1 Supplementary Material - Journal of Molecular and Cellular

Supplementary Material
Figure S1. ASncmtRNA-2 basal level expression in vivo and in vitro. (A) The relative quantification of the
basal ASncmtRNA-2 levels in aorta, heart and skeletal muscle of young mice shows preferential expression in
the aorta. (B) ASncmtRNA-2 expression is about 7-fold higher in EC than VSMC cultivated in vitro at P5
(** P<0.01).
1
Figure S2. Cell Cycle analysis of ASncmtRNA-2-over-expressing EC. (A) The EC transfection efficiency was
assessed for each of the three methods with a plasmid expressing GFP. Representative images are shown on
the left panel, whereas the transfection efficiencies, calculated as the fluorescent cell number on the total
number of cells, are reported on the right. (B) ASncmtRNA-2 induction 24 hours after pAS2 transfection with the
three methods was evaluated by qPCR. (C) The cell cycle distribution was assayed 48h after pAS2 transfection
in EC by lipofectamine 2000, TransIT-X2 and electroporation (* P<0.05; ** P<0.01).
2
Figure S3. A model for the ASncmtRNA-2 synthesis. A proposed model for the synthesis of ASncmtRNA-2 is
outlined. The Short Homologous Sequence (SHS, red) on the short arm of the nascent ASncmtRNA-2 would
anneal to the 3’ end of the 16S sense RNA, where both human and murine have a perfect match (9 and 6bp,
respectively). The SHS would be extended by a RNA-dependent RNA Polymerase (RdRP) to the 5’ end to form
the mature ASncmtRNA-2.
3
Table S1
hsa-miR-4485
target
TP53TG3
VAN3
RAB35
HNRPUL1
score
0.939
0.873
0.870
0.862
DNAH1
0.852
WHSC1L1
0.833
RNF41
RP1159H1.3
0.829
function/interaction
Target of p53 and supposed to have a role in the p53 pathway.
Induced in psoriatic epidermis.
Involved in the abscission of daughter cells.
Interacts with p53 and inhibits its transcription activity.
Involved in sperm cell flagellum function and in microtubule-based movement.
Proposed role in mitosis.
Over-expressed in tumoral tissue. Its knock-down induces cell cycle arrest at the
G(2)/M phase.
Involved in hematopoiesis.
0.824
Uncharacterized gene.
WNK1
0.821
MSH6
FBXO32
ZNF726
LACTB2
UNR
0.813
0.792
0.792
0.756
0.751
c-KIT
0.743
TJP1
REPS1
WDFY3
HARS2
VASH1
MAPRE2
RP11476E15.3
OPCML
C14orf37
PPAPDC3
ZNF606
TEX19
0.742
0.740
0.734
0.731
0.720
0.717
Serine/threonine protein kinase. Mutations are associated to hypertension.
Important for mitosis.
Protein involved in DNA double-strand break repair.
Member of F-box protein family, is a negative regulator of p21.
Zinc finger protein.
Beta 2 lactamase.
RNA binding protein, involved in mitosis and G2/M phase transition.
Type 3 trans-membrane receptor. Its knock-down induces cell cycle arrest at the
G2/M phase.
Tight junction protein with a role in cell cycle control.
Down-stream effector of Ras. It plays a role in cell cycle.
Phosphatidylinositol 3-phosphate-binding protein involved in autophagy.
Aminoacyl-tRNA synthetase involved in the Perrault syndrome.
EC-specific factor that inhibits angiogenesis.
Protein involved in microtubule reorganization during mitosis.
0.717
Uncharacterized protein.
0.717
0.711
0.709
0.704
0.701
Member of the IgLON subfamily with an opioid receptor function.
Uncharacterized gene.
Transmembrane protein involved in myoblast differentiation.
Transcriptional repressor of SRE and AP-1 genes.
Protein specifically expressed in testis.
reference
[1]
[2]
[3]
[4]
[5, 6]
[7]
[8]
[9]
[10]
[11]
[12, 13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
hsa-miR-1973
target
IRF2BPL
score
0.968
FGFRL1
0.963
TBX15
0.921
AZIN1
0.906
function/interaction
Transcription factor with a proposed role in female reproductive function.
Member of the fibroblast growth factor receptor family and target of the oncosuppressor miR-210. Its knock-down induces cell cycle arrest in the G1 phase.
Transcription factor of the T-box family, known to have a function in cell cycle control
and senescence.
Antizyme inhibitor with a proliferation induction role, in part through pRb activation.
reference
[25]
[26]
[27, 28]
[29]
4
Table S1 (continues)
CERS5
KCNB2
C2CD3
F8A3
SHC4
CRABP1
0.896
0.875
0.869
0.858
0.839
0.828
KPNB1
0.817
GZF1
0.804
TMEM198 0.800
DCUN1D4
MBNL2
BARD1
F8A2
MAFF
RPS6KA5
0.794
0.787
0.786
0.774
0.770
0.768
CXCL8
0.767
CSE1L
0.765
HOXB6
0.762
RPRM
0.750
PIN4
WIPF3
SHANK2
0.750
0.747
0.734
ARF3
0.728
CTAGE5
RAB40C
0.719
0.705
Member of the ceramide synthase family.
Potassium channel.
Involved in centriole and microtuble arrangement.
Coagulation factor VIII-associated 3.
Member of the Shc family with a role in cell proliferation and differentiation.
Modulates the G1 to S transition.
Member of the importin beta family. Its knock-down correlates with an activation of
p21 and p53.
Zinc finger protein that negatively regulates HOXA10, an activator of p21.
Trans-membrane protein activating the Wnt signalling pathways, which is involved in
the G2/M phase progression.
Required for covalent modification of cullins by the ubiquitin-like molecule Nedd8.
Involved in pre-mRNA spicing and is dysregulated in diabetes.
Binds BRCA1 oncosuppressor and participates in mitosis.
Coagulation factor VIII-associated 2.
Basic-leucine zipper transcription factor.
Kinase required for the G1 to S phase transition.
Member of the CXC chemokine family. Its knock-down induces G1 phase arrest in
cancer cells.
Protein involved in nuclear import. Its knock-down induces G1 phase arrest in cancer
cells.
Involved in the generation and proliferation of erythroid progenitor cell.
Glycosylated cytoplasmic protein. If over-expressed induces cell cycle arrest in G2
phase.
Protein with PPIase activity. Its inhibition results in cell cycle arrest.
Protein connected to male infertility.
Synaptic proteins connected with autism.
Member of ADP-ribosylation factor family, involved in vesicular trafficking. In yeast
changes subcellular localization in a cell cycle-dependent manner.
Tumor-associated antigen. Its alternative splicing is controlled by MALAT1.
Member of Rab family of small GTPases expressed in the nervous system.
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38, 39]
[40]
[41]
[42]
[33]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
Table S1. Potential targets of hsa-miR-4485 and hsa-miR-1973. A list of putative targets of hsa-miR-4485-3p
and hsa-miR-1973, as suggested by the miRNA target prediction tool microT-CDS (http://diana.imis.athenainnovation.gr/DianaTools/index.php?r=site/index) [55, 56], is reported together with their function. The genes
with a proven role in cell cycle control are highlighted in grey.
5
Table S2
primer
name
qPCR
5'-3' sequence
species
gene/target
p21-F
GACCAGCCTGACAGATTTCTATC
p21-R
CAGGCAGCGTATATACAGGAGA
p16-F
TCGTGCGATATTTGCGTTCCG
p16-R
GCTCTGCTCTTGGGATTGGC
18S-F
ATGGCCGTTCTTAGTTGGTG
CGCTGAGCCAGTCAGTGTAG
mouse
mouse
mouse
mouse
mouse
mouse
mouse
mouse
human
human
human
human
human
human
human
p21
p21
p16
p16
18S
18S
ASncmtRNA-2
ASncmtRNA-2
ASncmtRNA-2
ASncmtRNA-2
telomere
telomere
36B
36B
p21
GTGGACCTGGCTGAGGAG
CTTTCAATCGGGGATGTCTG
AGAGCTACGAGCTGCCTGAC
CGTGGATGCCACAGGACT
ACTTTGCAAGGAGAGCCAAA
TGGACAACCAGCTATCACCA
GGGAAAATCGACGGAGGA
GGGCGATTTCCTTCAAAGAC
human
human
human
human
human
human
human
human
human
p21
p16
p16
beta actin
beta actin
16S
16S
GPI
GPI
AAAAggatccTACCTAAAAAATCCCAAACATATAACTGAACTC
CACgggcccAAGAACAGGGTTTGTTAGGTAC
CTTgggcccGTGTGGGTATAATACTAAG
AAAAgaattcGCTAAACCTAGCCCCAAACCCACTC
human
human
human
human
ASncmtRNA-2
ASncmtRNA-2
ASncmtRNA-2
ASncmtRNA-2
18S-R
mmASN2-2F
mmASN2-6R
ASN2-2F
ASN2-4R
TeloF
TeloR
36B4f
36B4r
h-p21F
GCTAAACGAGGGTCCAACTG
h-p21R
h-p16-2F
h-p16-2R
bactF
bactR
16S-F
16S-R
GPIF
GPIR
GGATTAGGGCTTCCTCTTGG
cloning
ASN2-frs-F
2-frs-apai
12-frl-apai
ASN2-frl-R
CTAACCTAGAGAAGGTTATTAGGTTT
ACCGTGCAAAGGTAGCATAATCACT
CCGTAAATGATATCATCTCAACT
CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT
GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT
CAGCAAGTGGGAAGGTGTAATCC
CCCATTCTATCATCAACGGGTACAA
GGAAGACCATGTGGACCTGT
reference/
notes
[57]
[57]
[57]
[57]
BamHI
ApaI
ApaI
EcoRI
Table S2. Primers used in this study. The primers used for qPCR and cloning are listed together with the
species (mouse or human), the target gene and the reference, if any. For cloning primers, the artificially inserted
restriction sites used for the pAS2 construction are indicated in lower case and in the note column.
6
Supplementary Materials and Methods
Experimental Animals
Male C57/BL6 mice were housed under a 12 hour light-dark cycle and fed a standard chow diet ad
libitum. Two and half and 21 month-old animals (young and old, respectively) were anesthetized with
an intraperitoneal injection of ketamine/medetomidine cocktail (100 mg/10 mg/Kg) and perfused with
PBS from the apex of the heart. Aortas, hearts and abductor skeletal muscles were dissected out and
processed as described below.
Protocols complied with national and international law and policies (4D.L. N.116, G.U., supplement 40,
18-2-1992; EEC Council Directive 86/609, OJ L 358,1,12-12-1987; The Guidelines of the National
Institutes of Health for the Care and Use of Laboratory Animals, and with the US National Research
Council 1996).
Immunohistochemistry (IHC)
Aorta samples were fixed with 10% formalin and embedded in paraffin. Six μm-sections were deparaffinized, re-hydrated and boiled in Dako Target Retrieval Solution for 20’ at pH 9 (Dako). After
washing in PBS-0.1% Triton X-100 (PBS-T) slides were incubated in 3% H2O2 (Sigma-Aldrich) for 10’,
and treated 1 hour at RT with 5% goat serum in PBS-T. The primary antibody against p16 raised in
rabbit (sc-1207, Santa Cruz Biotechnology) was diluted in 1% goat serum PBS-T and incubated at 4°C
overnight. Sections were incubated with biotin-conjugated goat anti-rabbit antibody (Vector
Laboratories) followed by streptavidin conjugated to horseradish peroxidase (HRP) (ABC kit, Vector
Laboratories) for 30’ at RT. Immunoreactions were detected using the 3.3'-Diaminobenzidine
chromogen (Vector Laboratories) and slides were counterstained with hematoxylin. For the mock
controls the α-p16 primary antibody was omitted.
An Axioskop II microscope (Zeiss) was used to acquire the α-p16 stained sections. The entire aorta
cross section was analyzed with Axiovision Software Rel 4.7 (Zeiss).
Cells and SIPS
Human Umbilical Vein Endothelial Cells (HUVEC, Lonza) were used as in vitro model of EC and were
cultured in EGM-2 complete medium (Lonza). Human Aortic Smooth Muscle Cells (HASMCs, Lonza)
were used as an in vitro model of VSMC and were cultured in SmGM-2 complete medium (Lonza).
Both cell types were cultured to the fifth passage (P5) and fifteenth passage (P15) to produce
proliferative and replicative senescent cells, respectively.
7
SIPS was induced in HUVEC by UV and H2O2 treatment. In both cases cells were plated in 6-well
plates at 3x104 cells/cm2. For the H2O2 treatment, cells were exposed to 200 µM H2O2 for 3.5 hours
followed by the replacement of culture media. For the UV treatment, cells were exposed to 2.2 J/cm2
UV, which corresponded to 30’’ on the Transilluminator 2000 (BioRad). Cells were cultivated for
another 5 days, with media refreshment after 2 days.
RNA extraction from tissue and cells
RNA was extracted from cells by RNeasy Plus Mini Kit (QIAGEN). For the tissue samples, RNA was
extracted with the TRIzol reagent (Life Technologies), after sample homogenization with the
TissueLyser (Qiagen, Hilden, Germany) and treated with the TURBO DNA-free Kit (Invitrogen).
Manufacturer’s protocols were followed. RNA was quantified by NanoDrop spectrophotometer
(Thermo Scientific).
DNA extraction
DNA was extracted from 2x105 cells using the Nucleo Spin Tissue kit (Macherey Nagel). DNA was
eluted in 100 µl of H2O and quantified by NanoDrop spectrophotometer (Thermo Scientific).
Primer design and validation
Primer 3 software (available online at http://bioinfo.ut.ee/primer3-0.4.0) was used to design primers for
qPCR (reported in Table S2). Primer pairs were validated by amplifying cDNA prepared with and
without the RT Enzyme Mix to exclude genomic background amplification. Primer pairs showing more
than a single pick as melting curve were excluded due to likely unspecific amplification. Primer pairs
amplifying ASncmtRNA-2 were designed spanning the short/long arm junction site to exclude
amplifications from the sense and antisense 16S gene. Moreover, primers were selected with the
support of the ePCR software (http://www.ncbi.nlm.nih.gov/tools/epcr/) to exclude amplification of
Nuclear Mitochondrial DNAs (NuMts), i.e. nuclear genes with a high level of homology to mitochondrial
transcripts. For the human ASncmtRNA-2, qPCRs were carried out on HUVEC treated with 100 ng/ml
EtBr, which induces mtDNA and mtDNA transcript depletion (Figure S2).
cDNA preparation and qPCR
The cDNA was prepared with the Superscript III kit (Life Technologies) according to the
manufacturer’s instructions with minor modifications. One-hundred ng of total RNA (up to 4 µl) was
incubated with 1 µl of RT Enzyme Mix and 5 µl 2X RT Reaction Mix at 25°C 10’, 50°C 30’ and 85°C 5’.
The qPCR mix was prepared as follows: 5 µl Iq Sybr Green Supermix (Biorad), 0.2 µl cDNA, 0.5 µM of
each primer and H2O to 10 µl. The thermal cycle was performed in an iCycler thermocycler (Biorad)
8
with the following program: 3’ 95°C followed by 40 cycles of 95°C 15’’ and 60°C 45’’. The amplification
phase was followed by slow denaturation to create a melting curve. Each qPCR assay was performed
in technical duplicate.
For the miRNA quantification, miRNA-specific retro-transcription and TaqMan qPCR assays (ASSAY
ID 462832 for hsa-mir-4485, ASSAY ID 245468 for hsa-mir-1973, ASSAY ID 001093 for RNU6B) were
used according to the manufacturer’s instructions (Life technologies). Taqman qPCRs were performed
in technical triplicates (AB7900, Life Technologies).
Gene expression was quantified with the ΔΔCt method. The Ct values of the genes under
investigation were subtracted (ΔCt) to that of the housekeeping gene (β actin for cells, 18S for tissues
and RNU6B for miRNAs), which were selected based on previous literature [58-61]. The fold induction
was calculated with the formula 2-(ΔΔCt), where ΔΔCt is the difference between the average ΔCt of the
proliferative/young (for RS and aorta) or untreated (for SIPS and EtBr-treatments) samples and the
ΔCt of each sample.
Telomere length and mtDNA/ncDNA ratio variation assessment
Telomere length was quantified by qPCR with 100ng DNA and primers annealing on the telomeric
repeated regions and on the 36B4 gene as an internal reference [57]. The relative telomere length
was calculated with the ΔΔCt method.
The mtDNA content relative to the nuclear DNA (mtDNA/ncDNA) variation was assessed by qPCR.
One-hundred ng DNA was used with primers annealing on the mtDNA (16S-F/16S-R) and on the
ncDNA (36B4f/36B4r). The variation in mtDNA/ncDNA was calculated with the ΔΔCt method.
Senescence-Associated-βGal (SA-βGal) assay
The SA-βGal test was performed using the Senescence β-galactosidase Staining Kit (Cell Signalling
Technology) according to the manufacturer’s protocol. Pictures were acquired using a Zeiss Axiovert
200M microscope on at least ten randomly selected fields per sample. Reported values are the
average number of positive (blue) cells expressed as a percentage of the total number of cells in each
field.
Reactive Oxygen Species (ROS) quantification
ROS production was determined using DCF (H2DCFDA, Life Technologies) and Flow Cytometry
(FACSCalibur, BD Biosciences) according to the manufacturer’s protocol. Briefly, 5-20x104 cells were
collected and incubated with 1 µM H2DCFDA for 1 hour at 37°C. Cells were washed, resuspended in
400 µl of PBS and read at the Flow Cytometry on the FL1 Channel. The mean value of at least 104
events was considered for each sample.
9
Lipofuscin quantification
The lipofuscin content was assessed with a Flow Cytometry (FACSCalibur, BD Biosciences) by
measuring cell autofluorescence on the FL1 channel on at least 104 events [62]. The mean value was
taken for each sample.
pAS2 vector construction
The ASncmtRNA-2 full length sequence was cloned under the CMV promoter in the pcDNA3.1 vector
to produce the pAS2 construct. Two fragments correspondingly roughly to the short and long arms of
ASncmtRNA-2 were amplified with the ASN2-frs-F/2-frs-apaI and 12-frl-apaI/ASN2-frl-R primer pairs,
respectively (Table S2). The BamHI and EcoRI restriction sites were inserted in the ASN2-frs-F and
ASN2-frl-R primers, respectively, while the 2-frs-apaI and 12-frl-apaI primers were mutated, with
respect to the wild type sequence of ASncmtRNA-2, to include an ApaI restriction site. The short arm
was cut by BamHI and ApaI, the long arm was cut with ApaI and EcoRI and the pcDNA3.1 vector was
cut with BamHI and EcoRI. The three fragments were assembled in one ligation reaction. All enzymes
and competent cells were purchased from New England Biolabs.
EC transfection with plasmid DNA
Chemical transfection. HUVEC at P5 were plated at 4x104 cells/cm2 in 12 well plates with 1ml of
complete EGM-2 medium (Lonza). One-hundred µl of Optimem serum free medium (Life
Technologies) were incubated with 1µl of 1µg/µl endotoxin-free plasmid DNA (pAS2 or pcDNA3.1) and
to 3µl of either TransIT-X2 (Mirus) or Lipofectamine 2000 (Life Technologies) transfection reagents for
30’ incubation at Room Temperature (RT). The mixture was added drop wise to the cells and
incubated at 37°C for 4 hours. The cells were then extensively washed with PBS and fresh media was
added. After 24 hours the cells were washed again and fresh media added. Forty-eight hours after
transfection cells were collected for analysis. The experiment was carried out on three independent
replicates.
Electroporation. About 7.5x105 HUVEC at P5 was used for each electroporation with 3 pulses at 1600
V for 10 ms, according the manufacturer’s instructions (Neon system, Life Technologies) followed by
plating in 3 wells of a 6- well plate with complete EGM-2 medium (Lonza). Media was changed after
24 hours and after 48 hours cells were collected from each well and analysed separately (technical
replicates). Each electroporation was carried out on two independent replicates.
The transfection efficiency was calculated as the ratio between the fluorescent cells and the total
number of cells 48 hours after transfection with pEGFP-N1 (a GFP expressing vector, Clontech
Laboratories).
10
EC transfection with miRNA
HUVEC at P5 were transfected with the mirVana® miRNA mimic hsa-miR-4485 or the hsa-miR-1973
or a combination 1:1 of hsa-miR-4485 and hsa-miR-1973 or the Negative Control #1 (Life
Technologies) using the siRNA Transfection Reagent (Santa Cruz Biotechnology) according to the
manufacturer’s protocol. Briefly, 100K were seeded in 12-well tissue culture plates and cultured in
complete medium overnight. The siRNA Transfection Reagent and the microRNA mimic were diluted
in optiMEM medium (Lonza) and incubated 30 minutes at room temperature. After changing the EGM2 with optiMEM medium, the nucleic acid-transfection reagent mixture was added to each well to a
final miRNA mimic concentration of 100 nM. After 5 hours medium was completely replaced with
EGM-2 and the cells were cultured for additional 48 hours.
Cell cycle analysis
At least 5x104 cells were collected and resuspended in 200 µl PBS. An equal amount of PI mix
(Propidium Iodide 50 µg/ml, Sodium Citrate 33 mM, Triton X-100 0.1%) was added and samples were
incubated at 37°C for 15’. At least 10k events per sample were then analysed by FACS on the FL2
channel. Cell cycle distribution analysis was carried out with the ModFit LT software (Verity Software
House).
Statistical analysis
If not otherwise stated, each experiment was conducted at least on a biological triplicate. For the
qPCR on the mouse samples four animals per group were analyzed separately. The Student’s twotailed t test was applied to determine the statistical significance. For the qPCR the ΔCt values of the
two groups under analysis were compared.
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