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SUPPLEMENTAL INFORMATION
Loss of microRNA-143/145 Disturbs Cellular Growth and Apoptosis of Human
Epithelial Cancers by Impairing the MDM2-p53 Feedback Loop
SUPPLEMENTAL SECTION INVENTORY
SUPPLEMENTAL TABLES:
Table S1
MDM2+ and miR-143/145- are associated with higher-grade tumors in HNSCCs patients.
pathologic grade
Combined
Total
1
2
3
MDM2-
miR-143-
miR-145-
6 (23.1%)
5 (18.5%)
1 (8.3%)
12
MDM2-
miR-143+
miR-145+
5 (19.2%)
1 (3.7%)
0 (0%)
6
MDM2+
miR-143-
miR-145-
11 (42.3%)
18 (66.7%)
11 (91.7%)
40
MDM2+
miR-143+
miR-145+
4 (15.4%)
3 (11.1%)
0 (0%)
7
26
27
12
65
Total
The relationship between the combined expression of miR-143/145 and MDM2 and tumor grade
of human head and neck squamous cell carcinoma specimens was analyzed using the χ-square
test.
SUPPLEMENTAL FIGURES
Figure S1 relates to manuscript Figure 1.
Figure S2 relates to manuscript Figure 2.
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Figure S3 relates to manuscript Figure 3.
Figure S4 relates to manuscript Figure 5.
Figure S5 relates to manuscript Figure 5.
Figure S6 relates to manuscript Figure 6.
Figure S7 relates to manuscript Figure 6.
Figure S8 relates to manuscript Figure 7.
Figure S1. Expression of MDM2 is negatively correlated with miR-143/145 expression. The
expression levels of MDM2 and miR-143/145 in the HNSCCs cell lines were determined using
real-time PCR. (A) The MDM2 mRNA level are increased in HNSCC cell lines compared to
normal tissues and the leukoplakia cell line Leuk-1. (B) The MDM2 protein level are increased in
HNSCC cell lines compared to normal tissues and the leukoplakia cell line Leuk-1. (C) The
miR-143 expression is decreased in the HNSCC cell lines. (D) The miR-145 expression is
decreased in the HNSCC cell lines. The assays were performed in triplicate. The results are shown
as the mean ± SD.
Figure S2. MiR-143/145 regulates MDM2 expression in HNSCCs cells. (A) The expression
levels of mature miR-143 in the HN30 cells were quantified by real-time PCR 48 hours after
transfection with miR-143 mimics or inhibitor. The results are shown as the mean ± SD. P < 0.05.
(B) The expression levels of mature miR-145 in the HN30 cells were quantified by real-time PCR
48 hours after transfection with miR-145 mimics or inhibitor. The results are shown as the mean ±
2
SD. P < 0.05. (C) A western blot analysis showing the MDM2 and p53 expression after miR-143
and miR-145 transfection using mimics for miR-143, miR-145, and NC in the HN30 cells.
Twenty-four hours post-transfection, the cells were treated with different concentrations of
Nutlin3 for 24 hours. (D) A densitometric analysis of the MDM2 and p53 protein expression in
the HN30 cells that were treated as described in panel C.
Figure S3. Alignment of putative MREs of the MDM2 3’UTR with miR-143 and miR-145 (A)
The miR-143 that were predicted to interact with the MDM2 gene at several putative binding sites
in its 3’ UTR according to RNA22 prediction software. (B) The miR-145 that were predicted to
interact with the MDM2 gene at several putative binding sites in its 3’ UTR according to RNA22
prediction software.
Figure S4. Kinetics of the miRNAs-MDM2-p53 feedback loop during ADM stimulation. (A)
An immunoblot analysis of the SCC-25 cells treated with ADM (0.2 μg/ml). Whole-cell lysates
were collected at the indicated times. (B) The expression levels of mature miR-143 and miR-145 in
the SCC-25 cells treated with ADM were analyzed as described in panel A. (C) Quantification of
the band intensity in (A). The p53 and MDM2 band intensities were normalized to that of GAPDH
and were normalized to the time =0 controls. Three independent sets of experiments were
performed to generate the error bars. The error bars represent ± SD.
Figure S5. Kinetics of miRNAs-MDM2-p53 feedback loop during IR stimulation. (a) An
immunoblot analysis of the HN30 cells exposed to X-ray (2Gy). (b) Expression of mature
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miR-143 and miR-145 in the HN30 cells exposed to X-ray (2Gy). (c) Quantification of the band
intensity in (a). The p53 and MDM2 band intensities were normalized to those of GAPDH and
normalized to the time = 0 controls. The results shown in subsequent panels used this approach for
protein quantification. (d) An immunoblot analysis of the HN30 cells transfected with the
siRNA-p53 oligos and exposed to X-ray (2Gy). (e) Expression of mature miR-143 and miR-145 in
the HN30 cells treated with siRNA and exposed to X-ray (2Gy). (f) Quantification of the band
intensity in (D). (g) An immunoblot analysis of the HN30 cells transfected with miRNA inhibitors
and exposed to X-ray (2Gy). (h) Expression of mature miR-143 and miR-145 in the HN30 cells
treated with miRNA inhibitors and X-ray. (i) Quantification of the band intensity in (g). Three
independent sets of experiments were performed to generate the error bars. The error bars
represent ± SD. IR: ionizing radiation.
Figure S6. Effect of miR-143 and miR-145 on the growth of squamous cell carcinomas cells.
An EdU assay performed in the HN30 (A), TE-12 (B), NCI-H226 (C), and SCC-25 (D) cell lines.
The cells were transfected with miR-143, miR-145, miR-pool (miR-143 combined with miR-145)
and a scrambled sequence (NC) and were subsequently harvested at 48 hours after transfection.
Figure S7. Effect of miR-143 and miR-145 on sensitizing WT p53 cells to cisplatin-induced
apoptosis. The HN30, TE-12, NCI-H226 and SCC-25 cell lines transfected with the miR-143 and
miR-145 mimics. Twenty-four hours post-transfection, the cells were treated with cisplatin
(1μg/ml) for 24 hours and were labeled with AnnexinV-FITC.
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Figure S8. MDM2 modulation accounts for miR-143 and miR-145 inhibition of cell growth
in vitro. (a) The western blotting analysis showing the MDM2 and p53 protein expression in the
NCI-H226 cells after 48 hours of miRNAs, siRNA and MDM2 expression vector transfection. (b)
The EdU assays detecting effect of miRNAs, siRNA and MDM2 expression vector on
proliferation of NCI-H226 cells. (c) A flow cytometry analysis identifying effect of miRNAs,
siRNA and MDM2 expression vector on cisplatin–induced apoptosis of NCI-H226 cells. (d) The
western blotting analysis showing the MDM2 and p53 protein expression in the SCC-25 cells after
48 hours of miRNAs, siRNA and MDM2 expression vector transfection. (e) The EdU assays
detecting effect of miRNAs, siRNA and MDM2 expression vector on proliferation of SCC-25
cells. (f) A flow cytometry analysis identifying effect of miRNAs, siRNA and MDM2 expression
vector on cisplatin–induced apoptosis of SCC-25 cells. miR-pool: miR-143 and miR-145;
siMDM2: siRNA MDM2; MDM2: MDM2 expression vector; sip53: siRNA p53.
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SUPPLEMENTAL METHODS
The RT-PCR primers.
The RT-PCR primer sequences are listed below.
Genes
Primer sequences
Reference
human mature miR-143
forward 5’-TGAGATGAAGCACTGTAGCTC-3’
human mature miR-145
forward 5’-GTCCAGTTTTCCCAGGAATCCCT-3’
forward 5’-TGAGGTGCAGTGCTGCATC-3’
human pre-miR-143
*
reverse 5’-GCTACAGTGCTTCATCTCAGACTC-3’
forward 5’-GTCCAGTTTTCCCAGGAATC-3’
human pre-miR-145
*
reverse 5’-AGAACAGTATTTCCAGGAAT-3’
forward 5’-GAAAGATGGAGCAAG-3’
human MDM2
reverse 5’-GAGGTGGTTACAGCA-3’
forward 5’-TCACCCACACTGTGCCCATCTACGA-3’
human β-actin
*
reverse 5’-CAGCGGAACCGCTCATTGCCAATGG-3’
forward 5’-CTCGCTTCGGCAGCACA-3’
human U6 snRNA
*
reverse 5’-AACGCTTCACGAATTTGCGT-3’
RT-PCR primers for evaluating unprocessed pri-miRNAs.
Gene
Primer sequence
Reference
forward 5’-GTGCTGCATCTCTGGTCAGTTG-3’
human pri-miR-143
reverse 5’-AGCACTTACCACTTCCAGGCTG-3’
*
forward 5’-GGGATTCCTGGAAATACTGT-3’
human pri-miR-145
*
reverse 5’-CCTCTTACCTCCAGGGACAG-3’
The primers used for cloning the pcDNA3.0-p53-Mut vector.
Gene
Primer sequence
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forward 5’-GGACACTTTGCGTTCGG-3’
human mutant type p53
reverse 5’-TGCCAGCATTTCACAGAT-3’
The primers used for cloning the ORF of MDM2 without 3’UTR
Gene
Primer sequence
forward 5’- GTCGGAAAGATGGAGCAA -3’
ORF of MDM2
reverse 5’- GTGGCAGATGACTGTAGGC -3’
The primers used for cloning the pGL3-MDM2 3’ UTR.
Gene
Primer sequence
forward 5’-TCTAGACCTACAGTCATCTGCCACC-3’
MDM2 3’UTR-P1
reverse 5’-TCTAGATCATTACTCCCATCCCTTA-3’
forward 5’-TCTAGAGTAACAAGCCTGTCAAAT-3’
MDM2 3’UTR-P2
reverse 5’-TCTAGAATGCTACCGATACTCCTC-3’
forward 5’-TCTAGA TGAGGAGTATCGGTAGCA-3’
MDM2 3’UTR-P3
reverse 5’-TCTAGA TTTCAAAGATGAGGCAGA-3’
forward 5’-TCTAGAACCGTACCACTTGTCAGC-3’
MDM2 3’UTR-P4
reverse 5’-TCTAGAATTCAGCATCCACCCATA-3’
* These primers were designed in a previous report (1).
Materials and Reagents
The HN4, HN6, HN12, HN13 and HN30 human HNSCCs cell lines were obtained
from the NIH. The SCC-4, SCC-9, SCC-25 and CAL-27 HNSCCs cell lines were
obtained from the ATCC. HN4, HN6, HN12, HN13, HN30 and CAL-27 cell lines
were maintained in DMEM containing 10% fetal calf serum; the SCC-4, SCC-9 and
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SCC-25 cell lines were maintained in DMEM/F12 containing 10% fetal calf serum.
All of the cells were cultured in a humidified atmosphere of 5% CO2 at 37°C.
The miR-143 mimics, miR-143 inhibitor, miR-145 mimics and miR-145 inhibitor were
purchased from GenePharma (Shanghai, China). The MDM2, p53, p21 and BAX
antibodies were purchased from ProteinTech Group (Chicago, USA).
Target Screening
In
this
study,
we
used
a
publicly
available
search
engine,
RNA22
(http://cbcsrv.watson.ibm.com/rna22.html), to obtain the putative targets. For sites
predicted by RNA22, we considered heteroduplexes with minimum folding energy
and the maximum number of paired bases.
Transfection
The transfection of the cells was performed using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s protocol. Briefly, the cells were seeded in 6-well
plates at 30% confluence the day before transfection. The miR-143/145
mimics/inhibitors and miRNA controls (100 nM each) were used for each
transfection.
Real-time PCR assay
Briefly, the total RNA was extracted with TRIzol (Invitrogen) and subjected to
reverse transcription. The miRcute miRNA qPCR detection kit (TIANGEN, Shanghai,
China) was used according to the manufacturer’s protocol for detecting mature
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miRNAs. The results were normalized to U6 snRNA.
Western blot assay
Cell lysates (30 μg) were electrophoresed through 10% polyacrylamide gels and
transferred to a nitrocellulose membrane. The membrane was incubated with MDM2
antibody and p53 antibody. The secondary antibodies were labeled with IRDyes. The
signals were observed using an Odyssey Infrared Imaging System.
Irradiation and preparation of conditioned medium.
X-irradiation was carried out with a Clinac 600 GMV Machine (Varian) at room
temperature. The applicated doses were 2 Gy and the dose rate was 1 Gy/min.
Cell proliferation assay
Cell proliferation was determined by the EdU assay. 5-ethynyl-2′-deoxyuridine (EdU)
is readily incorporated into cellular DNA during DNA replication. The terminal
alkyne group is then detected through its reaction with fluorescent azides in a Cu
(I)-catalyzed [3 + 2] cycloaddition.
Cell apoptosis assay
The cancer cells were transfected with 100 nmol miR-143/145 mimics for 24 hours
and were treated with 1 μg/ml cisplatin for 24 hours. The cells were harvested and
resuspended in 500 μl of a binding buffer. The cell suspension (100 μl) was incubated
with 5 μl annexin-V and propidium iodide at room temperature for 20 minutes. The
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stained cells were analyzed with fluorescent-activated cell sorting (FACS) using BD
LSR II flow cytometry.
SUPPLEMENTAL REFERENCE
1.
Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K. Modulation of microRNA
processing by p53. Nature. 2009 Jul 23;460(7254):529-33.
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