Carcinogenesis vol.31 no.4 pp.559–566, 2010
doi:10.1093/carcin/bgp335
Advance Access publication January 6, 2010
microRNA-141 is involved in a nasopharyngeal carcinoma-related genes network
Liming Zhangy, Tan Dengy, Xiayu Liy, Huaying Liu, Houde
Zhou, Jian Ma, Minghua Wu, Ming Zhou, Shourong
Shen1, Xiaoling Li, Zhaoxia Niu, Wenling Zhang, Lei Shi,
Bo Xiang, Jianhong Lu, Li Wang, Dan Li, Hailin Tang and
Guiyuan Li
Cancer Research Institute, Central South University, Changsha, Hunan
410078, China and 1Third Xiangya Hospital, Central South University,
Changsha, Hunan 410013, China
To whom correspondence should be addressed. Tel: þ86 731 84805383;
Fax: þ86 731 84805446;
Email: [email protected]
Correspondence may also be addressed to Jian Ma. Tel: þ86 731 84805383;
Fax: þ86 731 84805446;
Email: [email protected]
microRNAs (miRNAs) are small non-coding RNAs and have been
implicated in the pathology of various diseases, including cancer.
Here we report that the miRNA profiles have been changed after
knockdown of one of the most important oncogene c-MYC or reexpression of a candidate tumor suppressor gene SPLUNC1 in
nasopharyngeal carcinoma (NPC) cells. Both c-MYC knockdown
and SPLUNC1 re-expression can down-regulate microRNA-141
(miR-141). miR-141 is up-regulated in NPC specimens in comparison with normal nasopharyngeal epithelium. Inhibition of
miR-141 could affect cell cycle, apoptosis, cell growth, migration
and invasion in NPC cells. We found that BRD3, UBAP1 and
PTEN are potential targets of miR-141, which had been confirmed following luciferase reporter assays and western blotting.
BRD3 and UBAP1 are both involved in NPC carcinogenesis as
confirmed through our previous studies and PTEN is a crucial
tumor suppressor in many tumor types. BRD3 is involved in the
regulation of the Rb/E2F pathway. Inhibition of miR-141 could
affect some important molecules in the Rb/E2F, JNK2 and AKT
pathways. It is well known that carcinogenesis of NPC is involved
in the networks of genetic and epigenetic alteration events. We
propose that miR-141- and tumor-related genes c-MYC, SPLUNC1,
BRD3, UBAP1 and PTEN may constitute a gene–miRNA network
to contribute to NPC development.
Introduction
It is well known that there will be more than one or even a network of
genetic or epigenetic alteration events that could have happened in
polygenetic inherited cancers such as nasopharyngeal carcinoma
(NPC) (1). Searching for the susceptibility genes or oncogenes that
are associated with NPC has attracted numerous research attentions.
We have been working on identification of NPC-related genes through
genome-wide scan, linkage analysis, expression profiling and position
cloning (2–6). Based on this experience and many other literature
evidence, we propose that NPC carcinogenesis is a stepwise, sequential process that involves multiple cancer-related genes.
By using suppression subtractive hybridization assay, we identified
that the SPLUNC1 gene is a tissue-specific gene of nasopharyngeal
epithelia and is down-regulated in NPC (5,7). SPLUNC1 protein is
Abbreviations: 141I, microRNA 141 inhibitor; 141M, microRNA 141 mimic;
INC, inhibitor negative control; miRNA, microRNA; miR-141, microRNA141; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide;
NPC, nasopharyngeal carcinoma; PCR, polymerase chain reaction; qRT-PCR,
quantitative reverse transcription–polymerase chain reaction; RT, reverse transcription; siRNA, small interfering RNA; UTR, untranslated region.
y
These authors contributed equally to this work
expressed in the serous glands and epithelium of the upper respiratory
tract. It is an innate immunity-defensive secretory protein and can
bind to bacterial lipopolysaccharide and inhibit Pseudomonas aeruginosa and Epstein-Barr virus, which is the most important environmental factor in NPC tumorigenesis (8).
BRD7 is a potential nuclear transcription factor and belongs to
a bromodomain family, which includes BRD2 and BRD3 (9). BRD7
is absent or low expressed in NPC biopsies and over-expression of it
can inhibit NPC cell growth and arrest cells in the G0–G1 phase (10).
BRD2 and BRD3 could interact with BRD7 and have distinct roles in
regulating cell apoptosis (11). Our previous study showed that c-MYC
is a negative regulator of BRD7 (12). c-MYC is over-expressed in
most human cancers and executes its multiple activities mostly
through transcriptional regulation of its target genes (13). Several
studies indicate that over-expression of c-MYC is a frequent genetic
abnormality in NPC (14,15).
UBAP1 gene is a putative NPC-related gene, which is located at
chromosome 9p21-22, where loss of heterozygosity frequently occurs
in NPC. UBAP1 is down-regulated in NPC specimens and contains
two ubiquitin-associated domains (16–18).
MicroRNAs (miRNAs) are short (20–23 nucleotides), endogenous, single-stranded non-coding RNA molecules and fundamental
regulatory elements of gene expression (19,20). miRNAs control
gene expression by inhibiting protein translation or degrading cognate target mRNAs through binding to their 3# untranslated regions
(UTRs) with varying degrees of sequence complementarity. Global
expression of miRNAs is seemingly deregulated in most cancer
types, according to research by recent high-throughput miRNA
microarray assays. The role of miRNAs in NPC carcinogenesis is
poorly investigated. Sengupta et al. (21) reported that mir-29c is
down-regulated in NPC and can up-regulate mRNAs encoding
extracellular matrix proteins. Chen et al. (22) recently identified
several classic tumor-related pathways that are targeted by dysregulated miRNAs in NPC. It becomes obvious that miRNAs,
oncogenes and tumor suppressors all are members of the complex
cancer pathway network.
In this study, we investigated the effect of c-MYC knockdown and
SPLUNC1 re-expression in an NPC cell line on global miRNA
expression profile and found some dramatic changes in miRNAs expression. One of them is microRNA-141 (miR-141), which is upregulated in NPC. Through computational and luciferase reporter
assays, we identified that BRD3, PTEN and UBAP1 are targets of
miR-141. This finding suggested that miR-141 is involved in an
NPC-related gene network.
Materials and Methods
Cell culture and stable transfection
5-8F cell lines (an NPC cell line with high tumorigenic and metastatic ability) were kindly provided by the Cancer Center of Sun Yet-Sen University
(Guangzhou, China). 5-8F/Si-c-MYC cells, which knockdown c-MYC
expression by stably expressed small interfering RNA (siRNA) targeting
c-MYC, and its negative control (NC) cells (5-8F/Si-control), which is a negative
mock siRNA vector, are introduced into 5-8F cells, as reported in previous
literature (12). pcDNA3.1/SPLUNC1 expression vector and pcDNA3.1/control
vector were stably expressed in 5-8F cells through Lipofectamine 2000 (Invitrogen, Carlsbad, CA)-mediated transfection according to the manufacturer’s
protocol. G418 selection (80 lg/ml) begun at 24 h post-transfection. Colonies
were picked after 2 weeks and expanded into cell lines. We used SPLUNC1
antibody (23) to verify the positive clone. These two cell lines were named as
5-8F/SPLUNC1 and 5-8F/control. All of these cell lines were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum. We also used the
same technique to establish 5-8F cells, which can stably express BRD3.
Antibody and western blotting
Western blotting was carried out as described previously (24). Anti-b-ACTIN
(A5441) was purchased from Sigma-Aldrich (St Louis, MO). Anti-CDK4
Ó The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
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(sc-163), cyclin D1 (sc-450), CDK2 (sc-260), phos-RB (Ser780), E2F3 (sc879), DP2 (sc-829), JNK2 (sc-56922) and PTEN (sc-7974) were from Santa
Cruz Biotechnology (Santa Cruz, CA). Anti-c-MYC (CBL434) was from Upstate Biotechnology (Chicago, IL). Anti-AKT and phos-AKT (Ser473) were
from Cell Signaling Technology (Danvers, MA). Anti-BRD3 and anti-UBAP1
were from Proteintec Group, Inc. (Wuhan, China). The loading control for
western blotting was b-ACTIN.
miRNA microarray analysis
miRNA microarrays were obtained from CapitalBio Corporation (Beijing,
China), corresponding to current release of Sanger miRNAs database
(http://www.mirbase.org/; miRBase Release 8.2). miRNAs were enriched
from total RNA-extracted cells (5-8F/Si-c-MYC and 5-8F/Si-control; 5-8F/
SPLUNC1 and 5-8F/control) with mirVana miRNA Isolation Kit (Ambion,
Foster City, CA) and labeled with mirVana Array Labeling Kit. Labeled
miRNAs were used for hybridization on each miRNA microarray containing 509 probes in triplicate, corresponding to 434 human (containing 122
predicted miRNA sequences from a published reference), 196 rat and 261
mouse miRNAs, to determine differential expression between the cell lines.
This procedure was repeated twice. The miRNA microarray from CapitalBio Corporation was a single-channel fluorescence chip; all oligonucleotide
probes were labeled with Cy3 fluorescent dyes (green color). Fluorescence
scanning was done using a double-channel laser scanner (LuxScan 10K/A;
CapitalBio). Then, the figure signals were transformed to digital signals
using image analysis software (LuxScan3.0; CapitalBio). Raw data were
normalized and analyzed using the Significance Analysis of Microassays
(SAM, version 2.1; Stanford University, CA) software.
Patient samples
For the miRNA expression study, we used 10 NPC tissues and 10 normal
nasopharyngeal epithelium samples from biopsy-negative cases as control.
Both samples were obtained from patients in the Second Xiangya Hospital
and Hunan Province Tumor Hospital. All tissue samples were snap frozen in
liquid nitrogen and stored at 80°C until laser-capture microdissection. We
used LEICA CM 1900 for frozen section and used LEICA CTR 6500 to obtain
the pure nasopharyngeal epithelium tissues. Written informed consent was
obtained from all study participants. Collections and use of tissue samples
were approved by the ethical review committees of the appropriate institutions.
Quantitative reverse transcription–polymerase chain reaction analysis
To confirm the miRNA microarray results, real-time reverse transcription
(RT)–polymerase chain reaction (PCR) was used to detect cellular miRNAs,
as described, but with minor modifications (25). The primers for RT–PCR to
detect miRNA were designed based on the miRNA sequences provided by the
Sanger Center miRNA Registry and were synthesized and purified by the
Shanghai Gene-Pharma Co. (Shanghai, China). The total RNAs were isolated
from cells or tissues with TRIZOL reagent (Invitrogen). RT reactions were
performed by means of the iScript cDNA synthesis kit (Bio-Rad, Hercules,
CA). Real-time PCR was performed on the BIO-RAD IQTM5 Multicolor RealTime PCR detection System (Bio-Rad). U6 RNA was used as an endogenous
control for miRNA detection.
were used to examine 10 000 cells per experiment. Data were analyzed with
Modfit software (Verity Software House, Topsham, ME). Values were expressed as mean and error deviation of three independent experiments.
Wound-healing assay
Cells were plated and grown overnight to confluence in a six-well plate. Monolayers of cells were wounded by dragging a 10 ll pipette tip. Cells were washed
to remove cellular debris and allowed to migrate for 24 h. Images were taken at
0 h and 24 h after wounding under the inverted microscope.
Transwell invasion assay
Cell invasion was measured by a Matrigel invasion chamber assay, which was
performed using 6.5 mm and 8 lm pore size Transwell chambers (Corning,
Corning, NY). 5-8F cells transfected with miRNA mimics or inhibitors were
plated 12 h post-transfection in serum-free medium (1 104 cells per Transwell) and allowed to migrate toward a 10% fetal bovine serum gradient for
48 h. Cells that remained on the top of the filter were scrubbed off and cells that
had migrated to the underside of the filter were fixed in methanol and stained
with hematoxylin and eosin. Whole filters were manually counted under the
inverted microscope.
Bioinformatics analysis
To find out the basic information about selected miRNAs, we chose miRBase
(http://www.mirbase.org/search.shtml) for further research. To select possible
targets to validate the significance of the detected miRNAs, we utilized both
TargetScan 5.0 (http://www.targetscan.org/) and Pictar (http://pictar.mdc-berlin
.de/), which identifies binding sites targeted by single miRNA, as well as those
that are co-regulated by several miRNAs in a coordinated manner. We used
DAVID 2008 Functional Annotation Bioinformatics Microarray Analysis Tools
(http://niaid.abcc.ncifcrf.gov/) to classify the function of the target genes, which
were predicted both from TargetScan 5.0 and Pictar. We used the Cancer Gene
Census (http://www.sanger.ac.uk/genetics/CGP/Census/) and Tumor Suppressor Gene Database (http://www.cise.ufl.edu/yy1/HTML-TSGDB/Homepage.html) to identify whether the target gene is an oncogene or a tumor
suppressor gene.
3#UTR luciferase reporter assays
The binding site of miRNAs and 3#UTR of target genes were predicted from
TargetScan 5.0 and Pictar. We synthesized two single strands of 3#UTR of
target genes that contained the binding site of miRNAs. Then two single
strands of 3#UTR of target genes that deleted five bases in the binding site
of miRNAs were synthesized as a mutant control. The oligonucleotides used in
these studies were as follows:
3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide assay
After transient transfection with miRNA mimics or inhibitors, the cells were
seeded into 96-well plates at 1500 cells per well. 3-(4,5-Dimethylthiazole-2yl)-2,5-biphenyl tetrazolium bromide (MTT; Sigma-Aldrich) assays were performed daily for 6 days. To this end, the medium was replaced with fresh
medium containing 0.5 mg/ml MTT for 4 h, and then carefully removed. Dimethyl sulfoxide (150 ll) was added to each well and mixed for 10 min, and
optical density at 490 nm was determined with an enzyme-linked immunosorbent assay reader.
3#UTR of BRD3 (which contained the binding site of miR-141)
5#-CTAGTTGCAGGTCTGCTTCTTAATTCAGTGTTATGATATCTTCCAGTTTTTGCA-3# and 5#-AGCTTGCAAAAACTGGAAGATATCATAACACTGAATTAAGAAGCAGACCTGCAA-3#
3#UTR of mutant BRD3 [which deleted five bases (AGTGT) in the binding
site of miR-141]
5#-CTAGTTGCAGGTCTGCTTCTTAATTC_____TATGATATCTTCCAGTTTTTGCA-3#
and
5#-AGCTTGCAAAAACTGGAAGATATCATA_____GAATTAAGAAGCAGACCTGCAA-3#
3#UTR of PTEN (which contained the binding site of miR-141)
5#-CTAGTTTTTTTTTTTTTTAAATGTGCAGTGTTGAATCATTTCTTCATAGTGCTA-3# and 5#-AGCTTAGCACTATGAAGAAATGATTCAACACTGCACATTTAAAAAAAAAAAAA A-3#
3#UTR of mutant PTEN [which deleted five bases (AGTGT) in the binding
site of miR-141]
5#-CTAGTTTTTTTTTTTTTTAAATGTGC_____TGAATCATTTCTTCATAGTGCTA-3# and 5#-AGCTTAGCACTATGAAGAAATGATTCA_____GCACATTTAAAAAAAAAAAAA A-3#
3#UTR of UBAP1 (which contained the binding site of miR-141)
5#-CTAGTCCCCTCCTAACAAAGTCTCATAGTGTTAACACTGGTTCTGCAATATCTA-3# and 5#-AGCTTAGATATTGCAGAACCAGTGTTAACACTATGAGACTTTGTTAGGAGGGGA-3#
3#UTR of mutant UBAP1 [which deleted five bases (AGTGT) in the binding
site of miR-141]
5#-CTAGTCCCCTCCTAACAAAGTCTCAT_____TAACACTGGTTCTGCAATATCTA-3#
and
5#-AGCTTAGATATTGCAGAACCAGTGTTA_____ATGAGACTTTGTTAGGAGGGGA-3#.
Flow cytometry analysis
Cells were collected, washed twice with phosphate-buffered saline and fixed in
70% ethanol overnight. Cells were centrifuged at 1500 g for 8 min, resuspended
in 50 lg/ml propidium iodide (Sigma-Aldrich) in phosphate-buffered saline
and immediately subjected to a FACStar flow cytometer (Beckton-Dickinson,
Mountain View, CA). Appropriate settings of forward and side scatter gates
The sense and anti-sense strands of the oligonucleotides were annealed,
digested with HindIII and SpeI and ligated into pmir-Report luciferase
vector (Ambion, Austin, TX). Seven 3#UTR luciferase reporters were constructed, which were named as LR-blank, LR-141/BRD3, LR-141/BRD3m,
LR-141/PTEN, LR-141/PTENm, LR-141/UBAP1 and LR-141/UBAP1m
(Figure 3b).
siRNAs transient transfection
siRNA duplex homologs in sequence with microRNA-141 mimics (141M) and
microRNA-141 inhibitors (141I) were synthesized and purified by Shanghai
Gene-Pharma Co. (Shanghai, China). siRNA duplexes with non-specific sequences were used as NC as well as inhibitor negative control (INC). Hundred
picomoles of different siRNAs (141M, 141I, NC and INC) were transfected
separately into cells by using Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s protocol.
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miR-141-related network in nasopharyngeal carcinoma
5-8F cells were seeded in 24-well plates 24 h prior to transfection.
The following day, 200 ng of reporter plasmid along with 10 pmol of
miRNA mimic or mimic-NC were co-transfected into cells using Lipofectamine 2000. Luciferase activity was measured in cell lysates 24 h after
transfection using a Luciferase Assay kit (Promega, Madison, WI).
b-Galactosidase activity was measured in cell lysates by b-galactosidase
Enzyme Assay System (Promega). Results are normalized against
b-galactosidase activity.
Results
Changes of miRNA profile induced by c-MYC inhibition or SPLUNC1
re-expression in NPC cells
To investigate the role of c-MYC in NPC, we have established
previously a stable NPC cell line 5-8F in which endogenous c-MYC
was inhibited through siRNA technique (12). Figure 1a shows that
endogenous c-MYC is significantly inhibited by siRNA-c-MYC stable
transfection. c-MYC is a helix-loop-helix leucine zipper transcription
factor that regulates an estimated 10–15% of genes in human genomes.
Both c-MYC and miRNAs control the expression of globe genes. We
thus sought to determine whether c-MYC is involved in NPC carcinogenesis through regulating miRNA expression in NPC cells.
An miRNA microarray capable of measuring expression of 434 human, 196 rat and 261 mouse miRNAs was used to analyze the miRNA
profile changes between 5-8F/Si-control and 5-8F/Si-c-MYC cell
lines (Figure 1b). Twelve human miRNAs were up-regulated and
seven miRNAs were down-regulated (with fold change .3) in
5-8F/Si-c-MYC. Among those miRNAs, miR-200C and miR-141
were dramatically down-regulated with fold change .10 (supplementary Table 1 is available at Carcinogenesis Online). In order to
exclude possibilities of false-positive results coming from miRNA
microarray analysis, we performed quantitative RT–PCR analysis
to verify the expression of miR-200C and miR-141. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) results
revealed that both miR-141 and miR-200c were down-regulated in
Fig. 1. Changes of miRNA profile induced by c-MYC inhibition or SPLUNC1 re-expression. (a) Western blotting confirmed that endogenous c-MYC was
significantly down-regulated in 5-8F/Si-c-MYC cells compared with 5-8F/Si-control and 5-8F. (b) The miRNA microarrays result of 5-8F/Si-c-MYC and 5-8F/Sicontrol cells. (c) The miRNA microarray results of fluorescence value of miR-141 and 200c. (d) qRT–PCR analysis of the relative expression levels of miR-141
and miR-200c in the same sample that were used for microarrays chip. Data were normalized to that of U6 and the relative levels are shown. (e) Assaying
SPLUNC1 protein expression levels in 5-8F/SPLUNC1 (S3, S4, S5, S6, S7), 5-8F/control (P1-P4) and 5-8F cell lines. The same membrane was first probed with
anti-SPLUNC1-antibody and then with anti-b-ACTIN. (f) The miRNA microarrays result of 5-8F/SPLUNC1 and 5-8F/control. (g) The miRNA microarray results
of fluorescence value of miR-141 and miR-205. (h) qRT–PCR analysis of the relative expression levels of miR-141 and miR-205 in the same sample that were used
for microarrays. Data were normalized to that of U6 and the relative levels are shown. The microarray results of fluorescence value show the same outcome as
qRT–PCR.
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the 5-8F/Si-c-MYC cells, which was in accordance with the results
from miRNA microarray (Figure 1c and d).
Since the SPLUNC1 gene is a tissue-specific gene of nasopharyngeal epithelia and down-regulated in NPC, we sought to explore the
downstream targets of the SPLUNC1 gene in NPC cells. We constructed an NPC cell line in which the SPLUNC1 gene was introduced
into 5-8F cells through stable transfection. From 31 SPLUNC1 transfected clones and 11 mock transfection clones, we verified two positive clones S4 and S5 that expressed high levels of SPLUNC1
protein, whereas in mock transfection clones (P1-P4), endogenous
SPLUNC1 was hardly to be detected (Figure 1e). We extracted total
RNA from S4 and P1 and performed miRNA microarray analysis
(Figure 1f). We found that 29 miRNAs were up-regulated and
25 miRNAs were down-regulated (with fold change .3) in Splunc1
re-expressed cells. Among those miRNAs, miR-202, miR-516-3P and
miR-520b were greatly up-regulated, whereas miR-122a, miR-141
and miR-205 were dramatically down-regulated with fold change
.10 (supplementary Table 2 is available at Carcinogenesis Online).
We also performed qRT–PCR to verify the expression of miR-141 and
miR-205 and confirmed that both miR-141 and miR-205 were downregulated in S4 cells and the differences were the same as the
anticipated results from miRNA microarray (Figure 1g).
miR-141 is down-regulated through c-MYC inhibition or SPLUNC1
re-expression and up-regulated in NPC specimens
We were surprised that both c-MYC inhibition and SPLUNC1 reexpression can down-regulate miR-141 in 5-8F cells. Since c-MYC
functions as an oncogene and SPLUNC1 is a candidate tumor suppressor gene in NPC, this suggests that miR-141 may be an important
regulator in 5-8F cells through controlling its downstream targets. To
explore the expression of miR-141 in the real world, 10 NPC specimens and 10 normal nasopharyngeal epithelium specimens were obtained to verify the miR-141 expression in these tissues by qRT–PCR.
All tissue specimens were purified by laser-capture microdissection
before the RNA extraction. Figure 2a showed that samples were mi-
crodissected by laser-capture microdissection system to remove the
lymphocytes and interstitial cells. Figure 2b showed that the average
expression levels of miR-141 are higher in 10 NPC specimens than in
normal nasopharyngeal epithelium tissues (P , 0.025).
miR-141 plays a role as oncogene in NPC cell through affecting cell
cycle, migration and invasion
To study the cellular function of miR-141 in NPC, we transfected
141M, 141I and siRNA duplexes with non-specific sequences as NC
and INC into NPC cells separately. As expected, the levels of
miR-141 were elevated by transfection of the mimics and decreased
by transfection of the inhibitors (Figure 3a). We then performed an
MTT assay to investigate whether miR-141 could affect the growth
of 5-F cells. As shown in Figure 3b, at day 6, miR-141 inhibitor
significantly decreased the viability of 5-8F cells, which suggested
that the miR-141 level could regulate NPC cell viability. The reason
141M did not increase the viability of NPC cells is possibly due to
there already being plenty of endogenous miR-141 in the 5-8F cells.
We next assayed the effect of miR-141 on cell cycle regulation.
Figure 3c shows that miR-141 inhibition resulted in a substantial
increase in the G0–G1 phase (proportion percentage from 42% to
69%) and a reduction in the number of cells in the S phase (from
30% to 12%). Little effect was noticed for the G2–M phase of the
cell cycle. We also found that miR-141 inhibition slightly increased
the apoptosis rate (Figure 3d).
In order to demonstrate the effect of miR-141 on the migration and
mobility of 5-8F cells, an in vitro cell invasion assay was performed
based on the principle of the Boyden chamber assay. Cells that had
migrated through the Matrigel matrix were counted and the numbers
are presented in Figure 3e. When treated with miR-141 inhibition, the
number of 5-8F cells migrating through the Matrigel decreased significantly compared with the control group (P , 0.05). To provide
further support on the effect of miR-141 on cell migration ability, the
in vitro scratch wound-healing assay was performed. As shown in
Figure 3f, 5-8F cells migrated significantly slower in miR-141
Fig. 2. miR-141 is up-regulated in NPC specimens. (a) Top panel, representative photographs (100) of normal nasopharyngeal epithelium microdissection;
bottom panel, NPC epithelium. Stained by methyl green. (b) qRT–PCR analysis of the relative expression levels of miR-141 with 10 normal tissues and
10 NPC tissues. Data were normalized to that of U6 and the relative levels are shown. Differences between groups were analyzed using an unpaired Student’s t-test
(P , 0.025).
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miR-141-related network in nasopharyngeal carcinoma
Fig. 3. Alteration of miR-141 can affect NPC cells’ growth, cell cycle, apoptosis and migration ability. (a) 141M, 141I or their NC miRNAs (NC or INC) were
transfected into 5-8F cells. Cells were harvested after 24 h transfection, and endogenous miR-141 level was measured by qRT–PCR. U6 RNA expression was
used for normalization. (b) After transfection with those miRNA mimics and inhibitors, the result of MTT assay was counted everyday for 6 days. Each value
represents mean ± SE for three observations at least. P , 0.05 compared with controls. (c) Graph showing the cell cycle status of each group by flow cytometry
analysis. (d) Graph showing the apoptosis rate of each group by flow cytometry analysis. (e) Graphical presentation of the number of migrated cells in each
group in the in vitro Transwell invasion assay. Data are mean ± SEM; n 5 3; P , 0.05 compared with controls. (f) Wound-healing assay. Upper panel, different
miRNA mimics or inhibitors groups at two time points (0, 24 h) are shown. The wound edge’s changes are shown with broken lines. Bottom panel, graphical
presentation of the percentage of the wound healing, which can be counted with (0 h width 24 h width of wound)/0 h width of wound. Data are mean ± SEM;
n 5 3; P , 0.05 compared with controls.
inhibition group (P , 0.05), whereas higher in 141M group
(P , 0.05). Those results suggested that miR-141 is an important
regulator of migration and invasion in 5-8F cells.
Bioinformatics and experiments verification of putative miR-141
targets: Brd3, Ubap1 and Pten are among them
Since miR-141 plays a role as oncogene in NPC, we sought to search
the possible targets of miR-141 by using Web-based bioinformatics
software. The basic information of miR-141 is collected from miRBase::Sequences database, and the possible targets are predicted by
the online software TargetScan and Pictar. TargetScan predicts that
429 genes are possible targets of miR-141, whereas Pictar predicts
that 465 genes are targets of miR-141. Then, we used DAVID 2008
Functional Annotation Bioinformatics Microarray Analysis Tools to
classify the function of the target genes, which are predicted from
both TargetScan 4.2 and Pictar. The results revealed that most
(.50%) target genes of miR-141 are involved in signaling pathways
such as mitogen-activated protein kinase, Wnt, JAK-STAT, tight junction, cell cycle and adherens junction, which are important for tumorigenesis (Figure 4a). We were interested to know whether tumor
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suppressor genes were potentially inhibited by miR-141. From the
Tumor Suppressor Gene Database, we found that some miR-141targeted genes, such as MAP2K4, MAP3K3 and DLC1, are considered
as classical tumor suppressor genes. In the list of miR-141-targeted
genes, three genes attracted our attention: PTEN, BRD3 and UBAP1.
PTEN inhibits PI3K activity and then acts as a vital tumor suppressor
gene involving multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth (26). BRD3 is a BRD7 (an
NPC candidate tumor suppressor gene cloned in our laboratory)
interacting protein and participates in the apoptosis process. UBAP1
was originally cloned in our laboratory and has been found to be
down-regulated in NPC tissues. As a candidate tumor suppressor
gene, the regulating mechanism of UBAP1 is largely unknown.
We cloned the 3#UTRs containing the miR-141-binding sites for
the PTEN, BRD3 and UBAP1 genes into a vector downstream of
a firefly luciferase gene. We named them as LR-blank, LR-PTEN
and LR-PTENm (mutant construct, predicted binding site of
miR-141 was deleted); LR-BRD3 and LR-BRD3m and LR-UBAP1,
LR-UBAP1m (Figure 4b). 5-8F cells were transfected with those constructs with 141M or NC. The 3#UTRs of PTEN, BRD3 and UBAP1
elicited significantly decreased luciferase activities (P , 0.05) in
141M transfected cells, whereas the mutant of PTEN, BRD3 and
UBAP1 (which deleted the binding sites) diminishes the regulating
function of miR-141. These results verified that the effect of miR-141
is due to direct interaction with the binding sites in 3#UTR of PTEN,
BRD3 and UBAP1 (Figure 4c).
Since miRNAs down-regulate a specific target by affecting mRNA
translation or mRNA stability, we want to detect whether transfection
with 141I could affect the PTEN, BRD3 and UBAP1 protein levels.
The result demonstrated that PTEN, BRD3 and UBAP1 were upregulated upon miR-141 inhibition (Figure 4d). We also noticed that
knockdown of c-MYC could up-regulate PTEN and BRD3, whereas
re-expression of SPLUNC1 could up-regulate UBAP1 expression
(Figure 4d). The above results confirmed that PTEN, BRD3 and
UBAP1 are direct targets of miR-141.
Fig. 4. Identification of targets of miR-141. (a) With the analysis of DAVID
2008 Functional Annotation Bioinformatics Microarray Analysis Tool, the
pie chart showed the percentage of predicted target genes that may involve in
different pathways. (b) Schematic representation of the seven reporter
constructs that were named as LR-blank, LR-141/BRD3, LR-141/BRD3m,
LR-141/PTEN, LR-141/PTENm, LR-141/UBAP1 and LR-141/UBAP1m.
‘m’ indicates the mutant construct that predicted that the binding site of
miR-141 was deleted. (c) The ‘200 ng’ indicated that firefly luciferase vector
were contransfected with indicated material (no treatment, 10 pmol NC,
10 pmol 141M) into 5-8F cells. Luciferase activities were measured after
48 h, and b-galactosidase is used to normalize for differences in transfection
efficiency. Data are confirmed in duplicate experiments. The data are mean ±
SEM of separate transfections (n 5 6); P , 0.05 compared with no
treatment group. (d) BRD3, PTEN and UBAP1 protein expression levels
were increased upon transfection with 141I compared with control group in
5-8F cells. Knockdown of c-MYC could up-regulate PTEN and BRD3 and
re-expression of SPLUNC1 could up-regulate UBAP1 expression.
564
Effect of miR-141 on Rb/E2F and PTEN/AKT pathway is possibly
through its targets
BRD3 is one of the BRD7 interacting proteins and BRD7 can regulate
Rb/E2F pathway (11). In order to know whether BRD3 may also regulate Rb/E2F pathway, BRD3 recombinant vector was transfected into
5-8F cells. Over-expression of BRD3 in 5-8F cells down-regulated
some critical molecules of Rb/E2F pathway including phos-Rb, cyclin D1 and E2F3 (Figure 5a). This result suggested that BRD3
negatively regulated the Rb/E2F pathway. We next explored the role
of miR-141 in Rb/E2F pathway since inhibition of miR-141 can
arrest NPC cells in the G0–G1 phase. Figure 5b showed that 141M
up-regulated cyclin D1, phos-Rb, E2F3 and DP2 in 5-8F cells, which
indicated that miR-141 is a promoter of cell cycle in 5-8F cells. We
also found that inhibition of miR-141 can up-regulate the levels of
JNK2 (Figure 5c). JNK2 activation is crucial for the induction of
cancer cell apoptosis (27). Since PTEN is a target of miR-141, we
then checked the phos-AKT level upon miR-141 inhibition.
Figure 5d shows that miR-141 inhibition down-regulated phosAKT expression. This result confirmed that miR-141 might regulate
PTEN/AKT at least partly.
Discussion
NPC is a malignancy with high occurrence in Southern China and
Southeast Asia. Previous studies indicate that multiple oncogenes,
including c-MYC, and multiple tumor suppressor candidates, such
as SPLUNC1, BRD7 and UBAP1, are abnormal in NPC. It is very
possible that there is a complex NPC-related oncogenes–tumor suppressor genes network contributing to NPC tumorigenesis. But the
mechanism of how those genes interact with each other to induce
NPC development is far from being fully known.
The continued presence of c-MYC was required for cancer development, whereas inactivation of c-MYC resulted in sustained
miR-141-related network in nasopharyngeal carcinoma
regression of tumors (28,29). Karlsson et al. (30) found that inactivation of c-MYC alone was sufficient to cause sustained tumor regression in c-Myc-induced hematopoietic tumors. Reducing c-MYC
expression in MCF-7 breast cells by RNAi could significantly inhibit
tumor growth both in vitro and in vivo (31). Several studies showed
that c-Myc also can regulate some miRNAs (32–34), but the mechanism of how c-MYC interacts with miRNAs that cause the tumorigenesis of NPC is not known.
In this study, we assayed whether alteration of c-MYC and SPLUNC1
could affect the miRNA–gene network in NPC cells by employing
miRNA microarray. A number of miRNAs were altered upon c-MYC
knockdown or SPLUNC1 re-expression (fold change .3). Among the
miRNAs with fold change .10, surprisingly, we found that miR-141
was down-regulated by both c-MYC knockdown and SPLUNC1
re-expression.
We then confirmed that miR-141 is expressed significantly higher in
NPC tissues compared with normal nasopharyngeal epithelial tissues
by qRT–PCR. We next found that inhibition of miR-141 significantly
decreases the viability of 5-8F cells and cell cycle arrest in the G0–G1
phase and decreases cell migration and invasion ability. We also noted
that miR-141 inhibition slightly increased apoptosis rate of 5-8F cells.
All of these results imply that miR-141 might play an important role as
oncogene in NPC tumorigenesis. The role of miR-141 in tumorigenesis
is controversial: it functioned as tumor suppressor in renal cell carcinoma (35) and breast cancer cell lines (36), whereas in our research, it
seemed to have the opposite effect on NPC cells. With the prediction of
online tools such as Pictar and TargetScan, one miRNA was often found
to target both oncogenes and tumor suppressor genes (37). As an example, miR-175 and miR-20a can behave as an oncogene (38) or
a tumor suppressor (33) in different cell types. A recent study showed
that miR-141, which belongs to the miR-200 family, was found to be
up-regulated in ovarian carcinoma (39), prostate cancer (40), adrenocorticotropic hormone pituitary tumor (41) and cholangiocarcinoma
(42) and down-regulated in renal cell carcinoma (35) and breast cancer
(36). Since miR-141 was either up-regulated or down-regulated in
Fig. 5. (a) BRD3 recombinant vector was constructed and transfected into
5-8F cells. Over-expression of BRD3 in 5-8F cells down-regulated some
critical molecules of Rb/E2F pathway including cyclin D1, phos-RB and
E2F3. But there are no changes of DP2, CDK2 and CDk4 observed by western
blotting. (b) 141M can up-regulate the protein expression levels of critical
molecules of Rb/E2F pathway including cyclin D1, pRB, E2F3 and DP2.
(c) miR-141 inhibition increased JNK2 protein expression. (d) miR-141
inhibition down-regulated phos-AKT expression in 5-8F cells.
different cancers, we could draw a conclusion that miR-141 may play
different roles as an oncogene or a tumor suppressor gene in different
cancer types.
MiR-141 inhibition can regulate the Rb/E2F pathway, which is
critical for normal cell cycle progression from G1 into S phase
(Figure 5b). In the presence of extracellular growth-stimulatory
signals, cyclin D and its kinase partner CDK4 form activated complex
and phosphorylate RB protein or RB family members, thus inactivating RB and allowing E2F/DP transcription factor to exert their transactivation activity. This leads to transcription of a number of genes
essential for DNA replication and entry into S phase (43).
Bioinformatics and experimental results confirmed that BRD3,
PTEN and UBAP1 act as direct targets of miR-141. Our previous
study indicated that BRD7 inhibited G1–S progression through Rb/
E2F and RAS/MEK/ERK pathways (10). Here, we also showed that
BRD3 also negatively regulated Rb/E2F pathway (Figure 5a).
Phosphorylation of AKT plays an important role to result in the
activation of cascade of different protein targets involved in cell
growth, proliferation and invasion, and promote tumorigenesis. PTEN
is the most important negative regulator of AKT signaling pathway
(44–47). MiR-141 might increase the AKT phosphorylation levels
through the inhibition of PTEN expression (Figure 5d).
Some bioinformatics tools employed in the PROSITE database
identified two tandem UBA domains within UBAP1 protein (18).
UBA domain is involved in conferring target specificity to multiple
enzymes of the ubiquitination system. The ubiquitin-dependent pathway has a decisive role in understanding pathological states including
abnormal cellular proliferation and tumor growth.
Here we draw a hypothesis schema of the relationship between
c-MYC, SPLUNC1, BRD3, UBAP1, PTEN and miR-141 as well as
the possible regulatory mechanism of NPC tumor inhibition when
knockdown of c-MYC or re-expression of SPLUNC1 occurs
(Figure 6). miR-141 played a role as an oncogene to affect NPC cell
cycle, migration and invasion by positive regulation of Rb/E2F and
AKT pathway. Inhibition of miR-141 also affected the JNK2 pathway
as well. BRD3, UBAP1 and PTEN are direct targets of miR-141,
which are involved in tumorigenesis. The possible relationship between miR-141- and tumor-related genes c-MYC, SPLUNC1, BRD3,
UBAP1 and PTEN may constitute a gene–miRNA network to contribute to NPC development. Our finding might shed light on a question:
how did miRNA and genes work together in tumorigenesis? The
research of miRNA may be a good method to solve the puzzle. However, to completely answer the puzzle, it is necessary to verify other
Fig. 6. Hypothetical model of miR-141-related genes network in NPC
carcinogenesis.
565
L.Zhang et al.
target genes and detect the expression of miRNAs in great amount of
tumor tissues in the future study.
Supplementary material
Supplementary Tables 1 and 2 can be found at http://carcin
.oxfordjournals.org/
Funding
China National Key Scientific Technology Programs (2006CB910502);
111 Project (111-2-12); National 863 High Technology Program
(2007AA02Z170); National Nature Scientific Foundation (30772481).
Conflict of Interest Statement: None declared.
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Received August 19, 2009; revised December 10, 2009;
accepted December 29, 2009