then transfected with the luciferase constructs of
the wild-type MeCP2 3′-UTR (MeCP2-miR-422a-WT) or a mutated
MeCP2 3′-UTR (MeCP2-miR-422a-mut). The luciferase activity was
analyzed. *t-test p < 0.05; **t-test p < 0.01; ***t-test p < 0.001
expression in hBMSC cultured in adipogenic medium without indomethacin (Fig. 4A), we assessed the amount of
secreted adiponectin in this conditioned medium and compared its concentration with that released in the medium
enriched with indomethacin (CTR +) or without miRNA
mimics and indomethacin (CTR−). Both mimics of miR422a and miR-483-5p significantly increased adiponectin
release compared to negative control. Accordingly, miRNA
inhibitors were able to reduce the amount of secreted adiponectin in the complete adipogenic medium (Fig. 4D).
As previously shown in Fig. 3, miR-422a is not modulated during hBMSC osteogenesis, contrary to miR-483-5p
which is strongly up-regulated. Accordingly, forced expression of miR-422a inhibitor did not affect the expression of
RUNX2, the master transcription factor for osteogenesis
[37], and two other osteogenic-related genes—bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN)—after
14 days of pro-osteogenic conditions. However, miR-422a
mimic caused a significant strong reduction of both BMP-2
and OCN gene expression (Fig. 4E). Interestingly, miR483-5p inhibition reduced RUNX2 expression.
MiR‑422a and ‑483‑5p are released
from differentiating adipocytes and are present
in plasma from subjects with osteoporosis
To assess whether miRNAs related to hBMSC adipogenesis
can be released in extracellular fluids we evaluated the levels
of miR-422a and -483-5p in conditioned media harvested in
the last 3 days out of the 14 days of culture in adipogenic and
Page 7 of 16 75
osteogenic medium. We observed that miR-422a level was
significantly higher in the AD-conditioned medium compared to both undifferentiated hBMSC and OS-conditioned
media, while miR-483-5p was similarly upregulated in both
AD and OS-conditioned media compared to hBMSCs one
(Fig. 5A).
To investigate the value of miR-422a and -483-5p as
promising non-invasive candidate biomarkers in osteoporosis, we checked their circulating levels in subjects with
primary type II osteoporosis (OP) and age-/gender-matched
control subjects (CTR). The clinical and biochemical characteristics of the subjects are listed in Table 1. The levels of
circulating miR-422a were higher in OP subjects compared
to CTR (p = 0.0007), whereas no significant difference was
shown for miR-483-5p (Fig. 5B). Finally, we analyzed the
correlation between circulating miR-422a levels and bone
mineral density assessed through dual-energy X-ray absorptiometry (DXA) in the whole cohort. Interestingly, we found
significant negative correlations between circulating miR422a and both T-score (p = 0.002) and Z-score (p < 0.001)
(Fig. 5C).
Discussion
Adipogenesis is intimately linked to osteogenesis in the
bone marrow milieu. Since bone and adipose tissue share
a common origin, the identification of factors driving the
hBMSC adipogenic program is of high relevance to human
diseases characterized by disruption to the differentiation
balance, such as osteoporosis and aging [38]. Interestingly,
it has been suggested that MeCP2 plays a role in regulating
both subcutaneous adipogenic process and, furthermore,
osteogenesis in a rodent model of Rett syndrome [10, 39,
40], therefore, we hypothesized that it may represent a key
factor addressing hBMSCs to one or the other differentiation
pathway within the bone marrow.
In this work, we show that MeCP2 expression is modulated in an opposite way in adipocytes and osteoblasts both
in vitro and in vivo and that its partial silencing in hBMSCs
results in the induction of a pro-adipogenic transcriptional
program. We demonstrate that MeCP2 modulation does
not depend on different expression levels of its mRNA and
accordingly on the methylation status of its gene, but on the
expression of specific miRNAs. In fact, using a microarray
approach, we have identified several differentially expressed
miRNAs in hBMSC-derived adipocytes compared to undifferentiated cells. We focused on the most upregulated miRNAs, which could act as post-transcriptional repressors of
MeCP2 expression during adipogenesis. Notably, miRNAmRNA target prediction analysis aimed at identifying which
of the most upregulated miRNAs could regulate MeCP2
expression showed that both miR-422a and miR-483-5p are
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Fig. 4 miR-422a and miR-483-5p promote adipogenesis in hBMSCs.
A Adipogenesis-related mRNA fold change in hBMSCs transfected
with miRNA mimics and negative miRNA mimic controls (CTR−
and CTR +). CTR- indicates hBMSCs induced to differentiation in
absence of indomethacin, while CTR + with indomethacin. MiRNA
mimics were added to the adipogenic medium without indomethacin.
* vs CTR−; # vs miR-422a mimic, ° vs miR-483-5p mimic. B Adipogenesis-related mRNA fold change in hBMSCs transfected with
miRNA inhibitors. MiRNA inhibitors were added to the complete
adipogenic medium. CTR + indicates hBMSCs treated with miRNA
inhibitor negative control #1 and induced to adipogenesis with a
complete adipogenic medium. * vs CTR + ; # vs miR-422a inhibi-
tor. C Representative images and densitometric quantification of cells
staining with Oil Red O. For mimic experiments: * vs CTR−; # vs
miR-422a mimic, ° vs miR-483-5p mimic. For inhibitor experiments:
* vs CTR + ; # vs miR-422a inhibitor. D Graph chart represents adiponectin released into the culture medium expressed in ng/ml. For
mimic experiments: * vs CTR−; # vs miR-422a mimic, ° vs miR483-5p mimic. For inhibitor experiments: * vs CTR + (E) Osteogenesis-related mRNA fold change in hBMSCs transfected with miRNA
mimics and inhibitors of miR-422a and miR-483-5p. * vs CTR + ; #
vs miR-422a mimic. Data are mean ± SD of three independent experiments. *, #, ° t-test p < 0.05; **, ##, °° t-test p < 0.01; ***, °°°t-test
p < 0.001
able to target MeCP2 mRNA. However, since miR-422a is
the only one modulated in adipogenesis but not in osteogenesis, we thought it may be the right candidate to reduce
MeCP2 expression in adipocytes. We found for the first
time that forced expression of miR-422a and miR-483-5p
in hBMSC can significantly downregulate MeCP2 expression and promote adipogenesis, with miR-422a being more
efficient in inducing the expression of adipogenic markers,
13
MiR‑422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal…
Page 9 of 16 75
Fig. 5 miR-422a has a higher expression in plasma of osteoporotic subjects compared with non-osteoporotic samples. A miRNA
fold change in the culture medium of cells induced to differentiate
into adipocytes (AD) and osteoblasts (OS) compared to hBMSCs. Data are mean ± SD of three independent experiments. *t-test
p < 0.05. B Violin plots showing miRNA relative expression in
plasma of non-osteoporotic (CTR) and osteoporotic (OP) subjects.
C Scatter plot showing correlations between relative miR-422a
expression levels (in arbitrary units, a.u.) and T-score or Z-score. Data
are expressed as a mean of 2−ΔCt normalized with cel-miR-39
including adiponectin. Albeit the concentration of adiponectin released by cells treated with miRNA mimics did not
reach the levels of positive control (adipogenic medium
with indomethacin), we observed a significant > fivefold
increase compared to cells treated with the medium without indomethacin. Indeed, indomethacin is the major driver
of adiponectin synthesis in in vitro models of adipogenesis [41]. Interestingly, data obtained with miR-483-5p are
in agreement with those found by others on its ability to
inhibit MeCP2 expression during fetal development [32] and
to regulate adipogenesis in the subcutaneous environment
[36]. On the contrary, miR-422a and miR-483-5p inhibition rescues MeCP2 expression in adipocytes and reduces
adipogenic marker expression. Interestingly, an in vitro
study on human visceral preadipocytes showed that metformin inhibits adipogenesis with a concomitant reduction of miR-422a levels [42]. More in general, miR-422a
increased during adipogenesis, exerted a greater impact
on the adipogenic process compared to miR-483-5p and
induced a significant inhibition of the osteogenic markers
BMP-2 and osteocalcin. Overall, these observations suggest
that miR-422a is specifically boosted in adipogenesis and
may possibly inhibit osteogenesis, a hypothesis that deserves
future investigations.
The great effect of miR-422a and miR-483-5p on adipogenesis may be related at least in part to the downregulation
of MeCP2 expression in differentiating hBMSCs. Indeed,
partial silencing of MeCP2 in undifferentiated mesenchymal cells led to the acquisition of an adipogenic profile with
upregulation of PPARγ and PLIN1 mRNAs, independently
from miR-422a and miR-483-5p. Of note, the expression
of miR-422a and miR-483-5p does not depend on MeCP2
itself because they are not modulated by its silencing. Furthermore, this effect is also confirmed by the upregulation of
adiponectin, leptin, FABP4, and PLIN1 mRNAs in silenced
cells harvested in an adipogenic medium compared to cells
transfected with an empty lentiviral vector. However, in this
condition, we did not observe an effect on the mRNA levels
of the transcription factor PPARγ, which may be covered
by the strong induction of the adipogenic cocktail. In agreement with our findings, RTT patients show high levels of
circulating leptin [43, 44] and adiponectin [45]. Adiponectin, one of the most extensively studied adipocyte-derived
factors, exerts a plethora of beneficial effects through its
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A. Giuliani et al.
Page 10 of 16
Table 1 Biochemical and anthropometric characteristics of subjects
enrolled for the study
Variables
Control subjects
(n = 16)
Age (years)
83.3 (5.9)
Gender (males, %)
7 (44%)
BMI (kg/m2)
24.8 (2.0)
Creatinine (mg/dL)
1.3 (0.5)
Sodium (meq/L)
141.6 (3.5)
Potassium (meq/L)
4 (0.4)
Calcium (mg/dL)
8.9 (0.5)
Hemoglobin (g/dL) 12.4 (1.7)
ESR (mm/h)
39.6 (21.1)
CRP (mg/L)
2.9 (2.7)
T-score
0.3 (0.7)
Z-score
2.4 (0.8)
BMD
256.3 (485.6)
Primary type
II osteoporosis
(n = 17)
86.3 (3.5)
5 (29%)
23.4 (2.5)
1.4 (0.7)
141.2 (4.9)
3.9 (0.3)
8.6 (0.7)
11.0 (1.3)
52.4 (31.8)
3.8 (2.7)
−2.3 (1)
−0.4 (0.8)
65.1 (249)
p-Value
0.083
0.392
0.087
0.642
0.790
0.421
0.169
0.012
0.186
0.346
< 0.001
< 0.001
0.161
Data are presented as mean (SD)
BMD bone mineral density; CRP c-reactive protein; ESR erythrocyte
sedimentation rate
p Values for unpaired t test (continuous variables) or Chi-squared test
(categorical variables)
insulin-sensitizing, anti-atherogenic, and anti-cancer properties [46, 47]. Since MAT appears to be the major contributor to circulating adiponectin, it has been suggested that its
increase may have beneficial effects in compromised health
conditions such as anorexia nervosa and chemotherapy
although this increase is related to a condition of osteoporosis [14].
In fact, one of the proposed mechanisms in the pathogenesis of osteoporosis is a shift of hBMSC differentiation
toward adipocyte rather than osteoblast [48]. For this reason,
it is important to characterize the broad range of mediators
in the bone marrow milieu that can regulate the commitment
of MSCs. Studies on RTT patients and different RTT mouse
models highlighted the existence of a direct relationship
between MeCP2 loss of function and the alteration of bone
homeostasis, which contributes to the onset of osteoporosis
and to a higher risk of bone fracture [10, 49–51]. Importantly, a mouse model in which MeCP2 has been reactivated
specifically in the nervous system but remained silenced
elsewhere showed that bone abnormalities are due to a loss
of MeCP2 in peripheral tissues [52], confirming a metabolic component in RTT syndrome. Interestingly, a recent
study showed that the overexpression of MeCP2 in BMSCs
enhanced the expression of osteogenic markers, including
RUNX2 and osteocalcin, and promoted calcium deposition
in a mouse model of osteoporosis [53]. Here, we showed that
not only MeCP2 genetic silencing can affect bone biology
but even that specific miRNAs affecting MeCP2 expression,
13
i.e. miR-422a and -483-5p are capable to influence osteogenesis when hBMSCs were cultured under proper conditions.
Besides MeCP2, miR-422a significantly reduced mRNA
levels of other specific targets involved in bone biology,
BMP2, and osteocalcin, while miR-483-5p inhibitor induced
a decline in RUNX2 mRNA levels. Accordingly, a recent
report showed that intra-articular injection of miR-483-5p
inhibitor delays the development of osteoarthritis through
the reduction in the number of RUNX2-positive chondrocytes [54]. Nonetheless, miR-483-5p resides in an intron
of the IGF2 gene and it has been shown to upregulate the
expression of its host gene [55], which is involved in longitudinal and appositional bone growth [56]. Overall, these
data suggest that both miRNAs are somehow involved in the
commitment of hBMSCs, with miR-422a exerting a more
pronounced effect toward the adipogenic lineage. In addition, miR-422a showed a higher expression in the plasma
of osteoporotic patients compared with non-osteoporotic
controls and was negatively related to T-score and Z-score.
A previous study showed that the search for circulating
miRNAs as minimally invasive biomarkers for osteoporosis revealed that miR-422a is upregulated in circulating
monocytes from low BMD postmenopausal women [57].
De-Ugarte and colleagues showed significant overexpression
of miR-483-5p through a microRNA array on bone samples
from postmenopausal women with a history of osteoporotic
fractures [58]. However, this latter observation was not replicated in our cohort, probably due to the limited number of
patients and to the different forms of osteoporosis (type II,
age-related vs. type I, postmenopausal) considered.
In conclusion, we showed that miR-422a and miR-483-5p
act as pro-differentiation factors in hBMSCs and that these
two miRNAs can affect the adipogenesis process by influencing MeCP2 expression. Our findings emphasize the need
to unravel MeCP2 expression and regulation in peripheral
tissues, especially in bone marrow stromal cells, with a look
at the potentially related diseases. A thorough comprehension of the factors capable to affect hBMSC differentiation
is important not only in the context of bone mineral disease. Many efforts have been devoted to preventing metabolic complications due to the accumulation and increased
secretory activity of visceral adipose tissue. Of note, BM
adipose tissue sustains its own integrity through the release
of extracellular vesicles (EVs) containing a typical adipocyte
signature, as well as anti-osteoblastic miRNAs exerting their
effects on the nearby hBMSCs [59]. It is tempting to speculate that such EVs could be secreted also in the bloodstream,
affecting adipose tissue homeostasis at the systemic level.
In this regard, the study of EVs as mediators of the extracellular dynamics of miR-422a and miR483-5p represents
an intriguing future perspective for the present research.
Future investigations are warranted to disentangle the roles
of miRNAs showing opposite patterns of modulation during
MiR‑422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal…
adipogenesis and to understand how these miRNAs integrate
into a complex network regulating adipose tissue formation
and function.
Page 11 of 16
75
analysis, ORO was extracted from the cells with isopropanol
and quantified spectrophotometrically at 520 nm.
RNA extraction and RNA expression
Materials and methods
Cell culture and differentiation
Human bone marrow stromal cells (hBMSCs) were purchased from Lonza (Allendale, NJ, USA) and maintained
in α-MEM (Euroclone, 20016, Milano, Italy) supplemented
with 10% fetal bovine serum (FBS) (Lonza), 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin.
Experiments were performed using different batches of
BMSCs up to the fifth passage.
For adipogenic differentiation, BMSCs were cultured as
shown in Rippo et al. [60]. Briefly, hBMSCs were seeded at
8 × 103 cells/cm2 on six-well plates in AD containing complete a-MEM, supplemented with 0.5 mM dexamethasone,
5 mg/ml insulin, 0.45 mM isobutylmethylxanthine (IBMX),
and 0.2 mM indomethacin (Sigma-Aldrich, St. Louis, MO,
USA). In specific miRNA mimics experiments, the adipogenic medium was supplemented with miR-422a and
miR-483-5p mimics instead of 0.2 mM indomethacin. In
these sets of experiments, we used two different adipogenic
cocktails as controls, with (CTR +) or without indomethacin
(CTR−).
Dedifferentiation of adipocytes obtained from three different subcutaneous fat tissue human samples was obtained
as previously described in Poloni et al. [33]. BM-derived
mesenchymal cells and adipocytes were collected from
patients undergoing hip surgery and maintained in their
growth medium until analysis.
All tissue samples were collected in accordance with local
ethics committee guidelines (300/DG), and all participants
provided their written informed consent to take part in the
study. During the dedifferentiation process, mature adipocytes lost their lineage gene expression profile, assumed the
typical mesenchymal morphology and immunophenotype,
and expressed stem cell genes.
Adipocyte staining
Adipocyte differentiation was assessed by Oil Red O (ORO)
staining. Briefly, cells were washed with PBS and fixed with
4% paraformaldehyde for 5 min. After fixation, samples were
washed twice in PBS, followed by incubation with freshly
filtered ORO staining solution (six parts Oil Red O stock
solution and four parts H2O; Oil Red O stock solution is
0.5% Oil Red O in isopropanol) for 30 min. For quantitative
Total RNA was recovered from hBMSCs using the Total
RNA Purification Kit purchased from Norgen (#37500,
Norgen Biotek, Thorold, ON, Canada) which allows the
isolation of both microRNAs and mRNAs. RNA was used
immediately or stored at −80 °C until analysis by Real-Time
(RT)-PCR.
mRNA analysis
1 µg of RNA was transcribed into cDNA using PrimeScript™ RT Reagent Kit with gDNA Eraser (RR047A,
Takara Bio) according to the manufacturer’s instructions.
One-twentieth of first-strand cDNA was used as a template
for RT-PCR amplification. RT-PCR was performed with TB
Green Premix Ex Taq (Tli RNase H Plus) (RR420A, Takara
Bio) in a reaction volume of 10 µl with specific primers
according to the protocol. β-actin or/and IPO8 was used
as reference gene. Primers were listed in Supplementary
Table 1.
MicroRNA analysis
MiRNAs were reverse transcribed following the manufacturer’s instructions (#4366596, Thermo Fisher Scientific) using
specific stem-loop primers for each miRNA. The RT-PCR
reaction mix included TaqMan MicroRNA assay (#4427975,
Thermo Fisher Scientific), TaqMan Universal Master mix no
UNG (4440040, Thermo Fisher Scientific) and RT product.
RNU48 was used as a reference gene. The 2−ΔCT method was
used to determine miRNA expression.
TaqMan MicroRNA array analysis of mature
microRNAs
Microarray analysis was performed as previously described
[61, 62]. In brief, the previously isolated RNA was reverse
transcribed by priming with a mixture of looped primers
using the manufacturer’s instructions (Megaplex RT primers
Human Pool A v2.1, Thermo Fisher Scientific). Pre-amplification of cDNA was performed using TaqMan Preamp Master Mix (#4384266, Thermo Fisher Scientific) and Megaplex
PreAmp Primers (10×), Human Pool A v2.1 (#4399233,
Thermo Fisher Scientific). Pre-amplified cDNA was used for
mature miRNA profiling by RT-PCR instrument equipped
with a 384-well reaction plate (7900 HT, Applied Biosystems) and TaqMan Array Human MicroRNA Cards v2.0
pool A (#4398977, Thermo Fisher Scientific) containing
367 different human miRNA assays in addition to selected
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A. Giuliani et al.
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small nucleolar RNAs. miRNAs expressed (Ct ≤ 30) in at
least one condition (hBMSCs, adipogenesis) were included
in the analysis. Data were presented as log2 fold change versus undifferentiated hBMSCs.
miRNA target prediction analysis
The open-source encyclopedia of RNA interactomes (http://
starbase.sysu.edu.cn/index.php) [63] was utilized to locate
target genes of miR-422a and miR-483-5p.
by recombination reaction, HEK293T cells were transfected
for 48 h with the pmirGLO-UTR reporter plasmid in combination with Negative Control (NC, sequence UUC UCC
GAACGUGUCACGUUU) mimics or miR-422a-5p mimics (sequence: ACUGGACUUAGGGUCAGAAGGC) at a
final concentration of 20 nM in 25 μl of pure DMEM. The
Firefly luciferase activity, normalized to Renilla luciferase
(for transfection efficiency), was determined with the dualluciferase reporter assay system (Promega), according to the
manufacturer’s instructions, and reported as % of the negative control mimic activity.
Cell transfection with miRNA mimics and inhibitors
Adiponectin production
Transfection of miRNA inhibitors and mimics was conducted as previously described [64]. Briefly, 1 × 105 hBMSCs were plated in six-well plates and incubated overnight
before transfection with miR-422a and miR-483-5p miRVANA miRNA mimics (MC12541, MC12629), miRVANA
miRNA inhibitors (MH12541, MH12629), miRVANA
miRNA inhibitor negative control #1 (4464077) or with
miRVANA miRNA mimic negative control #1 (4464058, all
from Thermo Fisher Scientific, San Jose, CA, USA) at a concentration of 30 nM. Transient transfection was performed
using TransIT-2020 transfection reagent (MIR 5404, Mirus
Bio LLC, Madison, WI, USA), according to the manufacturer’s protocol. The ratio of transfection reagent (µl)/miR
(µg) equal to 2:1 was found to be optimal. Transfection was
carried out concomitantly with the induction of differentiation and repeated at every medium replacement. Analyses on
adipogenesis induction were performed after 14 days after
transfection.
Luciferase reporter assay
The luciferase reporter assay was performed by Creative
Biogene Biotechnology (Shirley, NY, USA). The wild-type
MeCP2 reporter (MeCP2-WT) and the mutant MeCP2
reporter (MeCP2-Mut) were generated by subcloning the
3′-UTR sequences of MeCP2 bracketing the predicted
miR-422a-5p binding site and the full-length sequences of
MeCP2-Mut into the XhoI/XbaI site located at 3′UTR of
pmirGLO (Promega) vectors. The MeCP2 3′-UTR sequences
were as follows: WT human MECP2 3′UTR sequence
(miRNA binding sites in italics), CGACCTTGACCTCAC
TCAGAAGT CCAGAGTC TAG CGTAGT GCAGCAGGG
CAGTAGCGGTAATACTTAGTCAAATGTAATGTGGCT
TCTGGAATCATTGTCCAGAGCTGCTTCCCCGTCAC;
mutant human MECP2 3′UTR sequence (mutant sites in italics), CGACCTTGACCTCACTCAGATCAGGTCTCAGATC
CGTAGTGCAGCAGGGCAGTAGCGGTAATACTTAGTC
AAATGTAATGTGGCTTCTGGAATCATTCAGGTCTGCT
GCT TCC CCG TCAC. After ligation of the WT/mutant
human MECP2 3′UTR fragments into linearized pmirGLO
13
Cell medium was collected at the end of the experiments,
centrifugated, and stored at −80 °C until used in the assay.
Adiponectin concentration was measured using a commercially available high-sensitivity enzyme-linked immunosorbent assay (ELISA) (AG-45A-0001YEK-KI01, AdipoGen).
Von Kossa staining
After osteogenic induction for 21 days, cells were fixed in
4% paraformaldehyde for 20 min before being stained with
5% aqueous silver nitrate solution for 45 min at room temperature under the light. Next, the samples were washed with
deionized water and stained with 5% sodium thiosulfate for
10 min.
Specimen collection and immunohistochemistry
Small fragments of human femoral bone were collected from
patients undergoing hip surgery and then fixed in buffered
formalin 10% for 24–48 h. All tissue samples were collected
in accordance with local ethics committee guidelines 300/
DG, and all participants provided their written informed
consent to take part in the study.
After a decalcification step in neutral EDTA-sodium
hydroxide, a conventional paraffin embedding procedure was performed. Inguinal and omental adipose tissue and femoral BM aspirates were obtained from adult
female Sprague–Dawley albino rats (n = 3, 190–220 g;
age, 3 months; Charles River, Milan, Italy). Experiments
were carried out in accordance with the Council Directive
2010/63EU of the European Parliament and the Council of
September 22, 2010, on the protection of animals used for
scientific purposes and approved by the local veterinary
service. Samples were fixed in 4% paraformaldehyde overnight at 4 °C, then paraffin-embedded. Subsequently, 3 µm
sections were obtained from all the specimens and used for
the detection of MeCP2 reactivity. Briefly, following antigen retrieval, tissues were blocked in 3% H2O2 for 15 min
at room temperature, washed, and then probed with rabbit
MiR‑422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal…
polyclonal anti-MeCP2 antibody (abcam #2828. Cambridge,
UK) 1:200 overnight at 4 °C in a humidified chamber. Tissues were washed extensively in PBS and detection was
performed using an HRP-conjugated secondary antibody
followed by DAB colorimetric detection using a kit (Cell
Signalling Technology. MA, USA). Tissues were counterstained with hematoxylin, dehydrated, and mounted. Images
were taken using a Nikon Eclipse 80i microscope.
Methylation analysis
Genomic DNA was extracted from hBMSCs and hBMSCderived adipocytes and osteoblasts using Qiagen’s QiAmp
mini kit following the manufacturer’s recommendations.
Each sample type was extracted and assessed for its DNA
methylation profile in triplicate. Briefly, 1 µg of DNA was
converted with bisulfite using the EZ DNA Methylation Kit
(#D5001, Zymo Research) and analyzed using the Infinium
HumanMethylationEPIC BeadChip (#20042130, Illumina),
which allows assessing the methylation status of more than
850,000 CpG sites across the genome. Raw data files were
extracted using the minfi Bioconductor package (CIT Preprocessing, normalization, and integration of the Illumina
HumanMethylationEPIC array with minfi). Quality check
resulted in the removal of 1536 probes having a detection
p-value < 0.05 in more than 1% of the samples and in the
removal of 98,855 potentially cross-reactive probes according to Zhou et al. (CIT Comprehensive characterization,
annotation and innovative use of Infinium DNA methylation
BeadChip probes). Normalization was performed using the
preprocessFunnorm function implemented in minfi and DNA
methylation was expressed as beta values ranging from 0 (0%
of methylation) to 1 (100% of methylation). DNA methylation values in hBMSCs and hBMSC-derived adipocytes and
osteoblasts were compared pairwise using the limma package and p-values were adjusted using the Benjamini–Hochberg procedure. Adjusted p-values < 0.01 were retained as
significant. For the analysis of MeCP2 DNA methylation,
the genomic region encompassing the gene plus 5000 bp
upstream and downstream (chrX:153282264–153368188,
hg19 assembly) was considered.
Lentivirus construction and infection
LentiLox 3.7 (pLL3.7) vector system was used to induce
RNA interference of MeCP2. Three different short hairpin RNA (shRNA) sequences targeting MeCP2 transcript
(MeCP2sh1, MeCP2sh2, and MeCP2sh3—see Supplementary Table 2 for target sequences) were cloned into pLL3.7
as described [65]. Lentiviruses were produced by co-transfecting 293T cells with shRNA-containing pLL3.7 plasmids, or pLL3.7 empty vector, in combination with packaging plasmids as described [66]. Supernatants of transiently
Page 13 of 16
75
transfected 293T cells were recovered after 36 h and two
cycles (6 h each) of infection of hBMSCs were performed
within 48 h with a pool of the three shRNA-containing lentiviral vectors. EGFP positivity of target cells was monitored
to verify the efficiency of infection which approximately
reached 90%.
Protein extraction and immunoblotting
Total protein was extracted using RIPA buffer (150 mM
NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100,
5 mM EDTA, pH 8.0) containing protease inhibitor cocktail
(Roche Applied Science, Indianapolis, IN, USA) and quantified using the Bradford method. Proteins were separated on
gradient SDS-PAGE gels and transferred to nitrocellulose
membranes (Whatman). Membranes were then incubated
with the primary antibodies overnight at 4 °C. The following
primary antibodies were used: MeCP2 (D4F3) XP Rabbit
(#3456, Cell Signaling Technology); β-Actin (8H10D10)
Mouse mAb (#3700, Cell Signaling Technology); PPARγ
(81B8) Rabbit (#2443, Cell Signaling Technology). After
incubation with the specific HRP-conjugated antibody (Vector; 1:10,000 dilution), the chemiluminescent signal was
detected using Clarity and/or Clarity Max (Bio-Rad, Italy)
and images were acquired with Alliance Mini HD9 (Uvitec,
Cambridge, UK). Densitometric analysis was performed
with ImageJ software (https://imagej.nih.gov/ij/download.
html). Full and uncropped Western Blots were provided as
Supplemental Material.
Plasma samples
Plasma samples were obtained from 16 healthy subjects
(CTR) and 17 osteoporotic patients (OS) enrolled in the
SAFARI study. Subjects were considered healthy if they did
not present osteoporosis, liver diseases, renal failure, history
of cancer, neurodegenerative disorders, infectious or autoimmune diseases. Samples were collected at the Ospedali
Riuniti Marche Nord (Fano, Italy) hospital facilities. The
procedure was approved by the Ethical Committee Regione
Marche (CERM). Written informed consent was collected
from all participants.
Statistical analysis
Data are presented as mean ± standard deviation (SD) of at
least three independent experiments. The Student’s t-test was
applied to determine differences between samples. The correlation between circulating miR-422a levels and the Z-score
and T-score score was assessed using Pearson’s correlation
coefficient. Probability (p) values lower than 0.05 were considered statistically significant. The reported p-values were
13
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A. Giuliani et al.
Page 14 of 16
two-tailed in all calculations. Data were analyzed with SPSS
25.0 (SPSS Inc., IBM, Chicago, IL, USA).
Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s00018-023-04719-6.
Acknowledgements The authors want to acknowledge Dr. Giorgia Fattorini, Università Politecnica delle Marche, for donating rats used for
bone marrow isolation.
Author contributions AG, AS, GM, DR, EM, FP, and LB performed
the cell culture and transfection experiments, qRT-PCR, and most
western blot analyses. AG and JS wrote the manuscript, performed the
statistical analysis, and prepared figures. SA and MF performed experiments and analyzed data on lentivirus construction and cell transfection. LG performed immunohistochemistry stainings. DM and EMB
isolated adipocytes and mesenchymal stromal cells from human bone
marrow samples. MGB performed MeCP2 DNA methylation analysis. EE provided plasma samples from patients with osteoporosis. FL,
ADP, FO, AP, and MF provided valuable help and advice due to their
experience in the field. MRR conceptualized, coordinated and designed
the study.
Funding Open access funding provided by Università Politecnica delle
Marche within the CRUI-CARE Agreement. The work was supported
by grants from Università Politecnica delle Marche (RSA grant) to
ADP, FO, and MRR, and by grants from the Italian Ministry of Health
(Ricerca corrente) to IRCCS INRCA.
Data availability Complete profiling results of hBMSCs miRNAs have
been deposited in NCBI’s Gene Expression Omnibus (GEO) (https://
www.ncbi.nlm.nih.gov/geo) with accession reference GSE189508.
2.
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Declarations
Conflict of interest The authors report no conflicts of interest.
Ethics statement This study use human tissue samples which were collected in accordance with local ethics committee guidelines (Comitato
Etico Regione Marche, 300/DG), and all participants provided their
written informed consent to take part in the study. Plasma samples were
obtained from healthy subjects and patients with osteoporosis enrolled
for the SAFARI study. The study was performed in accordance with
the Declaration of Helsinki.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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