MicroRNA-125a-5p partly regulates the inflammatory
response, lipid uptake, and ORP9 expression in
Ting Chen1, Zhouqing Huang1, Liansheng Wang1, Yue Wang1, Feizhen Wu2, Shu Meng1,
and Changqian Wang1*
1Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road,
Shanghai 200092, Peoples Republic of China; and2Department of Bio-electronic Center, ShangHai University, Shanghai,
Peoples Republic of China
Received 9 January 2009; revised 3 April 2009; accepted 7 April 2009; online publish-ahead-of-print 17 April 2009
Time for primary review: 26 days
Aims The inflammatory responses of monocytes/macrophages and the stimulation of lipid uptake into
these cells by oxidized low density lipoprotein (oxLDL) are critical to the initiation and development
of atherosclerosis. Increasing evidence has demonstrated that many microRNAs play important roles
in the cell proliferation, apoptosis, and differentiation that accompany inflammatory responses.
However, whether microRNAs are associated with monocyte/macrophage inflammatory responses or
oxLDL stimulation is not yet known. The aim of the present study is to investigate microRNAs in mono-
cytes/macrophages and their potential role in oxLDL-stimulation of lipid uptake and other atherosclero-
Methods and results Microarrays were used to analyse the global expression of microRNAs in oxLDL-
stimulated human primary peripheral blood monocytes. Expression profiles of the microRNAs were ver-
ified using TaqMan real-time PCR. Five microRNAs (microRNA-125a-5p, microRNA-9, microRNA-146a,
microRNA-146b-5p, and microRNA-155) were aberrantly expressed after oxLDL treatment of human
primary monocytes. Bioinformatics analysis suggested that microRNA-125a-5p is related to a protein
similar to ORP9 (oxysterol binding protein-like 9) and this was confirmed by a luciferase reporter
assay. MicroRNA-125a-5p was found to mediate lipid uptake and to decrease the secretion of some
inflammatory cytokines (interleukin-2, interleukin-6, tumour necrosis factor-a, transforming growth
factor-beta) in oxLDL-stimulated monocyte-derived macrophages.
Conclusion MicroRNA-125a-5p may partly provide post-transcriptional regulation of the proinflamma-
tory response, lipid uptake, and expression of ORP9 in oxLDL-stimulated monocyte/macrophages.
Atherosclerosis is a chronic inflammatory disease of the
arterial wall with enormous epidemiological relevance1,2
and monocytes/macrophages play important roles in the for-
mation of atherosclerotic lesions.3,4Circulating monocytes
migrate under the subendothelial space and differentiate
into macrophages as one of the key steps in the develop-
ment of atherosclerosis. Macrophages then take up oxidized
low density lipoprotein (oxLDL), which leads to their conver-
sion into foam cells,5–7and these foam cells can in turn
secrete many proinflammatory factors, such as transforming
growth factor (TNF)-a and interleukins (ILs).8Cholesterol
loaded macrophage foam cells are the hallmark of early
atherosclerosis and eventually undergo secondary necrosis
to form the lipid core of advanced atherosclerotic plaques.
When exposed by rupture or erosion, the core triggers
acute thrombotic events leading to myocardial infarction
In addition, oxLDL has long been known to contain lipids
with chemotactic activity toward human monocytes, there-
fore, its accumulation also increases monocyte recruitment
to the vascular wall.10Thus, monocyte/macrophage involve-
ment in atherosclerosis appears to involve a range of func-
tions, including the expression of multiple proinflammatory
factors, adhesion molecules, chemotactic factors, and sca-
venger receptors that are regulated by oxLDL-stimulation.11
*Corresponding author. Tel: þ86 21 3377 6115; fax: þ86 21 65790000 7058;
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Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2009.
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Cardiovascular Research (2009) 83, 131–139
However, little is known regarding the complex upstream
regulators of gene expression and translation involved in
MiRNAs, an emerging class of highly conserved, non-
coding small RNAs, regulate gene expression at the post-
transcriptional level by inhibiting the translation of protein
from mRNA or by promoting the degradation of mRNA.
MiRNAs are transcribed by RNA polymerase II as part of a
primary transcript12,13that is processed by Drosha and
DGCR8 into a smaller RNA molecule.14Mature miRNAs
specifically bind to 30-UTRs of target cellular mRNAs,
leading to either mRNA degradation or inhibition of trans-
lation.15More than 500 human miRNAs have been identified
so far, and increasing evidence indicates that miRNAs have
distinct expression profiles and play essential roles in
various physiological and pathological processes, including
cardiogenesis, haematopoietic lineage differentiation, and
A few specific miRNAs that regulate endothelial cell func-
tions and angiogenesis have been described.16For example,
Let7-f, miR-27b, and miR-130a have been identified as
pro-angiogenic miRNAs. Others, such as miR-221 and
miR-222, have been shown to inhibit in vitro endothelial
cell migration, proliferation, and angiogenesis.16
studies also indicate that specific miRNAs (e.g. miR-155,
miR-21, and miR-126) are responsible for vascular inflam-
mation and diseases.16Thus, the identification of miRNAs
and their respective targets may offer new therapeutic
strategies for the treatment of a number of vascular dis-
eases such as atherosclerosis, for improvement of neovascu-
larization after ischaemia, or for the prevention of
atherosclerotic inflammation. Therefore, the aim of the
present study was to use a combination of microarray and
TaqMan real-time PCR to analyse miRNA expression profiles
in oxLDL-stimulated human primary peripheral blood mono-
cytes, to study the possible roles of miRNAs in atherosclero-
tic processes in these cells.
The investigation conformed with the principles outlined in the
Declaration of Helsinki for use of human blood and was approved
by the Ethics Committee of Experimental Research, JiaoTong Uni-
versity Shanghai Medical College.
2.1 Human primary peripheral blood monocyte
isolation and culture with oxLDL
Peripheral human blood was obtained from healthy donors. Mono-
nuclearcells were isolated
Ficoll-isopaque (Sigma, St Louis, MO, USA) density gradient.17To
obtain the monocytes, the mononuclear cells were allowed to
adhere to six-well plates with 5% autologous serum for 2 h at 378C,
in a 5% CO2incubator. Non-adherent cells were removed and the
adherent cells were co-cultured with oxLDL (30 mg mL21) for 6–12 h.
2.2 Analysis of the expression of miRNAs by
mParafloTMMicroRNA microarray assay
Total RNA was extracted from monocytes using Trizol reagent
according to the manufacturer’s instructions (Qiagen kit). Microar-
ray assay was performed with the mParafloTMMicroRNA microarray
assay system (LC Sciences). The assay was initiated with 2–5 mg
total RNA, and the small RNAs (,300 nt) isolated were 30-extended
with a poly(A) tail using poly(A) polymerase. Thereafter, an
oligonucleotide tag was ligated to the poly(A) tail for later fluor-
escent dye staining; two different tags were used for the different
RNA samples. The detection probes were made by in situ synthesis
using photogenerated reagent chemistry. The detection used fluor-
escence labelling by tag-specific Cy3 and Cy5 dyes, whereas the
images were collected using a laser scanner (GenePix 4000B, Mol-
ecular Device) and were digitized using the Array-Pro image analysis
software (MediaCybernetics). Each miRNA probe was repeated in
septa-replicate on the chip. Microarray data were analysed by
first subtracting the background (the median of 5–25% of the
lower signal intensities) and then the signal was normalized using
a LOWESS (locally weighted regression) filter.18For each sample, tri-
plicates were analysed at each time point and significant differ-
ences between 0, 6, 12 h for a given detectable miRNA signal
were calculated. The ratio of the two sets of detected signals (log
2 transformed, balanced) and the P-values of the t-test and
ANOVA were calculated. The differentially detected signals with
P , 0.05 were analysed using gene hierarchical clustering of the
log2 value of each different time group and then were displayed
in a heatmap. Clustering was performed using Cluster 3.0 created
by Michiel de Hoon, Seiya Imoto, and Satoru Miyano, University of
Tokyo, Human Genome Center (Euclidean distance, links using the
average) and viewed in heatmap using Java TreeView 1.0.13
2.3 miRNA real-time quantitative PCR
As determined by the microarray results, the five most notable aber-
miR-146b-5p, and miR-155) were further measured using TaqMan
real-time PCR. The PCR reaction was directly monitored by the
ABI PRISM 7000 Sequence Detection System (Applied Biosystems,
CA, USA). Briefly, cDNA was made from enriched miRNA using the
TaqMan MicroRNA RT kit. U6 RNAwas used as an endogenous control.
2.4 THP-1 cell culture and anti-miRNA transfection
The human monocytic cell line THP-1 was obtained from the Amer-
ican Type Culture Collection (ATCC, Rockville, MD, USA). This was
maintained at a density of 106/mL in RPMI 1640 medium sup-
plemented with 10% foetal bovine serum, 10 mM HEPES (Sigma),
1% pen/strep solution, and incubated in a 5% CO2 incubator at
378C. To induce monocyte differentiation to macrophages, THP-1
cells were cultured with 100 nM phorbol 12-myristate 13-acetate
(PMA) (Calbiochem, San Diego, CA, USA) for 24 h.19
MiR-125a-5p inhibitor (Ambion) was transfected into PMA-induced
THP-1 using the fast transfection protocol recommended by Qiagen.
The miR-125a-5p inhibitor was diluted in the medium at a final con-
centration of 50 nM. To confirm the efficiency of transfection, the
same amount of Cy3-labelled negative control (Ambion) was also
transfected. Cells were incubated with miR-125a-5p inhibitor for
24 h and then exposed to oxLDL (50 mg mL21) for 24 h.
2.5 HPLC analysis of the lipid levels
PMA-differentiated THP-1 cells were transfected with miRNA inhibi-
tor, oxLDL was added 24 h after transfection, and cells were further
incubated for 24 h. The sterol analyses were performed using a high
performance liquid chromatography (HPLC) system (model 2790,
controlled with Empower Pro software; Waters Corp., Milford, MA,
USA). Sterols were detected using a photodiode array detector
equipped with a 4 mL cell (model 996; Waters Corp.). Analysis of
cholesterol and cholesteryl esters was performed after elution
with acetonitrile-isopropanol 30:70 (v/v)20and detection by absor-
bance at 210 nm.
2.6 Flow cytometry
PMA-differentiated THP-1 cells were transfected with miRNA inhibi-
tor for 24 h, then fluorescent-tagged Dil-oxLDL was added, and the
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Atherosclerotic proinflammatory response associated microRNAs139