Supplemental Materials
Methods and Materials
Cell culture
Human aortic SMCs (Lonza, Walkersville, MD, passage #3) were cultured in
growth media SmGM-2 (Lonza) in 5% fetal bovine serum and used between
passages 5 and 6. HEK293 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
Microarray analysis of miRNA expression
RNA samples for miRNA microarray were isolated by using mirVana miRNA
isolation kit (Ambion) according to the manufacturer's protocol. RNAs obtained
from human aortic SMCs treated with PDGF-BB (20 ng/ml) at 0 h, 3 h, 6 h, and
24 h were subjected to MicroRNA Microarray Service (LC Sciences, Houston).
Hybridization was performed on a μParaflo microfluidic chips containing
detection probes consisting of a chemically modified nucleotide coding
segment complementary to target microRNA according to miRBase software.
Version 9.1 arrays were used to detect >470 unique mature human miRNAs
and contained a total of 46 positive and negative control probes designed by
LC Sciences to determine uniformity of sample labeling and assay conditions.
After hybridization, RNA samples for comparison were labeled with
tag-specific dendrimer Cy3 and Cy5 fluorescent dyes. Raw data were
analyzed by subtracting the background and normalizing the signals with a
cyclic LOWESS filter (Locally-weighted Regression). A miRNA to be listed as
detectable when it met at least three criteria: 1) signal intensity higher than 3×
the background standard deviation, 2) spot coefficient of variation < 0.5, in
which coefficient of variation is calculated as (standard deviation)/(signal
intensity) and 3) at least 50% of the repeating probes had a signal 3-times
higher than background standard deviation. For two color experiments, the
ratio of the two sets of detected signals (Cy3/Cy5, log2 transformed, balanced)
and all differentially expressed transcripts with p value< 0.01 were listed.
PCR analysis, RNA analysis by quantitative RT-PCR (qRT-PCR)
MiRNAs and mRNAs were isolated with miRNeasy Mini Kit (QIAGEN) and
RNeasy kit (QIAGEN). qRT-PCR for miRNA was performed on cDNA from 20
ng of total RNA using the protocol of the miRCURY LNA TM Universal cDNA
Synthesis and SYBR® Green Master Mix Kit (Exqion). PCR analysis for
miR-638 was performed using the primers (forward: GAG AGG ATC CTG
CCG CAG ATC GCT G; reverse: GAG TAA GCT TCA GGG AGT CCT CTG
CC). qRT-PCR for NOR1, Nurr1, Nur77, smooth muscle 22α (SM22α), smooth
muscle α actin (SMA), Calponin, and smooth muscle myosin heavy chain
(MYH11) were performed on cDNA generated from 250 ng of total RNA using
the protocol of the HotStart-IT® SYBR® Green qPCR Master Mix with UDG
(2X) Tested User FriendlyTM kit (USB Corporation). Primer sequences for
human NOR1, Nurr1, Nur77, SM22α, SMA, Calponin, MYH11 and 18S are
presented in Table 2. Expression of miR-638 relative to U6 and that of relative
to NOR1, Nurr1, Nur77, SM22α, SMA, Calponin, and MYH11 relative to 18S
were determined using the 2－ΔΔCt method.
Oligonucleotide Transfection
For miR-638 overexpression, miR-638 mimic (QIAGEN) was added to the
complexes at the final concentration of 30 nM. For miR-638 knockdown, the
miR-638 inhibitor (QIAGEN) was added to the complexes at the final
concentration of 100 nM. mirVana™ miRNA mimic Negative Control (Life
Technologies) was applied as a control. 2 hours after seeded into the 6 well
plates, cells were transfected using HiPerFect Transfection Reagent (QIAGEN)
based on the manufacture’s protocol. Transfection medium was replaced by
regular cell culture medium after 24 hours of transfection.
Luciferase Reporter Assay
HEK293 cells were seeded in a 96-well plate and reached to ~80% confluence
at time of transfection. The 3’-UTR of NR4A3 (NOR-1) variant 1 was used in
our study and the ready to use reporter vector pLight Switch containing human
NR4A3 3-UTR (3150 bp, starting from
5’-CTTCCTGGACACCCTACCTTTCTAA) was purchased from SwitchGear
Genomics (Menlo Park, CA). The SwitchGear GoClone reporter construct was
cotransfected with either vehicle (vehicle control), miR-638 mimic (50 nM),
anti-miR-638 (50 nM), or a non-targeting control miRNA (50 nM) into HEK293
cells with following the transfection procedures provided by Switchgear
Genomics. 48 hours after transfection, add LightSwitch Luciferase Assay
Reagent and read signal on luminometer. Calculate knockdown by taking the
ratio of the signal of the miR-638 mimic over the non-targeting control. To
construct MUT-NR4A3 3’UTR, the QuickChange II Site-Directed Mutagenesis
Kit (Agilent Technologies, Santa Clara, CA) was used to induce four point
mutations in the 3’-UTR region of WT-NR4A3 as shown in Figure 4A. The
following primers were used for the mutagenesis of the miR-638 target site in
the human NR4A3 3’UTR (1st forward: 5’-GTT CAC CTT CCC TGT CCG TCC
CTT CTG AGG TAT G-3’, 1st reverse: 5’-C ATA CCT CAG AAG CCA CGG
ACA GGG AAG GTG AAC-3’; 2nd forward: 5’-GTT CAC CTT CCC TGT CCG
TGG CTT CTG AGG TAT G-3’, 2nd reverse: 5’-C ATA CCT CAG AAG CCA
CGG ACA GGG AAG GTG AAC-3’).
Construction of adenovirus
The adenovirus expressing miR-638 (Ad-miR-638) was made using RAPAd®
Universal Adenoviral Expression System (CELL BIOLABS, INC) according to
the manufacturer’s instructions. Briefly, pacAd5 9.2-100 Ad backbone vector
was cotransfected with the pacAd5 K-NpA shuttle vector containing the
miR-638 sequence into Ad293 cells using FuGene 6 Transfection Reagent
(Roche, Indianapolis, IN). Adenoviruses harboring wild-type NOR1 (Ad-NOR1)
and green fluorescent portein (Ad-GFP) were made using AdMax (Microbix)
according to the protocol provided by manufacture. The viruses were
propagated on Ad293 cells and purified using Cscl2 banding followed by
dialysis against 20mmol/l Tris-buffered saline with 2% glycerol. Titering was
performed on Ad293 cells using Adeno-X Rapid Titer kit (Clontech) according
to the instructions of the manufacturer. For adenovirus-mediated GFP,
miR-638 or NOR1 gene transduction in vitro, human aortic SMCs were grown
to ~60% confluence and infected with adenovirus at indicated multiplicity of
infection (MOI). 24 hr after transduction, the viral suspension was removed
and the cells were incubated for another 48 hrs.
Western blot
Cells were washed with PBS and lysed into Pierce® RIPA Buffer. After 30 mins
extraction at 4℃ with rocking, insoluble material was removed by
centrifugation. Cell lyses were boiled to elute immune complexes and resolved
by SDS/PAGE, transferred to nitrocellulose. Blots were blocked with 5% nonfat
milk in PBS with 0.1% Tween20 (PBST) and developed with diluted antibodies
for PCNA (1:2000 dilution; Cell Signaling), Cyclin D1 (1:1000 dilution; Cell
Signaling), a-tubulin (1:1000 dilution; Cell Signaling), NOR1 (1:500 dilution;
R&D), SM22α (1:500 dilution; Santa Cruz), SMA (1:800 dilution; Sigma) and
GAPDH (1:1000 dilution; Santa Cruz), followed by incubating with either IRDye
700 or 800 secondary antibodies and visualized using Odyssey Infrared
Imaging System software (Li-Cor, Lincoln, NE).
Smooth muscle Cell scratch wound assay
In gain of function experiment, human SMCs were seeded in 6-well plates at a
concentration of 2.5×105 cells per well and transduced with Ad-miR-638
(100MOI), Ad-NOR1 (100MOI), or Ad-GFP (100MOI). In loss of function
experiment, miR-638 inhibitors (100nM), or negative control (100nM) and
HiPerFect Transfection Reagent (QIAGEN) was mixed following the
manufacturer’s instructions and added to cells in 6-well plates at a density of
2.5×105 cells per well. After incubation with serum-deprived in 0.5% FBS for 48
hrs, a linear wound was gently introduced in the center of the cell monolayer
using 200ul tip, followed by washing with PBS to remove the cellular debris.
Then the cells were subjected to stimulation with or without human PDGF-BB
(PEPROTECH) at a final concentration of 20ng/ml and monitored for 24 hrs.
Photos Images were captured using an Olympus IX 71 microscope equipped
with a diagnostic instrument RT SPOT Digital Camera (Canon).
VSMC proliferation in vitro
VSMC proliferation was determined by cell counting and MTT assay using
Vybrant® MTT Cell Proliferation Assay Kit (InvitrogenTM). For cell counting, the
cells were detached by trypsinizatioin and re-suspended in PBS. The cells
were then counted under a microscope. For MTT assay, SMCs were seeded
at 1×104 cells per well in 96-well culture plates for 24 hrs. The cells were
transduced with Ad-miR-638, Ad-NOR1, or Ad-GFP and incubated with
serum-deprived in 0.5% FBS for 48 hrs. Then MTT labeling reagent was
added and incubated for 4h, and solubilized in 0.01N HCl containing 10% SDS.
Absorbance was measured at 570 nm on an ELASA plate reader.
Cell cycle analysis by Flow Cytometry
VSMCs (1×106) were seeded onto P60 dishes and were transduced with
Ad-miR-638 or Ad-GFP. After 48 hrs incubation with serum-deprived in 0.5%
FBS, VSMCs were treated with or without PDGF-BB (20ng/ml). Finally, the
cells were fixed with 70% ethanol at 4℃ overnight, treated with RNaseA in
PBS, stained with propidium iodide (Sigma, St. Louis, MO) and subjected to
flow cytometric DNA content analysis using FACSCalibur (Beckman Coulter,
Co., USA). The percentages of cells in G1, S, and G2/M phases were
analyzed.
Immunofluorescent detection of microRNA-638 in human aortic SMC
The human aortic SMCs were seeded into 2 well glass slide chamber and
grown to confluence of 60%. Then the cells were made quiescent by 48 hours
culturing in starvation medium containing 0.5% FBS. After that, PDGF-BB (20
ng/ml) was added into medium and cultured for 24 hrs. Then the cells were
fixed in paraformaldehyde (4%). After washed with PBST, slides were digested
with proteinase K (10 g/ml) for 1 min, postfixed with 4% paraformaldehyde
and followed by washes in PBST. Then slides were pre-hybridized in
hybridization buffer (50% Formamide, 5×SSC,0.1% Tween-20, 50μg/ml
heparin and 500μg/ml yeast tRNA) at 55 °C for 2 hrs and hybridized with
hsa-miR-638 probes (20 nM) (LNA mercury probe, Exiqon) at 55°C overnight.
Then sections were washed in 0.2% SSCT at 55 °C, followed by blocking for 1
hour at room temperature with PBST/5% horse serum. Subsequently,
anti-digoxigenin-POD antibody (Roche) was added at dilution of 1:1000 and
incubated at room temperature for 2 hrs. After post-hybridization washes in
PBST, the signals were detected using the tyramide signal amplification
system (PerkinElmer) according to the manufacturer’s protocol. Cell nuclear
was stained by DRQ5 and imaged with an Olympus IX81 confocal-scanning
microscope equipped with Olympus FV5-PSU/IX2-UCB camera.