miR-34a Inhibits Migration and Invasion of Tongue Squamous Cell Carcinoma via Targeting MMP9 and MMP14
et al. (2014) miR-34a Inhibits Migration and Invasion of Tongue Squamous Cell Carcinoma via Targeting
MMP9 and MMP14. PLoS ONE 9(9): e108435. doi:10.1371/journal.pone.0108435
miR-34a Inhibits Migration and Invasion of Tongue Squamous Cell Carcinoma via Targeting MMP9 and MMP14
Ling-fei Jia 0
Su-bi Wei 0
Keith Mitchelson 0
Yan Gao 0
Yun-fei Zheng 0
Zhen Meng 0
Ye-hua Gan 0
Guang-yan Yu 0
Alfons Navarro, University of Barcelona, Spain
0 1 Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology , Beijing, China, 2 Medical Systems Biology Research Center, Tsinghua University , Beijing , China , 3 Department of Oral Pathology, Peking University School and Hospital of Stomatology , Beijing, China, 4 Central Laboratory , Peking University School and Hospital of Stomatology , Beijing, China, 5 CapitalBio Corporation, Changping District, Beijing , China
Background: miR-34a is an important tumor suppressor gene in various cancer types. But little is known about the dysregulation of miR-34a in tongue squamous cell carcinoma (TSCC). In this study, we investigate the expression and potential role of miR-34a in TSCC. Methods: We evaluated miR-34a expression and its relationship with clinicopathological characters in 75 pairs of TSCC samples, and confirmed the role of miR-34a for predicting lymph node metastases from a further 15 pairs of paraffinembedded TSCC specimens with stringent clinicopathological recruitment criteria using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The effects of miR-34a on cell proliferation, migration and invasion were examined in TSCC cell lines using Cell Counting Kit-8 assay, wound healing assay and transwell assay, respectively. The effects of miR-34a on the expression of matrix metalloproteinase (MMP) 9 and 14 were detected by luciferase reporter assays and Western blot analysis. The expression of miR-34a, MMP9 and MMP14 were also confirmed in TSCC samples by in situ hybridization and immunohistochemistry. Results: miR-34a expression in tumor tissues from TSCC patients with positive lymph node metastases was significantly lower than that with negative lymph node metastases. Overexpression of miR-34a significantly suppressed migration and invasion in TSCC cells and simultaneously inhibited the expression of MMP9 and MMP14 through targeting the coding region and the 39untranslated region, respectively. Moreover, miR-34a expression in TSCC was inversely correlated with protein expression of MMP9 and MMP14 in the TSCC samples. Conclusions: miR-34a plays an important role in lymph node metastases of TSCC through targeting MMP9 and MMP14 and may have potential applications in prognosis prediction and gene therapy for lymph node metastases of TSCC patients.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This study was supported by National Natural Science Foundation of China (81402235, 81472527), and Foundation of Peking University School and
Hospital of Stomatology (PKUSS20140104). Co-author Keith Mitchelson is employed by CapitalBio Corporation, Beijing, China. CapitalBio Corporation provided
support in the form of salary for author KM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or
preparation of the manuscript. The specific role of this author is articulated in the author contributions section.
Competing Interests: The author Keith Mitchelson is employed by CapitalBio Corporation, Beijing, China. There are no patents, products in development or
marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials.
Tongue squamous cell carcinoma (TSCC) is the most common
type of oral cancer and is characterized by its high rate of
proliferation and lymph nodal metastases [1,2]. The presence of
cervical lymph node metastases is one of the most important
prognostic factors for patients with TSCC [3,4]. In clinical
practice, if cervical lymph node metastases of TSCC are apparent
at presentation of patients, neck dissection is necessary. However,
the treatment of early-stage TSCC patients with clinically negative
cervical lymph node is still controversial . Although
clinicopathological characteristics often guide the clinicians treatment
choices, biomarkers of cervical lymph node metastases in TSCC
would greatly assist the decision-making for appropriate clinical
treatment . Therefore, understanding the molecular pathways
of TSCC lymph nodal metastases in TSCC would be helpful in
improving diagnosis, and potentially therapy of this disease.
MicroRNAs (miRNAs) are endogenously expressed small
noncoding RNAs of 1924 nucleotides (nt) that regulate gene
expression by either inhibiting translation or by inducing
degradation of their target messenger RNAs (mRNAs) [7,8].
Several miRNAs, such as miR-184/138/21/195, have been
shown to have the critical roles in the development and
progression of TSCC [9,10,11,12]. miR-34a is a well recognized
tumor suppressor gene in various cancer types, in which it can
inhibit cell proliferation, induce cell apoptosis and senescence by
targeting to CDK4/6, Cyclin E2, Cyclin D1, E2F3, Bcl-2,
MYCN, Notch1/2 and SIRT1 [13,14,15,16], and can inhibit cell
migration and invasion by targeting c-Met, Notch1, Jagged1 and
Fra-1 [14,17,18,19]. Although a previous study demonstrated that
miR-34a has anti-angiogenic functions in head and neck
squamous cell carcinoma (HNSCC) , the relationship between
the expression of miR-34a and lymph node metastases of TSCC
patients remains to be investigated and whether there are other
targets of miR-34a that regulate cancer cell migration and
invasion needs to be elucidated. Matrix metalloproteinases
(MMPs) are secreted during the growth, invasion, metastases,
and angiogenesis of tumors, and can affect the surrounding
microenvironment, causing dynamic changes of bio-behavior of
the tumor . However, the precise molecular mechanism
relationships between miR-34a and MMPs in the cellular
malignant and invasive phenotypes are not fully understood.
Here, we reported that miR-34a expression in tumor tissues
from TSCC patients with positive lymph node metastases was
significantly lower than that with negative lymph node metastases.
Mechanistic analysis showed that miR-34a inhibited migration
and invasion of TSCC cell lines via targeting the coding region of
MMP9 and the 39untranslated region (UTR) of MMP14.
Materials and Methods
These experiments were approved by the Institutional Ethics
Committee of Peking University School of Stomatology (Approval
number PKUSSIRB-2012010) and all samples were obtained
from patients who signed informed consent forms approving the
use of their tissues for research purposes after surgery.
Human tissue specimens
All tissue specimens were collected from the Department of
Oral and Maxillofacial Surgery, Peking University School of
Stomatology. For freshly frozen tissues, paired primary TSCC
samples from anterior portions of the tongue and adjacent
nonmalignant tissues that were at least 1.5 cm distal to the tumor
margins were obtained from 75 TSCC patients who underwent
operations between May 2008 and August 2011. These freshly
frozen specimens were also used in our previous study . The
median duration of follow-up was 25 months (range, 948
months). Tumor tissues and the matched nonmalignant tissues
were snap-frozen in liquid nitrogen and then stored at 280uC
until use. For clinical stage, information obtained from clinical
examination and radiologic imaging was used to stratify the
patients into I-IV clinical stage (cTNM). For lymph node
metastases, the pathologic stage (pTNM) information derived
from histopathologic examination of the regional lymph nodes was
used to judge the patients lymph node metastases status. The
clinicopathological characteristics of patients were summarized in
The formalin-fixed paraffin-embedded (FFPE) tissues were
obtained from 30 patients who were selected from amongst about
1,000 TSCC patients who had undergone operations at the
hospital between May 2000 and October 2007. The inclusion
criteria were: the clinical data showed that the carcinoma was
located in the body (anterior portions) of the tongue, clinical
negative cervical lymph node (cN0), no distant metastasis (M0),
tumor size limited in T2 and T3. Histological examination showed
moderate differentiation. The patients received removal of the
primary carcinoma and neck dissection without preoperative
radiotherapy or chemotherapy, and were followed-up for at least 5
years post-operation. These 30 patients were divided into 2 groups
according to the histological examination and the results of
followup. One group of 15 patients called the fortunate group:
histological examination of the operative specimen did not show
cervical lymph node metastasis, all of the patients were alive for
more than 5 years after operation. The second group of 15
patients was called the unfortunate group, histological
examination showed cervical lymph node metastasis, all of the patients
died within 4 years after operation. Age and gender were also
matched individually between the 2 groups (Table S1).
For both freshly frozen and FFPE samples, tumor size and
clinical stage were classified according to the TNM staging system
 and each of the surgical samples we examined was confirmed
by the pathologists using H & E-stained sections.
RNA isolation and quantitative reverse transcription PCR
For freshly frozen tissues, total RNA from tumor and normal
tissue samples was isolated by using TRIzol reagent (Invitrogen,
Carlsbad, CA) according to the manufacturers instructions. For
each of the FFPE samples, total RNA was isolated from five
sections (10 mm thickness/section) using miRNeasy for FFPE kit
(Qiagen, Valencia, CA) according to the manufacturers
instructions. The expression of miR-34a was determined by quantitative
stem-loop reverse transcription qRT-PCR . Primers for
qRTPCR of miR-34a, MMP9, MMP14,U6 (internal control for
miRNAs) and b-actin (internal control for mRNAs) are listed in
Table S1. Quantitative PCR was conducted at 95uC for 10 min
followed by 40 cycles of 95uC for 15 sec and 60uC for 60 sec in an
ABI 7500 real-time PCR system. The relative expression levels of
miR-34a, MMP9 and MMP14 were calculated using the 22ggCt
Construction of expression vectors and RNA
The empty vector, pcDNA3.0, and a miR-34a expression
vector, pcDNA3.0-miR-34a, were gifts from Professor Moshe
Oren (Weizmann Institute of Science, Israel) . Similar to a
previous study, the miR-34a inhibitor (anti-miR-34a), with a
sequence complementary to miR-34a, and the anti-miR-NC used
as a negative control for anti-miR-34a were synthesized by
Genepharma (Shanghai, China) . Human MMP9 and
MMP14 full-length cDNA cloned into pcDNA3.0 vector
(Invitrogen, Carlsbad, CA) was purchased from Integrated Biotech
Solutions Company (Ibsbio, Co., Ltd., Shanghai, China) and
named as pcDNA3.0-MMP9 and pcDNA3.0-MMP14. The small
interfering RNAs (siRNAs) targeting human MMP9 and MMP14
transcripts named as siMMP9-1, siMMP9-2, siMMP14-1, and
siMMP14-2, respectively, and siRNA control were all purchased
from Integrated Biotech Solutions Company (Ibsbio, Shanghai,
China). Sequences of miR-34a inhibitor and the siRNAs were
listed in Table S2.
Cell cultures and transfection
Primary normal human oral keratinocyte (HOK) cells were
purchased from ScienCell Research Laboratories (San Diego, CA,
USA) and were cultured in a keratinocyte growth medium
(ScienCell Research Laboratories Inc.) following the manufacturers
Abbreviations: T, tumor; N, nonmalignant tissue; T1T4: T stage of TNM classification system.
instructions. Human tongue cancer cell lines SCC-15 and CAL27
were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA), cultured as described previously . Cells
were seeded onto 6-well plates the day before transfection to ensure
80% confluence at the time of transfection. Transfection with 4 mg
of expression vectors or 50 nM siRNAs was performed using
Lipofectamine 2000 (Invitrogen) in accordance with the
manufacturers instrction. One well was used to assess the equal efficiency of
transfection with an empty pcDNA3.0 vector containing the gene
for green fluorescent protein (GFP) or 59carboxyfluorescein
(FAM)labeled oligonucleotides (Ibsbio, Co., Ltd.), from which we
consistently observed .80% transfection efficiency.
Establishment of miR-34a stable overexpressing
The empty lentivirus vector contains the gene for GFP, pLL3.7,
and a miR-34a expression lentivirus vector, pLL3.7-miR-34a were
purchased from Integrated Biotech Solutions Company (Ibsbio,
Co., Ltd.). Vesicular stomatitis virus G envelope protein
pseudotyped viruses were prepared by packaging the retroviral
or lentiviral vectors in 293T cells by transient transfection using
the calcium phosphate method, as previously described . After
infection of CAL27 cells with either pLL3.7 or pLL3.7-miR-34a,
the infected cells were screened and sorted by a FACS based on
the expression of GFP which indicated the presence of the
plasmids. The sorted pLL3.7 or pLL3.7-miR-34a infected CAL27
cells were then used as the stable NC or miR-34a overexpression
model as previously described  (Figure S1). We referred to the
infected cells as either NC or miR-34a groups respectively, in the
Cell proliferation assay
The effects of miRNA-34a overexpression and deregulation of
MMP9 or MMP14 on cell proliferation were assessed using the
Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan).
Briefly, SCC-15 and CAL27 cells were seeded onto 96-well plates
(26103 cells/well) and transfected with pcDNA3.0 (NC) and
pcDNA3.0-miR-34a (miR-34a) as indicated. CCK-8 (10 ml) was
added to each well at various time points and incubated at 37uC
for 3 h. The absorbance at 450 nm was measured using a
microplate spectrophotometer (Bio-Tek Instruments Inc,
Wound healing assay
SCC-15 and CAL27 cells were cultured in a 6-well culture plate
and transfected with miR-34a, siMMP9 and siMMP14 using
Lipofectamine 2000. Subsequently, wounds were created in the
confluent cells using a 200 ml pipette tip. The debris was removed
by washing with serum-free medium. After 24 h of incubation, the
cells that migrated into the wounded area or protruded from the
border of the wound were photographed under an inverted
microscope. Wound healing was quantified by measurement of the
average linear speed of movement of the wound edges. Each
experiment was independently performed at least three times.
The 8-mm pore size membrane, plain (for migration) or
matrigel-coated (for invasion), transwell inserts (Costar, High
Wycombe, UK) were placed in the wells of 24-well culture plates.
600 ul DMEM containing 10% fetal bovine serum was added to
the lower chamber. SCC-15 and CAL27 cells were resuspended in
100 ml serum-free DMEM (16105 cells) and added to the upper
chamber. After 24 h of incubation at 37uC with 5% CO2, cells on
the topside of the filter were manually removed with a cotton
swab. Cells adherent to the undersurface of the filter were fixed in
cold methanol for 10 min and then stained with 0.01% crystal
violet in 20% ethanol. After 10 min of incubation, the filters were
washed thoroughly in water and images were taken and counted (5
random 2006 fields per well).
miRNA target validation
Predicted miR-34a target genes and their target binding sites
regions were investigated using the RNA22 software, which does
not need validated targets for training, and neither requires nor
relies on cross-species conservation, and thus is an ideal tool for
determining miRNA targets sites beyond the 39UTR . The
potential binding sites of miR-34a from MMP1 to MMP14 were
pedictied by RNA22 software. The results showed that only
MMP9 (GenBank accession number, NM_004994.2) and MMP14
(GenBank accession number, NM_004995.3) mRNA contain
putative miR-34a target sites. The miR-34a target sequences in
the coding region of MMP9 were amplified by PCR and then
cloned into a modified version of pcDNA3.1(+) that contained a
firefly luciferase reporter gene (gift from Brigid L.M. Hogan, Duke
University, Durham, NC, USA) , at a position downstream of
a luciferase open reading frame similar to the method previously
described . The fragment of 39UTR of human MMP14, which
contained predicted target site of miR-34a, was amplified by PCR
and cloned into the downstream of the modified pcDNA3.1(+)
luciferase reporter, between the EcoRI and XhoI cloning sites.
These vectors were named wild type coding region or 39UTR.
The site-directed mutagenesis of the miR-34a binding site in
MMP9 coding region and MMP14 39UTR were performed using
Site-Directed Mutagenesis Kit (SBS Genetech, Beijing, China) and
named as mutant coding region or mutant 39UTR. All constructs
were confirmed by DNA sequencing. CAL27 cells grown in a
48well plate were co-transfected with 400 ng of either pcDNA3.0 or
pcDNA3.0-miR-34a, 40 ng of the firefly luciferase reporter
plasmid including the wild type or mutant 39UTR of MMP9 or
MMP14, and 4 ng of pRL-TK, a plasmid expressing Renilla
luciferase (Promega, Madison, WI). Luciferase activity was
measured 24 h after transfection using the Dual-Luciferase
Reporter Assay System (Promega).
Western blot analysis
Western blot was performed as described previously . The
primary antibodies against MMP9 (Cell Signaling Technology,
Beverly, MA), MMP14 (Abgent, San Diego, CA), Cyclin D1
(Santa Cruz Biotechnology, Santa Cruz, CA),CDK6 (Cell
Signaling Technology), Bcl-2 and b-actin (Santa Cruz) were
diluted 1:1,000. To quantify the intensity of the bands obtained
through Western blot assay, ImageJ software-based analysis
(http://rsb.info.nih.gov/ij/) was applied. The background was
subtracted, and the signals of the detected bands were normalized
to that of loading control b-actin bands.
Immunohistochemistry and in situ hybridization
Immunohistochemical staining was performed with a two-step
detection kit (Zhongshan Goldenbridge, Beijing, China) as
previously described . The primary antibodies were MMP9
(Cell Signaling Technology, 1:150 dilution) and MMP14 (Abgent,
1:100 dilution). The intensity of MMP9 and MMP14
immunoreactions was scored as follows: score 0, negative; score 1, weak;
score 2 moderate; score 3, strong. miRNAs in situ hybridization
assay were performed essentially as previously described .
Dual-DIG-labelled LNA probes miR-34a detection probe or
Scramble-miR (negative control) were obtained from Exiqon
(Exiqon, Vedbaek, Denmark) and the hybridizations were
performed at 42uC. The probe sequences are listed in Table S1.
Cell cycle and apoptosis analysis
At 48 h post-transfection, TSCC cells were harvested by
trypsinization and washed with phosphate-buffered saline (PBS).
Then, the cell cycle and apoptosis analysis were performed as
described previously .
All statistical analyses were performed using SPSS for Windows
version 16.0. Students t test and one-way ANOVA were used to
analyze the relationship between miR-34a expression and
clinicopathological characteristics. Correlation between miR-34a
expression and MMP9 or MMP14 protein levels was analyzed
using Spearmans rank correlation coefficient analysis with r and P
values as indicated. Experiments with cell cultures were done at
least in triplicate. Data were expressed as mean 6 standard
deviation (SD). A two-tailed value of P,0.05 was considered to be
miR-34a expression was reduced in TSCC and correlated
with clinicopathological features
Expression of miR-34a was reduced in 63 of 75 (84%) tumor
samples compared with their nonmalignant counterparts
(Figure 1A). The average expression level of miR-34a was decreased
significantly in tumor tissues compared with the nonmalignant
tissues (Figure 1B). Moreover, miR-34a was also reduced
significantly in the TSCC cell lines SCC-15 and CAL27, compared
with HOK cells (Figure 1C). By normalizing miR-34a expression
levels in the tumor tissues with those in adjacent nonmalignant
tissues (Tumor/Nonmalignant, T/N), we observed statistically
significant relationships between miR-34a expression (T/N) and
several clinicopathological features of TSCC (Table 1), including
tumor size (P = 0.030), node metastases (P,0.001) and patient
mortality (P = 0.030).
Decreased miR-34a expression was significantly
correlated with lymph node metastases
We further analyzed the expression levels of miR-34a from
FFPE tissues including a group of 15 TSCC patients with negative
lymph node metastases (fortunate group) compared with
another group of 15 TSCC patients with positive lymph node
metastases (unfortunate group) matched for age, gender,
location of the primary carcinoma, pathologic differentiation,
TNM stage, and treatment modality (Table S2). The average
expression level of miR-34a was statistically significantly decreased
in the group with positive node metastases compared with that of
the group with negative node metastases (Figure 1D, P = 0.005).
Decreased miR-34a expression was significantly
correlated with the survival of the patients
As shown in Table S2, 30 patients were divided into 2 groups
according to their prognosis. One group had favorable prognosis
with survival of at least 5 years (fortunate group) and another
group had worse prognosis with death within 4 years
(unfortunate group). The expression level of miR-34a in the unfortunate
group was significantly lower than that in the fortunate group
(Figure 1D, P = 0.005).
CAL27 cell lines and primary normal human oral keratinocyte (HOK) cells. Data were presented as 22gCt (miR-34a-U6) values (**P,0.01). (D), Means of
miR-34a relative levels from formalin-fixed paraffin-embedded tissues including a group of 15 TSCC patients with positive lymph node metastases
compared with another group of 15 TSCC patients with negative lymph node metastases matched for age, gender, pathologic differentiation, and
TNM stage. Data were presented as 22gCt (miR-34a-U6) values (**P,0.01). (E), Cell viability was measured using CCK-8 assays, the data were presented
as means 6SD as indicated (*P,0.05, **P,0.01). (F) and (G) Representative photomicrographs of wound healing and transwell assays results for
SCC15 and CAL27 cells transfected with pcDNA3.0-miR-34a (miR-34a) or pcDNA3.0 (NC) (6200 magnification, **P,0.01).
miR-34a inhibited migration and invasion of TSCC cells
To investigate the effects of miR-34a on cell migration and
invasion, miR-34a was transfected in SCC-15 and CAL27. After
transfection, miR-34a expression was increased in both cell lines
(Figure S2). Since ectopic expression of miR-34a in HNSCC cell
lines is known to significantly inhibit tumor cell proliferation ,
and therefore may also affect migration and invasion assays. We
investigated whether overexpression of miR-34a could inhibit cell
proliferation of the two TSCC cell lines. Overexpression of
miR34a inhibited the viability of SCC-15 and CAL27 cells (Figure 1E),
induced substantial accumulation of the cell population at the G1
stage of the cell cycle and promoted apoptosis in both cell lines at
48 h after transfection (Figure S3A and B). In addition, the
endogenous expression of Cyclin D1, CDK6 and Bcl-2 proteins
were significantly decreased in TSCC cells in which miR-34a was
overexpressed (Figure S4). However, there was no significant
difference in proliferation between miR-34a-transfected cells and
the control at 24 h post-transfection (Figure 1E). We then
performed cell migration and invasion assays within 24 h after
transfection of miR-34a. The wound healing assay showed that
overexpression of miR-34a significantly inhibited migration of
TSCC cells compared with the negative control (Figure 1F).
Similarly, both transwell migration and transwell invasion assays
also demonstrated that migration and invasion of TSCC cell lines
were inhibited by miR-34a overexpression (Figure 1G). Moreover,
we used SCC-15 cell line, which showed a relatively higher level of
miR-34a than CAL27 cells, to further evaluate the effects of
miR34a inhibition. The results showed that silencing of miR-34a by
transfection of miR-34a inhibitor into SCC-15 cells increased their
migration and invasion (Figure S5).
miR-34a inhibited expression of MMP9 and MMP14
through targeting the coding region and 39UTR,
According to predictions by RNA22 software, MMP9 mRNA
contains a putative miR-34a target site in the coding region (1876
1898 nt) and MMP14 mRNA also a putative miR-34a target sites
in the 39UTR region (22702292 nt) (Figure 2A). We constructed
luciferase reporter plasmids to contain the putative sequences for
MMP9 or MMP14 or their corresponding mutant sequences as
controls. Overexpression of miR-34a significantly suppressed
luciferase activity of the reporter containing the miR-34a targeted
wildtype sequences of MMP9 or MMP14, but not their
corresponding mutant sequences (Figure 2B). Moreover,
overexpression of miR-34a inhibited endogenous MMP9 mRNA
expression (P,0.01), but not MMP14 mRNA expression (P.
0.05) (Figure 2C). The protein levels of MMP9 and MMP14 were
both reduced by miR-34a in SCC-15 and CAL27 cell lines
(Figure 2D). Furthermore, after transfection of miR-34a inhibitor
into SCC-15 cells, the protein levels of MMP9 and MMP14 were
significantly increased (Figure S6). These results suggested that
MMP9 and MMP14 could be direct targets of miR-34a.
Knockdown of endogenous MMP9 or MMP14 inhibited
migration and invasion of the TSCC cell lines
To confirm whether downregulation of MMP9 and MMP14 by
miR-34a could result in inhibition of migration and invasion of
TSCC cells, we knocked down the expression of endogenous
MMP9 or MMP14 by their siRNAs to mimic the effects of
miR34a overexpression. When the protein levels of both MMP9 and
MMP14 were significantly reduced by siRNAs in CAL27 cell
(Figure S7), migration and invasion of the cells were
correspondingly significantly inhibited (Figure 3A and 3B), suggesting that the
inhibitory effects of miR-34a on cells migration and invasion
could, at least partially, act through its inhibition of MMP9 and
Overexpression of MMP9 or MMP14 partially reversed
inhibitory effects of miR-34a on TSC migration and
Moreover, we used CAL27 cells stably transfected with
miR34a to test whether overexpression of MMP9 or MMP14 could
reverse the inhibitory effects of miR-34a on migration and
invasion of TSCC cells. The expression of miR-34a was
significantly increased in the miR-34a stably-transfected cells
(miR-34a) compared to cells stably-transfected with control vector
(Figure S8). As shown in Figure 3C and 3D, overexpression of
MMP9 or MMP14 partially rescued migration and invasion
capacities of CAL27 cells stably transfected with miR-34a.
Expression of MMP9 and MMP14 proteins inversely
correlated with miR-34a expression in TSCC
Since MMP9 and MMP14 transcripts were identified as direct
targets of miR-34a, we examined the relationship between their
protein expression and miR-34a expression in the paraffin sections
of 75 TSCC samples and their matched nonmalignant samples
using immunohistochemistry. Immunostaining of MMP9 and
MMP14 in the tumor tissues were both inversely correlated with
miR-34a expression measured by qRT-PCR (Figure 4A).
Moreover, we also examined miR-34a expression in TSCC and
matched nonmalignant tissues in consecutive sections using in
situ hybridization. Both the immunohistochemistry and the in situ
hybridization analysis showed that miR-34a expression was
inversely correlated with immunostaining intensity of MMP9
and MMP14 in all TSCC specimens examined. Moreover, the
immunostaining of MMP9 and MMP14 in TSCC adjacent
nonmalignant tissues was generally of reduced intensity, coincident
with the relatively high miR-34a expression (Figure 4B and 4C).
The present study demonstrated that the reduced expression of
miR-34a in freshly frozen TSCC specimens was significantly
correlated with tumor size, lymph node metastases and patients
mortality. Among the three clinical factors, lymph node metastases
of TSCC patients had the most significant statistical correlation
with miR-34a expression. Then, we designed a case-control study
in 30 TSCC FFPE samples to exclude the influence of some
Figure 2. miR-34a targets MMP9 and MMP14. (A), The sequence of miR-34a (middle) matches the coding sequence (CD) of MMP9 and
39untranslated region (UTR) of MMP14 (top). Bottom, mutations of the CD of MMP9 and 39UTR of MMP14. (B), miR-34a inhibited wild-type, but not
mutated MMP9 CD and MMP14 39UTR luciferase reporter activity. CAL27 cells were co-transfected with firefly luciferase reporter plasmids containing
wild type or mutant MMP9 CD and MMP14 39UTR, and pRL-TK plasmid (a plasmid expressing rellina luciferase) and pcDNA3.0-miR-34a (miR-34a) or
pcDNA3.0 (NC) as indicated. After 24 h, firefly luciferase activities were measured and normalized by use of renilla luciferase activities. Data were
presented as mean 6SD (*P,0.05). (C), The relative MMP9 and MMP14 mRNA levels determined by qRT-PCR in miR-34a or NC transfected CAL27 cells
(*P,0.01). (D), Inhibition of the expression of MMP9 and MMP14 proteins. Representative Western blotting image (top) and the quantification
(bottom) of MMP9 and MMP14 proteins in miR-34a transfected SCC-15 and CAL27 cells. b-actin was used as internal control and was also detected by
Western blot (**P,0.01).
Figure 3. Inhibition of MMP9 and MMP14 was responsible for the TSCC cells migration and invasion effects of miR-34a. (A) and (B),
Inhibition of cell migration and invasion by knockdown of MMP9 and MMP14. CAL27 cells were transfected with siRNA control, siMMP9-1, siMMP9-2,
siMMP14-1, and siMMP14-2 as indicated. (C) and (D), MMP9 and MMP14 overexpression partially rescues miR-34a-reduced cell migration and
invasion. Representative photomicrographs of wound healing and transwell assays results for stable expression miR-34a CAL27 cells transfected with
pcDNA3.0-MMP9 (MMP9) or pcDNA3.0-MMP14 (MMP14) (6200 magnification, **P,0.01).
indicated. (B), The concurrence of high miR-34a expression and corresponding low expression of MMP9 or MMP14 were confirmed in TSCC and
nonmalignant specimens by ISH with miR-34a detection probe or Scramble-miR and IHC, Primary antibodies were omitted in negative control for IHC
(2006magnification). (C), The concurrence of low miR-34a expression and corresponding high expression of MMP9 or MMP14 was confirmed in TSCC
and high miR-34a expression and corresponding low expression of MMP9 or MMP14 were confirmed in the matched nonmalignant specimens by ISH
with miR-34a detection probe or Scramble-miR and IHC. Primary antibodies were omitted in negative control for IHC (2006 magnification).
clinical factors, such as age, gender, location of the tumor,
pathologic differentiation, TNM stage, and treatment modalities,
which may influence the metastasis rate of cervical lymph nodes
and the prognosis of the patients . According to the
pathological results on cervical lymph nodes and prognosis of
the these patients, the patients were divided into a fortunate
group and an unfortunate group. All the clinicopathological
data on above mentioned factors influencing cervical lymph node
metastasis and patients prognosis were strictly controlled case by
case. The results showed that the expression level of miR-34a in
the unfortunate group with positive cervical lymph node
metastasis was significantly lower than that in the fortunate
group with negative cervical lymph node metastasis. These results
provided further evidence that reduced expression of miR-34a is
closely related to metastasis rate of cervical lymph nodes and
prognosis of TSCC patients. More importantly, all the 30 FFPE
TSCC samples were selected from the TSCC patients with the
clinical node-negative (cN0) neck. In clinical practice, whether or
not neck dissection should be performed to the TSCC patients
with cN0 neck is still controversial [32,33]. Currently, it is also
difficult to accurately assess the metastatic status of the cervical
lymph node . Our results showed that miR-34a expression was
significantly correlated to lymph node metastases of cN0 TSCC
patients. Thus, the level of expression of miR-34a detected from
TSCC biopsy samples could provide useful information for
clinicians to make a choice whether or not neck dissection is
As a potential functional explanation for miR-34a effects in
lymph node metastases of TSCC, we demonstrated that miR-34a
inhibited migration and invasion of TSCC cells and the results
were consistent with a previous study . Although cell migration
and invasion occur as normal events in a number of physiological
processes, uncontrolled migration and invasion are two key
elements lead to metastases, which causes as high as 90% of
human cancer deaths [31,34]. Furthermore, we investigated the
mechanism of the inhibitory effects of miR-34a on TSCC cells
migration and invasion. MMP9 and MMP14, which have been
regarded as regulators of tumor migration and invasion, are
known to be involved in cell migration and invasion and frequently
found to be upregulated in HNSCC [5,33]. We performed a series
of experiments using one TSCC cell line (CAL27) to demonstrate
that the inhibition of MMP9 and MMP14 by miR-34a may at
least partially account for the suppressing migration and invasion
effect of miR-34a in TSCC cells.
Moreover, miR-34a targeted MMP9 at its coding region. In
animals, miRNAs usually inhibit mRNA translation and decrease
mRNA stability by binding sequences in the 39UTR .
However, MMP9 transcripts have short 39UTR (,250 nt),
miRNA recognition elements that can hardly be predicted in this
restricted space. To the best of our knowledge, there is only one
previous report showing that MMP9 can be regulated directly by
miR-218 in osteosarcoma . On the other hand, genes with
shorter 39UTRs have significantly more miRNA recognition
elements in the coding region . Our results showed that the
predicted miR-34a target region of MMP9 has the potential to
interact with 18 nt of miR-34a and miR-34a could regulate
MMP9 through targeting at its coding region. Several studies have
demonstrated that miRNAs can down-regulate their target genes
via direct targeting the coding regions in animals [30,38,39].
However, the mechanism by which miRNAs mediate this manner
of repression is not completely understood. Standart et al.
suggested that miRNAs prevent the closed loop mRNA
configuration induced by interaction of poly(A)-binding proteins
with initiation factors at the 59cap . The predicted target
region of miR-34a in MMP9 is located just around 240 nt before
the stop codon, which is near the 39UTR and the poly (A) tail.
Therefore, it might allow for miRNA-mediated repression in a
similar way as 39UTR binding miRNAs. In addition, our results
show that miR-34a directly down-regulated MMP9 that seems
different from other studies, in which miR-34a indirectly
downregulates MMP9 expression through suppression of Fra-1 in colon
cancer  and DLL1 in choriocarcinoma . It appears that
miR-34a may down-regulate MMP9 expression through both
direct regulatory mechanism and indirect trans-regulatory
mechanism. The current study also demonstrated that miR-34a
downregulated MMP9 by targeting the its 39UTR. Besides, MMP14 has
also been shown to be a direct target of miR-10b in glioma ,
miR-9 in neuroblastoma  and miR-133a in esophageal cancer
. These studies revealed that multiple miRNAs probably
contribute to the loss of MMP14 expression in specific types of
Our in vitro experiments with human tongue cancer cell line
SCC-15 and CAL27 also showed that miR-34a induced the
G1phase arrest and promoted cell apoptosis. Cyclin D1 and CDK6
are two of the key proteins involved in cell cycle control and are
essential for G1 to S transition . Bcl-2 is also one of the key
regulators of cell apoptosis . The present study confirmed that
the protein expression of Cyclin D1, CDK6 and Bcl-2 were
reduced by overexpression of miR-34a, which is consistent with
previous studies in non-small cell lung cancer A549 cells  and
neuroblastoma NLF cells . These data suggested the potential
tumor suppressor roles of miR-34a in TSCC.
In conclusion, our results demonstrated that miR-34a
expression was significantly correlated with lymph node metastasis and
prognosis of TSCC patients and could inhibit migration and
invasion of TSCC cell lines via targeting MMP9 and MMP14.
Overexpression of miR-34a in TSCC cells can also inhibit cell
cycle progression and promoted cell apoptosis. The present study
suggests that miR-34a have potential applications in prognosis
prediction and gene therapy for lymph node metastases of TSCC
Figure S1 The transduction efficiency of the sorted
lentivirus vectors pLL3.7 or pLL3.7-miR-34a infected
CAL27 cells were.90% (1006 magnification).
Figure S2 The expression of miR-34a was significantly
increased in SCC-15 and CAL27 cells after transfection
with pcDNA3.0 -miR-34a (miR-34a) and compared to
transfection with pcDNA3.0 as negative control (NC).
Data was presented as mean 6SD (**P,0.01).
Figure S3 Overexpression of miR-34a inhibited cell
cycle progression and promoted cell apoptosis. (A),
Inhibition of cell cycle progression by overexpression of
miR34a. SCC-15 and CAL27 cells were transfected with pcDNA3.0, a
negative control (NC) or with pcDNA3.0-miR-34a (miR-34a), as
indicated. Cells were stained with propidium iodide (PI) at 48 h
post-transfection and analyzed with FACS (**P,0.01). (B),
Promotion of apoptosis by overexpression of miR-34a. SCC-15
or CAL27 cells were transfected for 48 h as in (A) and apoptotic
cells were monitored with FACS after Annexin V and PI staining
Figure S4 Overexpression of miR-34a decreased the
endogenous protein expression of Cyclin D1, CDK6 and
Bcl-2 in TSCC cell lines. SCC-15 and CAL27 cells were
transfected with pcDNA3.0 as a negative control (NC) or with
pcDNA3.0-miR-34a (miR-34a) as indicated. After 48 h, Cyclin
D1, CDK6 and Bcl-2 and internal control b-actin were detected
by Western blot.
Figure S5 Inhibition of miR-34a in SCC-15 significantly
increased cell migration and invasion. Representative
photomicrographs of wound healing and transwell assays results
for SCC-15 cells transfected with miR-34a inhibitor
(anti-miR34a) or the negative control (anti-miR-NC) (6200 magnification,
Figure S6 Inhibition of miR-34a in SCC-15 significantly
increased protein levels of MMP9 and MMP14. SCC-15
Figure S7 Inhibition of the expression of MMP9 and
MMP14 by siRNAs targeting MMP9 and MMP14
transcripts. CAL27 cells were transfected with siRNA control,
siMMP9-1, siMMP9-2, siMMP14-1, and siMMP14-2 as indicated.
After 24 h, MMP9, MMP14 and internal control b-actin were
detected by Western blot.
Figure S8 The expression of miR-34a was significantly
increased in the miR-34a stably-transfected cells
(miR34a) compared to that stably-transfected with control
vectors (NC). Data was presented as mean 6SD (**P,0.01).
Sequences of RNA and DNA Oligonucleotides.
Conceived and designed the experiments: LFJ GYY YHG. Performed the
experiments: LFJ SBW YG YFZ KM ZM. Analyzed the data: LFJ SBW
YG. Contributed reagents/materials/analysis tools: LFJ SBW YFZ. Wrote
the paper: LFJ GYY YHG KM.
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