miR-324-3p suppresses migration and invasion by targeting WNT2B in nasopharyngeal carcinoma
Liu et al. Cancer Cell Int
miR-324-3p suppresses migration and invasion by targeting WNT2B in nasopharyngeal carcinoma
Chao Liu 1 2
Guo Li 1 2
Nianting Yang 1 2
Zhongwu Su 1 2
Shuiting Zhang 1 2
Tengbo Deng 1 2
Shuling Ren 1 2
Shanhong Lu 1 2
Yongquan Tian 1 2
Yong Liu 0 1 2
Yuanzheng Qiu 0 1 2
0 Yong Liu and Yuanzheng Qiu are the co-
1 Department of Otolaryngology Head and Neck Surgery, Xiangya Hospital, Central South University , 87 Xiangya Road, Changsha 410008, Hunan , China
2 Otolaryngology Major Disease Research Key Laboratory of Hunan Province , Changsha 410008, Hunan , China
Background: Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma of the head and neck with strong ability of invasion and metastasis. Our previous study indicated that miR-324-3p, as a tumor-suppressive factor, could regulate radioresistance of NPC cells by targeting WNT2B. The purpose of this study is to investigate the role of miR324-3p on migration and invasion in NPC cells. Methods: Quantitative real time PCR was applied to measure the expression level of miR-324-3p and WNT2B mRNA in both cells and tissues, and the expression level of WNT2B protein was determined by western blotting. The capacity of migration and invasion were tested by using wound healing and transwell invasion assay. Results: Ectopic expression of miR-324-3p or silencing its target gene WNT2B could dramatically suppress migration and invasion capacity of NPC cells. Meanwhile, the alterations of miR-324-3p in NPC cells could influence the expression level of the biomarkers of epithelial-mesenchymal transition (EMT), including E-cadherin and Vimentin. Moreover, the expression of miR-324-3p was obviously downregulated and WNT2B was significantly upregulated in NPC tissues. The expression levels of miR-324-3p and WNT2B were closely correlated with T stage, clinic stage and cervical lymph node metastasis of NPC (P < 0.05). Conclusion: miR-324-3p could suppress the migration and invasion of NPC by targeting WNT2B and the miR-324-3p/ WNT2B pathway possibly provide new potential therapeutic clues for NPC.
Nasopharyngeal carcinoma; miR-324-3p; WNT2B; Invasion
Nasopharyngeal carcinoma (NPC) is an Epstein-Barr
virus associated cancer that mostly occurs in Southern
China and South-Eastern Asia . Despite great
improvements in chemoradiotherapy over the past few decades,
the overall 5-year survival rate for NPC still remains poor
. Strong ability to migrate and invade is a leading cause
for the dismay prognosis of advanced NPC patients .
For this reason, exploring the molecular mechanisms
underlying NPC migration and invasion is essential for
the development of novel therapeutic strategies.
microRNAs (miRNAs) are a kind of small
non-protein-coding RNA, which consist of 19–25 nucleotides
. They can inhibit target gene expression at the
posttranscriptional level by pairing with the 3′ untranslated
regions (3′ UTRs), 5′ UTRs or coding region of mRNAs
[5, 6]. Accumulated evidences prove that dysregulated
miRNAs can function as oncogenes or tumour
suppressors in the initiation and progression of various human
cancers . In NPC, miRNAs are also reported to play
important regulatory roles in many critical biological
processes including cell proliferation, apoptosis,
radiosensitivity and chemosensitivity [8–10]. As to migration
and invasion of NPC, a series of miRNAs like miR-145 or
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miR-744 can inhibit or enhance NPC migration and
invasion by targeting SMAD3 or ARHGAP5, respectively [11,
12]. These above findings indicated that miRNAs provide
a new perspective for the investigation of migration and
invasion in NPC.
In our preliminary study, we have found that
miR324-3p could regulate the radioresistance of NPC cells
and further confirmed WNT2B was the target gene of
miR-324-3p . Here, we focused on the roles of
miR324-3p involved in NPC migration and invasion. Our
results showed that miR-324-3p inhibited migration and
invasion of NPC cells and affected
epithelial-mesenchymal transition (EMT) biomolecules, and the target gene
WNT2B could enhance the migration and invasion
ability of NPC. Currently, in NPC tissue specimens,
miR324-3p was found to be downregulated while WNT2B
was upregulated. Moreover, the expression levels of
miR324-3p and WNT2B were associated with stages of NPC,
as well as with lymph node metastasis. These results
provide valuable clues toward understanding the molecular
mechanisms of NPC migration and invasion.
NPC cell lines 5-8F and 6-10B were purchased from
the Cell Center of Central South University, Changsha,
China. The cells were cultured in RPMI medium 1640
(Hyclone, Logan, UT, USA) with 10% foetal bovine serum
(Gibco BRL, Gaithersburg, MD, USA) and were
incubated at 37 °C in a humidified chamber with 5% CO2.
Cells in an exponential growth state were used for
Patients and tissue preparation
Primary NPC (n = 39) and normal nasopharyngeal
epithelium (NPE) (n = 21) tissues were obtained from the
the Department of Otolaryngology Head and Neck
Surgery, Xiangya Hospital, Central South University,
Changsha, China. All patients had no history of
previous malignancies. Staging was performed by the 2008
NPC staging system of China. The study protocol was
approved by the Research Ethics Committee of the
Central South University, Changsha, China. Informed
consents were obtained from all of the patients.
Oligonucleotides and transfection
miR-324-3p mimics, WNT2B siRNA and negative
control (NC) were chemically synthesized from
GenePharma Co., Shanghai, China. The NPC cells 5-8F and
6-10B were transfected with Lipofectamine 2000 reagent
(Invitrogen, Burlington, ON, Canada) according to the
manufacturer’s instructions. The transfection efficiency
was observed by fluorescence microscopy, and the
expression level of miR-324-3p and WNT2B were
evaluated using the qRT-PCR examination System (Bio-Rad,
Hercules, CA, USA).
RNA extraction and quantitative real‑time PCR (qRT‑PCR)
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used
to extract the total RNA from NPC cells and tissues. The
All-in-One™ miRNA qRT-PCR Detection Kit (GeneCo
poeia Inc., MD, USA) was applied in reverse transcription
and quantitative detection of miRNAs according to the
user manual. The detection of mRNAs was carried out
with TaqMan Reverse Transcription Reagents and SYBR
Green PCR Master Mix (Applied Biosystems, CA, USA).
PCR quantification was conducted using the 2−ΔΔCT
method and normalized to U6 for miRNA or GAPDH for
mRNA. The sequences of the primers used for the PCR
are as follows: WNT2B forward, 5′-TGG CGT GCA CTC
TCA GAT TT-3′ and reverse, 5′-GAC AAG ATC AGT
CCG GGT GG-3′; GAPDH forward, 5′-TCC AAA ATC
AAG TGG GGC GA-3′, and reverse, 5′-AGT AGA GGC
AGG GAT GAT GT-3′. The technical documentation of
qRT-PCR was listed in Additional file 1, and
representative data of standard and melt curves of the premirs were
listed in Additional file 2: Figure S1. And the validation
of the stability of the reference genes between NPC and
NPE was showed in Additional file 2: Figure S2.
Wound healing assay
Wound-healing assay was used to examine the cell
migration activity. When the cells were grown to reach
almost total confluence (nearly 36–48 h after
transfection), a 10 μl plastic pipette tip was adopted to creat an
artificial wound. Then the cells were cultured in
serumfree medium. The initial gap length (0 h) and the residual
gap length 24–72 h after scratching were observed under
the inverted microscope. Experiments were performed in
Cell invasion assay
A transwell invasion assay was performed according
to the operating instruction. Briefly, 1 × 104 cells with
serum-free medium were seeded to the top chamber
with Matrigel-coated membrane (BD Biosciences,
Bedford, MA, USA). After a 48 h incubation period,
noninvading cells on the surface of the upper chamber were
removed with a cotton swab. Cells on the lower side of
the chamber were fixed in paraformaldehyde, stained
with crystal violet, and then counted in five random
fields under microscope. Each experiment was done
Western blotting analysis
Western blotting was carried out as described
previously. Total cell or tissue proteins were extracted and
separated on 10% SDS-PAGE gels, and electroblotted
onto PVDF (polyvinylidene fluoride) membranes
(Millipore, Billerica, MA, USA). Then, the protein expression
was detected by incubation with the relevant primary
antibody followed by an appropriate secondary
antibody. The primary antibody in current study included
anti-WNT2B (1:800, Boiss Inc., Woburn, MA, USA),
anti-E-cadherin and anti-Vimentin (1:800, Cell Signaling
Technology, Danvers, MA, USA). Anti-β-Actin or
antiGAPDH (1:1000, Beyotime, Shanghai, China) were used
as the loading control. The relative expression of WNT2B
protein was calculated by the image intensity of the ratio
of WNT2B and β-Actin.
All data shown are representative results of at least
three independent experiments and were expressed as
mean ± standard deviation (SD). Statistical analyses
were performed using a two-sided unpaired Student’s t
test (for equal variances) or Mann–Whitney U test (for
unequal variances) with SPSS 18.0 software. P < 0.05 was
considered statistically significant.
Ectopic expression of miR‑324‑3p inhibits migration
and invasion of NPC cells
To investigate the role of miR-324-3p on NPC
migration and invasion, we upregulated the expression of
miR324-3p by transfecting miR-324-3p mimic into NPC 5-8F
and 6-10B cells. A transfection efficiency of 93.8 ± 2.1
and 92.7 ± 3.3% was observed under fluorescence
microscopy in 5-8F and 6-10B cells, respectively, and
the expression of miR-324-3p was successfully increased
(P < 0.01; Fig. 1a, b). The wound healing assay showed
that cell migratory abilities of miR-324-3p
overexpressing cells were greatly inhibited compared with uninfected
mock and control cells (P < 0.01, Fig. 1c, d). The transwell
invasion assay showed that the number of invading cells
was significantly decreased in miR-324-3p upregulated
cells (P < 0.01, Fig. 1e, f ). These results indicated that
ectopic expression of miR-324-3p could inhibit migration
and invasion of NPC cells.
Silencing expression of WNT2B suppresses migration
and invasion of NPC cells
Our previous study had shown that WNT2B was the
target gene of miR-324-3p, so we transfected WNT2B
siRNA plasmids into NPC cells to confirm its role on
migration and invasion of NPC. The expression of
WNT2B was obviously decreased in 5-8F and 6-10B
cells by qRT-PCR detection (Fig. 2a, b). The wound
healing assay demonstrated that cell migration ability was
inhibited when WNT2B was downregulated (P < 0.01,
Fig. 2c, d). The transwell invasion assay showed that
the number of invading cells was significantly reduced
in WNT2B siRNA transfected cells (P < 0.01, Fig. 2e,
f ). These data confirmed that the target gene WNT2B
could also affect the migration and invasion of NPC
miR‑324‑3p alters the expression of EMT biomarkers
E‑cadherin and Vimentin
EMT was a classical mechanism of tumour invasion and
metastasis, and we had confirmed that WNT2B could
affect the expression of EMT biomarkers in previous
study . Here, we found following the upregulation
of miR-324-3p in both NPC 5-8F and 6-10B cells, the
expression of epithelial markers E-cadherin was
upregulated while mesenchymal markers Vimentin was
downregulated (Fig. 3), which suggested that EMT might
participate in the process of miR-324-3p mediated
migration and invasion of NPC.
miR‑324‑3p is downregulated and its target gene WNT2B is
upregulated in NPC specimens
We have validated that both miR-324-3p and target gene
WNT2B could affect migration and invasion of NPC,
so we further quantified the expressions of miR-324-3p
and WNT2B in 39 freshly frozen NPC and 21 normal
NPE tissues. The results showed that in NPC tissues, the
expression of miR-324-3p was significantly decreased
and both WNT2B mRNA and protein were upregulated
(Fig. 4). And the expression of miR-324-3p was negative
correlated with WNT2B mRNA and protein (Additional
file 2: Figure S3).
Correlationship between miR‑324‑3p/WNT2B expression
and clinicopathological parameters
The relationship between miR-324-3p/WNT2B
expression and clinicopathological characteristics of NPC was
explored. As summarized in Table 1, miR-324-3p lower
expression was associated with tumour T classification,
clinic stage and lymph node metastasis (P < 0.05),
respectively. And WNT2B mRNA/protein overexpression was
also associated with tumour T classification, clinic stage
and lymph node metastasis (P < 0.05). However, no
significant relationship existed between these markers and
variables such as gender and age (P > 0.05). More
expression data of NPE and NPC with two differential
stratifications of T and clinic stage forms was shown in Additional
file 2: Figure S4.
Fig. 1 Ectopic expression of miR-324-3p inhibits migration and invasion of NPC cells. The transfection efficiency and expression of miR-324-3p
were determined under fluorescent microscope and qRT-PCR in 5-8F (a) and 6-10B (b) cells. The migration of different cell groups was examined by
wound healing experiments in 5-8F (c) and 6-10B (d) cells. The invasion of different cell groups was detected by transwell invasion assays in 5-8F (e)
and 6-10B (f) cells. The results represent the average of three independent experiments ± standard deviation (S.D). (**P < 0.01)
To understand the underlying molecular mechanisms
involved in NPC migration and invasion is beneficial to
develop better therapeutic strategies for NPC patients.
Recently, as important regulatory factors, miRNAs have
been shown to play crucial roles in various malignant
biobehaviours of tumours, including NPC migration and
invasion. Our previous study indicated that miR-324-3p
could target WNT2B to affect the radioresistance of NPC
in vitro . In this study, we further demonstrated that
miR-324-3p could also regulate the migration and
invasion of NPC, which contributes to illustrate the complex
molecular mechanisms of migration and invasion in
miR-324-3p was located in the region of human
chromosome 17p13.1 and firstly identified in mammalian
neurons . Up to now, with the rapid development of
microarray and sequencing technology, some researchers
have found that miR-324-3p was dysregulated in a variety
of tumours such as breast cancer , hepatocellular
carcinoma  and pancreatic cancer . However, none
of these studies further explored the roles of miR-324-3p
on tumour maglinant biobehaviours. More specifically,
previous investigations into the function of miR-324-3p
were limited, and only several reports showed
miR324-3p was a target of ACE inhibition to promote renal
fibrosis and miR-324-3p could target RelA promoter to
induce its expression in an Ago2 dependent manner in
cells of neural origin [19, 20]. Functional analyses of
miR324-3p on tumour were just seen in our preceding study
 and another similar report of NPC radioresistance,
in which SMAD7 was validated as the target of
miR324-3p . In this study, we further confirmed the role
of miR-324-3p on NPC migration and invasion, which
suggests miR-324-3p as an anti-tumour miRNA.
miRNAs usually exert their function by interacting
with their target genes via base pairing. In our previous
research, we found that WNT2B was a direct target gene
of miR-324-3p and confirmed WNT2B could affect
radioresistance of NPC cells . WNT2B was known to
stimulate the canonical WNT/β-catenin pathway and affected
various malignant tumour progression . In head and
Fig. 2 Silencing expression of WNT2B suppresses migration and invasion of NPC cells. WNT2B was successfully downregulated by WNT2B siRNA
in 5-8F (a) and 6-10B (b) cells. The migration of different cell groups was examined by wound healing experiments in 5-8F (c) and 6-10B (d) cells.
The invasion of different cell groups was examined using matrigel invasion assays in 5-8F (e) and 6-10B (f) cells. The results represent the average of
three independent experiments ± standard deviation (S.D). (**P < 0.01)
Fig. 3 miR-324-3p alters the expression of EMT biomarkers
E-cadherin and Vimentin. Following ectopic expression of miR-324-3p,
the expression of E-cadherin was upregulated and Vimentin was
downgrelated, shown by Western blotting
neck squamous cell carcinoma, WNT2B played a role in
tumourigenesis and chemotherapy resistance in vivo and
in vitro . Intratumoural WNT2B was reported to be
correlated with the expression of Survivin and c-Myc,
tumour proliferation and prognosis in malignant pleural
mesothelioma . Jiang et al. also found that the
highexpression level of WNT2B was associated with the
progression and worse outcome of pancreatic cancer .
In addition, WNT2B can increase the ability of
metastasis and chemoresistance of ovarian cancer through the
caspase-9/BCL2/BCL-xL pathway and EMT/p-AKT
pathways . All these studies highlighted the role of
WNT2B on tumour malignant processes. However, the
function of WNT2B on NPC was only seen in our
preliminary work, and here we further found that WNT2B played
roles on migration and invasion of NPC, and revealed that
both WNT2B mRNA and protein was positively
correlated with higher stages of NPC, which indicated the
possibility of WNT2B to be the novel biomarker of NPC.
EMT was defined as a process that epithelial cells
transformed into a mesenchymal phenotype, in which
the expression of epithelial marker E-cadherin and
Fig. 4 The expression of miR-324-3p and WNT2B in NPC specimens. a miR-324-3p was downregulated in NPC tissues compared with NPE tissues.
b WNT2B mRNA was upregulated in NPC tissues compared with NPE tissues. c WNT2B protein was upregulated in NPC tissues compared with NPE
tissues. miR-324-3p, WNT2B mRNA and protein were normalized with the internal control U6, GAPDH and β-Actin, respectively. (**P < 0.01)
Table 1 Clinicopathological features of the cases of NPC
Variables Cases miRNA‑324‑3p
mesenchymal marker Vimentin was considered as the
classical indicators . And EMT had been widely
investigated to be classical mechanism of tumour
invasion and metastasis [27, 28]. Moreover, recent
researchers reported that miR-544a could induce EMT through
the activation of WNT signaling pathway in gastric
cancer . In other tumours such as ovarian ,
colorectal  and tongue cancer , EMT was also associated
with the activation of the WNT signalling pathway. With
regard to NPC, we found that WNT2B was able to change
the expression of E-cadherin and Vimentin . In this
study, we validated that miR-324-3p also had the
ability of altering the expression of EMT biomarkers, which
showed that miR-324-3p might regulate the migration
and invasion of NPC through EMT.
In summary, we have demonstrated that miR-324-3p
can target WNT2B to regulate migration and invasion
in NPC, and both miR-324-3p and target gene WNT2B
were associated with T stage, clinic stage and cervical
lymph node metastasis. Therefore, miR-324-3p/WNT2B
axis may be potential therapeutic target for the treatment
of patients with NPC.
miR-324-3p is a tumor-suppressive miRNA in NPC
and inhibit NPC cell migration and invasion by
targeting WNT2B. Complete understanding of the
miR-3243p/WNT2B pathway might contribute to discover new
potential therapeutic clues for NPC.
Additional file 1. qRT-PCR technical documentation.
Additional file 2: Figure S1. Representative data of standard and melt
curves of the premirs. (A) Standard curves of GAPDH and WNT2B. (B) Melt
curves of GAPDH. (C) Melt curves of WNT2B. (D) Standard curves of U6 and
miR-324-3p. (E) Melt curves of U6. (F) Melt curves of miR-324-3p. Figure
S2. Validation of the stability of the reference genes. (A) Amplification plot
and CT values of U6 between NPC and NPE. (B) Amplification plot and CT
values of GAPDH between NPC and NPE. Figure S3. Spearman
correlation analysis between the expression of miR-324-3p and WNT2B. (A)
miR324-3p was negative correlated with WNT2B mRNA. (B) miR-324-3p was
negative correlated with WNT2B protein. Figure S4. The expression data
of NPE and NPC with two differential stratifications of T and clinic stage
forms. (A) The expression of miR-324-3p between NPE and NPC. (B) The
expression of WNT2B mRNA between NPE and NPC. (C) The expression of
WNT2B mRNA between NPE and NPC.
NPC: nasopharyngeal carcinoma; NPE: nasopharyngeal epithelium; NC:
negative control; EMT: epithelial-mesenchymal transition; UTR: untranslated
regions; qRT-PCR: quantitative real time PCR.
LC, LY and QYZ conceived and designed the experiments. LC, LY, QYZ, LG and
YNT performed the experiments. LC, SZW, ZST and DTB analyzed the data. RSL,
LSH and TYQ contributed reagents and tissues collection. LC and LY wrote the
paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This research was reviewed and approved by the Ethic Committee of Xiangya
Hospital, Central South University (Study ID number 201303151). Informed
consent was obtained from all individual participants included in the study.
1. Torre LA , Bray F , Siegel RL , Ferlay J , Lortet-Tieulent J , Jemal A. Global cancer statistics 2012 . CA Cancer J Clin . 2015 ; 65 : 87 - 108 .
2. Razak AR , Siu LL , Liu FF , Ito E , O'Sullivan B , Chan K. Nasopharyngeal carcinoma: the next challenges . Eur J Cancer . 2010 ; 46 : 1967 - 78 .
3. Bensouda Y , Kaikani W , Ahbeddou N , Rahhali R , Jabri M , Mrabti H , Boussen H , Errihani H. Treatment for metastatic nasopharyngeal carcinoma . Eur Ann Otorhinolaryngol Head Neck Dis . 2011 ; 128 : 79 - 85 .
4. Bartel DP . MicroRNAs: genomics , biogenesis, mechanism, and function. Cell . 2004 ; 116 : 281 - 97 .
5. Lytle JR , Yario TA , Steitz JA . Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR . Proc Natl Acad Sci USA . 2007 ; 104 : 9667 - 72 .
6. Gu W , Wang X , Zhai C , Zhou T , Xie X. Biological basis of miRNA action when their targets are located in human protein coding region . PLoS ONE . 2013 ; 8 : e63403 .
7. Di Leva G , Garofalo M , Croce CM . MicroRNAs in cancer . Annu Rev Pathol . 2014 ; 9 : 287 - 314 .
8. Ou H , Li Y , Kang M. Activation of miR-21 by STAT3 induces proliferation and suppresses apoptosis in nasopharyngeal carcinoma by targeting PTEN gene . PLoS ONE . 2014 ; 9 : e109929 .
9. Qu JQ , Yi HM , Ye X , Li LN , Zhu JF , Xiao T , Yuan L , Li JY , Wang YY , Feng J , He QY , Lu SS , Yi H , Xiao ZQ. MiR-23a sensitizes nasopharyngeal carcinoma to irradiation by targeting IL-8/Stat3 pathway . Oncotarget . 2015 ; 6 : 28341 - 56 .
10. Peng X , Cao P , Li J , He D , Han S , Zhou J , Tan G , Li W , Yu F , Yu J , Li Z , Cao K. MiR-1204 sensitizes nasopharyngeal carcinoma cells to paclitaxel both in vitro and in vivo. Cancer Biol Ther . 2015 ; 16 : 261 - 7 .
11. Huang H , Sun P , Lei Z , Li M , Wang Y , Zhang HT , Liu J. MiR -145 inhibits invasion and metastasis by directly targeting Smad3 in nasopharyngeal cancer . Tumour Biol . 2015 ; 36 : 4123 - 31 .
12. Fang Y , Zhu X , Wang J , Li N , Li D , Sakib N , Sha Z , Song W. MiR -744 functions as a proto-oncogene in nasopharyngeal carcinoma progression and metastasis via transcriptional control of ARHGAP5 . Oncotarget . 2015 ; 6 : 13164 - 75 .
13. Li G , Liu Y , Su Z , Ren S , Zhu G , Tian Y , Qiu Y. MicroRNA-324-3p regulates nasopharyngeal carcinoma radioresistance by directly targeting WNT2B . Eur J Cancer . 2013 ; 49 : 2596 - 607 .
14. Li G , Wang Y , Liu Y , Su Z , Liu C , Ren S , Deng T , Huang D , Tian Y , Qiu Y. MiR185-3p regulates nasopharyngeal carcinoma radioresistance by targeting WNT2B in vitro . Cancer Sci . 2014 ; 105 : 1560 - 8 .
15. Kim J , Krichevsky A , Grad Y , Hayes GD , Kosik KS , Church GM , Ruvkun G . Identification of many microRNAs that copurify with polyribosomes in mammalian neurons . Proc Natl Acad Sci USA . 2004 ; 101 : 360 - 5 .
16. Hu Z , Dong J , Wang LE , Ma H , Liu J , Zhao Y , Tang J , Chen X , Dai J , Wei Q , Zhang C , Shen H. Serum microRNA profiling and breast cancer risk: the use of miR-484/191 as endogenous controls . Carcinogenesis . 2012 ; 33 : 828 - 34 .
17. Wen Y , Han J , Chen J , Dong J , Xia Y , Liu J , Jiang Y , Dai J , Lu J , Jin G , Han J , Wei Q , Shen H , Sun B , Hu Z. Plasma miRNAs as early biomarkers for detecting hepatocellular carcinoma . Int J Cancer . 2015 ; 137 : 1679 - 90 .
18. Namkung J , Kwon W , Choi Y , Yi SG , Han S , Kang MJ , Kim SW , Park T , Jang JY . Molecular subtypes of pancreatic cancer based on miRNA expression profiles have independent prognostic value . J Gastroenterol Hepatol . 2016 ; 31 : 1160 - 7 .
19. Macconi D , Tomasoni S , Romagnani P , Trionfini P , Sangalli F , Mazzinghi B , Rizzo P , Lazzeri E , Abbate M , Remuzzi G , Benigni A. MicroRNA-324-3p promotes renal fibrosis and is a target of ACE inhibition . J Am Soc Nephrol . 2012 ; 23 : 1496 - 505 .
20. Dharap A , Pokrzywa C , Murali S , Pandi G , Vemuganti R. MicroRNA miR324-3p induces promoter-mediated expression of RelA gene . PLoS ONE . 2013 ; 8 : e79467 .
21. Xu J , Ai Q , Cao H , Liu Q. MiR-185-3p and miR-324-3p predict radiosensitivity of nasopharyngeal carcinoma and modulate cancer cell growth and apoptosis by targeting SMAD7 . Med Sci Monit . 2015 ; 21 : 2828 - 36 .
22. Katoh M. Differential regulation of WNT2 and WNT2B expression in human cancer . Int J Mol Med . 2001 ; 8 : 657 - 60 .
23. Li SJ , Yang XN , Qian HY . Antitumor effects of WNT2B silencing in GLUT1 overexpressing cisplatin resistant head and neck squamous cell carcinoma . Am J Cancer Res . 2015 ; 5 : 300 - 8 .
24. Kobayashi M , Huang CL , Sonobe M , Kikuchi R , Ishikawa M , Kitamura J , Miyahara R , Menju T , Iwakiri S , Itoi K , Yasumizu R , Date H. Intratumoral Wnt2B expression affects tumor proliferation and survival in malignant pleural mesothelioma patients . Exp Ther Med . 2012 ; 3 : 952 - 8 .
25. Jiang H , Li F , He C , Wang X , Li Q , Gao H. Expression of Gli1 and Wnt2B correlates with progression and clinical outcome of pancreatic cancer . Int J Clin Exp Pathol . 2014 ; 7 : 4531 - 8 .
26. Wang H , Fan L , Xia X , Rao Y , Ma Q , Yang J , Lu Y , Wang C , Ma D , Huang X. Silencing Wnt2B by siRNA interference inhibits metastasis and enhances chemotherapy sensitivity in ovarian cancer . Int J Gynecol Cancer . 2012 ; 22 : 755 - 61 .
27. Iwatsuki M , Mimori K , Yokobori T , Ishi H , Beppu T , Nakamori S , Baba H , Mori M. Epithelial-mesenchymal transition in cancer development and its clinical significance . Cancer Sci . 2010 ; 101 : 293 - 9 .
28. Guarino M , Rubino B , Ballabio G. The role of epithelial-mesenchymal transition in cancer pathology . Pathology . 2007 ; 39 : 305 - 18 .
29. Yanaka Y , Muramatsu T , Uetake H , Kozaki K , Inazawa J. MiR -544a induces epithelial-mesenchymal transition through the activation of WNT signaling pathway in gastric cancer . Carcinogenesis . 2015 ; 36 : 1363 - 71 .
30. Baldwin LA , Hoff JT , Lefringhouse J , Zhang M , Jia C , Liu Z , Erfani S , Jin H , Xu M , She QB , van Nagell JR , Wang C , Chen L , Plattner R , Kaetzel DM , Luo J , Lu M , West D , Liu C , Ueland FR , Drapkin R , Zhou BP , Yang XH. CD151- alpha3beta1 integrin complexes suppress ovarian tumor growth by repressing slug-mediated EMT and canonical Wnt signaling . Oncotarget . 2014 ; 5 : 12203 - 17 .
31. Vincan E , Barker N. The upstream components of the Wnt signalling pathway in the dynamic EMT and MET associated with colorectal cancer progression . Clin Exp Metastasis . 2008 ; 25 : 657 - 63 .
32. Liang J , Liang L , Ouyang K , Li Z , Yi X. MALAT1 induces tongue cancer cells' EMT and inhibits apoptosis through Wnt/beta-catenin signaling pathway . J Oral Pathol Med . 2016 . doi:10.1111/jop.12466.