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MicroRNA-195 regulates vascular smooth muscle cell phenotypes and prevents
neointimal formation
Yung-Song Wang; Hay-Yan J. Wang; Yi-Chu Liao; Pei-Chien Tsai; Ku-Chung Chen;
Hsin-Yun Cheng; Ruey-Tay Lin and Suh-Hang Hank Juo
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Materials and Methods
MicroRNA transfection
miR-195 precursor, miR-195 inhibitor and negative control miRNA(NC-miR) were
purchased from Ambion Inc. (TX, USA) with the sequences information shown as
miR-195 precursor, 5’-UAGCAGCACAGAAAUAUUGGC-3’; miR-195 inhibitor,
5’-GCCAATATTTCTGTGCTGCTA-3’; negative control sequence,
5’-AGUACUGCUUACGAUACGG-3’. The NC-miR is a random sequence Pre-miR
molecule that has been extensively tested in human cell lines and tissues and validated
to produce no identifiable effects on known miRNA function. By using Lipofectamine
2000 reagent, the precursor miR-195 and Pre-miR Negative Control were transfected
into HASMC for 24 hours.
BrdU incorporation
HASMCs were first plated in a 60 mm3 plate and transfected at 24th hour with
miR-195 precursor or NC miR. 24 hours post-treatment, cells were pulsed for 1 hour
with 10 μM BrdU (BD Bioscience, CA, USA). After treatment, the cells were
prepared and stained with anti-BrdU monoclonal antibody (BD Bioscience, CA, USA)
and counter-stained with Hoechst33342 (Sigma) according to manufactures
instruction.
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Preparation of stable cells
We have developed a pEP-miR-195 clone for the stable expression of miR-195 in
VSMCs (CRL-1999). We have developed a pEP-miR-195 clone for the stable
expression of miR-195 in VSMCs (CRL-1999). The disadvantage of this technology
is that only a limited number of mammalian cells can be developed into stable clones
after transfection of a foreign gene. Since vascular SMCs are highly heterogeneous in
lineage sources, the technique used in this study to create cell line may lead to a
selection of cells with different original sources cross the comparison groups.
Transcriptome
Total RNA was obtained from CRL-1999 cells over-expressing miR-195 and the
normal CRL-1999 cells, respectively. The cDNA microarrays were performed using
the Human Whole Genome OneArray (Phalanx Biotech group, Taiwan). The Cy5
fluorescent intensities of each spot were analyzed by GenePix 4.1 software (Molecular
Devices). The signal intensity of each spot was loaded into Rosetta Resolver System®
(Rosetta Biosoftware) for data process and data analysis. The array experiments were
repeated to check the result reproducibility using Pearson correlation (R value > 0.95).
Normalized spot intensities were transformed to gene expression (using log2 ratios)
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between the control and treatment groups. The spots with log 2 ratio ≥ 1 or log 2 ratio
≤ -1 and P value < 0.05 were selected for further analysis. The functional annotations
of genes were analyzed by Ingenuity Pathways Analysis (IPA; Ingenuity Systems,
Redwood City, CA, www.ingenuity.com).
RNA interference
We used shRNA to perform RNA interference. The lenti-viral plasmids expressing
21-mer shRNAs against Cdc42 (clone ID: TRCN0000047632), CCND1 (clone ID:
TRCN0000040039) and FGF1 mRNA (clone ID: TRCN0000072524) were obtained
from the National RNAi Core Facility in Academia Sinica (Taipei, Taiwan).
HASMCs were transfected with plasmids expressing shRNA (6 μg DNA per 105 cells
for 48 h) using Lipofectamine 2000. Scramble shRNA in pLKO.1 plasmids was used
as a control for the RNA interference experiments. The expression levels of the Cdc42,
CCND1 and FGF1 genes were determined by real-time PCR. The procedures to
extract and measure transcripts were as follows: Trizol reagent (Invitrogen) for RNA
extraction and real-time PCR by SYBR green Master Mix (Applied Biosystems) with
specific primers (Cdc42: 5'-GCCCGTGACCTGAAGGCTGTCA-3' and
3'-ACCACAGCCGTAGTATGATTTTCGT-5'; CCND1:
5'-AGTTCATTTCCAATCCGCCCTCCA-3' and
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3'-TCAGGGAGGAGAGGCCTCGTAA-5'; FGF1:
5'-CACATTCAGCTGCAGCTCAG-3' and 3'-CTGAGTGATACCGGTCTTTCGT-5';
GAPDH 5'-GAAGGTGAAGGTCGGAGTC-3' and
3'-CTTTAGGGTAGTGGTAGAAG-5'). GAPDH was used as a housekeeping control.
Expression levels of mRNA were quantified by employing the 2-ΔΔCt relative
quantification method.
CDC42 3’-UTR reporter construct
Cdc42 3’-UTR plasmid constructs were created for experimental confirmation as a
miR-195 binding site. A 1425-bp segment of PCR production from the wild-type
3′-UTR containing one miR-195 binding site was cloned into the Mlu I/Hind III site
of the pMIR-REPORT Luciferase vector (Applied Biosystems). The mutant 3′-UTR
was also generated by site-directed mutagenesis based on the two-step PCR
megaprimer method as described previously 1. The sequence of the mutant 3’-UTR of
Cdc42 contains 5'-GTTGAACGACGACCCTGGTTC-3' (the six mutated nucleotides
were underscored). A construct (either wild or mutant type) and miR-195 precursor
were co-transfected into the HEK293 cells. Cells were cultured for 2 days and the
luciferase activity was measured by the Dual-Luciferase Reporter Assay System
(Promega, WI, USA).
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Enzyme-linked immunosorbent assay for cytokine levels
HASMCs transfected with miR-195 precursor or miR-195 inhibitor were incubated
with or without 40 μg/ml oxLDL for 24 h and the supernatants of conditioned medium
were collected for the measure of IL-1β, IL-6 and IL-8 levels by using the ELISA kits
(BD Bioscience, CA, USA) according to the manufacturer’s protocol.
Western blot for protein levels
The antibody to fibroblast growth factor (FGF1) was purchased from GeneTex
Inc. (CA, USA). The antibody to cell division cycle 42 (Cdc42) was purchased from
BD Biosciences (Pharmingen, CA, USA). The antibody to cyclin D1 (CCND1) and
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Millipore
Inc.
MicroRNA in situ hybridization
In situ hybridization of rat carotid artery was performed with a digoxigenin-labeled
miR-195 locked nucleic acid (LNA) probe (probe sequence:
5’-DIGN-GCCAATATTTCTGTGCTGCTA-3’; Exiqon, Vedbaek, Denmark). A LNA
U6 small nuclear RNA (RNU6B) positive control probe (5’-
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DIGN-CACGAATTTGCGTGTCATCCTT-3’) have been also purchased from Exiqon.
Digoxigenin was detected by a digoxigenin-specific, horseradish peroxidase-labeled
antibody (IsHyb in situ Hybridization kit, Biochain, CA, USA).
Immunohistochemistry
Rat arteries were perfused with 4% paraformaldehyde in PBS (pH 7.4).
Immunohistochemistry (IHC) was performed on 10-μm sections using rabbit
polyclonal anti-mouse Cdc42 (1:100; Product Description, IL, USA) as primary
antibodies. Antibodies against SM alpha actin (M0851,DAKO) were used as a
positive control for HASMCs. IHC staining was performed according to the
manufacturer's protocol (IHC Select® Immunoperoxidase Secondary Detection
System; Millipore, MA, USA). After extensive washing with a rinse buffer, the
sample was incubated with 3,3′ diaminobenzidine until the appearance of a brownish
color. The sample was counter-stained with hematoxylin for 1 min. For
immunoabsorption control tests, the SM alpha actin antibodies were first preincubated
with Amicon Ultra-2 Centrifugal Filter Devices (Millipore, MA, USA) for 24 hours
and the remaining staining procedures were completed as described above. We did not
see any non-specific immunolabeling in the sections used for these control tests.
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Morphometric analysis for neointimal lesion formation
Morphometric analysis was performed in sections by using the computerized image
analysis system (Image J; National Institutes of Health). Sections (5μm thick)
obtained at equally spaced intervals in the middle of injured and control common
carotid artery were stained with hematoxylin and eosin. The intimal (I) to medial (M)
area ratio of each section was calculated. The average I/M of the six sections were
used as the final I/M of each animal.
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Supplemental Table 1. oxLDL treatment to HASMCs caused suppression of several
cardiovascular related microRNAs. The presented data were from one single
experiment of microarray array without replication. The citation number indicates the
reference reporting these microRNAs are cardiovascular related.
OxLDL/saline treatment
References
miRNA
Fold change
miR-146b
0.346
2, 3
miR-16
0.294
4, 5
miR-125a
0.271
6, 7
miR-222
0.174
8, 9
miR-31
0.153
10
miR-195
0.076
11, 12
miR-145
0.072
13-17
miR-146a
0.049
18
miR-221
0.032
8
miR-125b
0.029
19
miR-210
0.026
20
miR-143
0.020
16, 17
miR-21
0.019
20-22
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Supplemental Figure 1. Transfection efficiency of miR-195 precursor and inhibitor
into HASMCs.
For determining the transfection efficiency of miR-195 precursor and inhibitor, RNA
isolated from transfected HASMCs was analyzed by qRT-PCR. miR-195 levels were
normalized to endogenous RNU6B levels. Expression data, represented as mean ± SD
of three independent experiments, are shown relative to the expression of miR-195 in
untreated control cells. Statistical significance (*P < 0.0001) tested by one-way
ANOVA with a Tukey-Kramer post hoc test.
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Supplemental Figure 2. miR-195 levels from the stable clone at 3th and 6th passages
were detected by quantitative RT-PCR. Data represents mean ± SD from 3
independent experiments performed in triplicates.
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Supplemental Figure 3. miR-195 downregulates FGF1 at the transcriptional levels
and decreases HASMC proliferation. OxLDL-treated HASMCs were transfected with
the miR-195 precursor or a negative control miR (NC-miR). FGF1 proteins in
HASMCs were measured by Western blot at 24 h post-transfection. OxLDL-treated
HASMCs were transfected with shRNAs (6 μg DNA per 105 cells) specific for FGF1.
The data were obtained at 24 h post-transfection of shRNAs. shRNAs inhibited the
proliferation of oxLDL-treated HASMCs. Data represents mean ± SD from 3
independent experiments performed in triplicates.
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Supplemental Figure 4. Sequence and evolutionary conservation of miR-195 among
different species.
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Supplemental Figure 5. The expression of miR-195 in HASMCs after being infected
by Ad-GFP or Ad-miR-195 was detected by real-time quantitative PCR. Data
represents mean ± SD from 3 independent experiments performed in triplicates.
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Supplemental Figure 6. shRNAs down-regulate FGF1 in HASMCs. The mRNA
levels of FGF1 were examined with real-time quantitative PCR. Data represents mean
± SD from 3 independent experiments performed in triplicates.
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