MiR-548c impairs migration and invasion of endometrial and ovarian cancer cells via downregulation of Twist
Sun et al. Journal of Experimental & Clinical Cancer Research
MiR-548c impairs migration and invasion of endometrial and ovarian cancer cells via downregulation of Twist
Xiaochun Sun 1
Manhua Cui 0
Aichen Zhang 1
Lingling Tong 1
Kun Wang 1
Kai Li 1
Xue Wang 1
Ziqian Sun 1
Hongye Zhang 1
0 Department of Obstetrics and Gynecology, the Second Hospital of Jilin University , Changchun 130041 , China
1 Department of Obstetrics and Gynecology, China-Japan Union Hospital of Jilin University , Changchun 130021 , China
Background: MicroRNAs (miRNAs) are a class of small non-coding RNAs, which post-transcriptionally repress the expression of genes involved in cancer initiation and progression. Although some miRNAs that target many signaling pathways (also called universe miRNAs) are supposed to play a global role in diverse human tumors, their regulatory functions in gynecological cancers remain largely unknown. We investigated the biological role and underlying mechanism of miR-548c (one universe miRNA) in endometrial and ovarian cancer. Methods: The effects of miR-548c overexpression on cell proliferation, migration and invasion were studied in endometrial and ovarian cancer cells. TWIST1 (Twist) was identified as a direct miR-548c target by western blot analysis and luciferase activity assay. The expression of miR-548c and Twist were examined by qRT-PCR in endometrial and ovarian cancer tissues. Results: Here, we report that miR-548c is down-regulated in endometrial and ovarian cancer tissues when compared to normal tissues, and our meta-analysis reveal that decreased miR-548c expression correlates with poor prognosis in endometrial cancer patients. We show that in endometrial and ovarian cancer cells, ectopic expression of miR-548c significantly inhibits whereas knockdown of miR-548c dramatically induces cancer cell proliferation, migration and invasion. By using luciferase reporter assay, we demonstrate that Twist, a known oncogene in endometrial and ovarian cancers, is a direct target of miR-548c. Furthermore, the expression of Twist partially abrogates the tumor suppressive effects of miR-548c on cell migration and invasion. Conclusion: These findings suggest that miR-548c directly downregulates Twist, and provide a novel mechanism for Twist upregulation in both endometrial and ovarian cancers. The use of miR-548c may hold therapeutic potential for the treatment of Twist-overexpressing tumors.
microRNA; miR-548c; EMT; Endometrial cancer; Ovarian cancer
Metastasis is the main cause of death among patients
with endometrial cancer (EC, the most frequently
diagnosed gynecologic malignancy) cbl1, 2] and ovarian
cancer (OC, the most lethal gynecological cancer) [
initial steps of metastasis include the detachment and
migration of cancer cells and subsequent invasion into
surrounding tissues [
transition (EMT) is considered a key step in metastasis
of endometrial and ovarian cancer [
1, 5, 6
enhances the migratory and invasive capacity of tumor
cells to facilitate malignant progression. Therefore,
understanding the molecular mechanisms that mediate
migration, invasion and EMT process of EC and OC cells
could identify novel molecular targets for future
treatments of these cancers.
Twist1 (Twist) is a critical oncogene that is
overexpressed and plays important roles in EMT induction in
EC and OC [
1, 7, 8
]. High Twist expression positively
correlated with deep myometrial invasion and poor
outcome in EC [
]. Aberrant expression of Twist in OC
correlated with advanced tumor stage and predicted a
poor clinical outcome [
]. MicroRNAs (miRNAs)
are a class of small non-coding RNA molecules that
regulate gene expression via interactions with the
3’-untranslated regions (UTRs) of mRNAs targets, causing
translational suppression or mRNA decay. MiRNAs
control metastatic progression [
], and act as either
tumor suppressors or oncogenes in EC and OC [
Although a number of miRNAs, such as miR-106b and
miR-543, have been reported to suppress the expression
of Twist and EC cell invasion [
], miRNAs affecting
Twist expression in EC and OC is still not completely
understood. Recently, two distinct classes of microRNAs:
universe miRNAs (for example, miR-34a) that regulate
many signaling pathways, as well as intra-pathway
miRNAs that target multiple genes within a single signaling
pathway, have been identified [
]. Universe microRNAs
are predicted to affect more targets/signaling pathways,
and might play a global role in tumor cells [
interest, several universe miRNAs (including miR-548a/
b/c, etc.) belong to the same miR-548 family [
have been implicated in tumorigenesis [
their regulatory functions in gynecological cancers
remain largely unknown.
In this study, we investigated the biological
functions and underlying mechanism of miR-548c in EC
and OC, and provide in vitro evidence that miR-548c
inhibits migration and invasion in both EC and OC
cells by directly targeting Twist. We further
demonstrate that miR-548c expression inversely correlates
with Twist expression in EC and OC tissues and the
downregulation of miR-548c is associated with poor
prognosis in EC patients.
Cell culture and transient transfection
The human EC cell lines (RL95-2 and HEC-1) and
human OC cell lines (SKOV-3 and OVCAR3) were
obtained from Chinese Academy of Sciences
Committee on Type Culture Collection Cell Bank, Shanghai,
China. These cells were cultured in DMEM/F12
medium (Invitrogen, Shanghai) or PRMI-1640 medium
(Gibco, Carlsbad, CA, USA) supplemented with 10 % fetal
bovine serum (FBS, Invitrogen, Shanghai). MiR-548c
mimic (40 nM), antisense miR-548c inhibitor (40 nM),
Twist siRNA (5 nM) and respective negative controls were
purchased from Ambion (TX, USA), and were transfected
using Lipofectamine 2000 (Invitrogen, CA, USA),
according to the manufacturer’s instructions. Transient
transfection of Twist cDNA plasmids (OriGene, MD, USA) were
performed with Lipofectamine Plus reagent (Invitrogen,
CA, USA) according to the manufacturer’s protocol.
RNA extraction and real-time qRT-PCR
Total RNA was extracted using TRIzol reagents
(Invitrogen, CA, USA). The miRNA qRT-PCR was performed
using the NCode miRNA qRT-PCR analysis (Invitrogen,
CA, USA). Forward primers for miRNA detection are
the exact sequence of mature miR-548c-3p. For mRNA
analysis, RT reaction was carried out using 100 ng total
RNA with the PrimeScript RT reagent kit (Takara,
Japan). Real-time qRT-PCR was performed using the
Takara SYBR Premix Ex Taq II (Takara, Japan). Primers
used for the amplification of E-cadherin [
], CD133 [
], MMP-9 [
] and GAPDH [
been previously described. All miRNA and mRNA
quantification data were normalized to GAPDH. Results were
given as the fold change relative to controls.
Cell proliferation assay and apoptosis assay
To evaluate cell proliferation, 5 × 103 cells were plated in
96-well plates for 24 h, and then transfected with
miRNA mimic/inhibitor with or without Twist siRNA or
Twist cDNA vector. At 72 h, cell proliferation was
determined by the Cell Counting Kit-8 (Dojindo, Japan). The
absorbance was determined at 450 nm using a
microplate reader, and data were expressed as the percentage
of absorbance relative to controls. For cell apoptosis
assay, cells were transfected for 72 h and apoptotic cells
were identified using the DeadEnd Colorimetric TUNEL
System kit (Promega, WI, USA), following the
manufacturer’s instructions. Cell apoptosis was quantified as the
numbers of apoptotic cells found in 15 random fields.
The ratio of dead/total cell number was calculated.
Cell migration and invasion assay
Cells were transfected for 24 h and then seeded into
upper chamber of Boyden chambers coated with or
without Matrigel as described previously [
incubation for 24 h, migration and invasion were stained
and counted under a light microscope. Relative
migration and invasion activities were expressed as the fold
change over respective controls.
Cells were harvested 48 h after transfections. Whole-cell
protein extracts were prepared using the M-Per
Mammalian Protein Extraction Reagent (Pierce Biotechnology,
MA, USA) according to the manufacturer’s instructions.
Total proteins (40 μg) were loaded onto 10 % SDS-PAGE
for immunoblots with antibodies to Twist (Abcam,
ab50887) and GAPDH (Santa Cruz, sc-47724). Primary
antibodies were used at a dilution of 1:1000.
Luciferase activity assay
The Twist 3’-UTR luciferase vectors was obtained from
OriGene (Rockville, MD, USA). A quick-change
sitedirected mutagenesis kit (Stratagene, CA, USA) was
used to mutate the miR-548c-binding site. Luciferase
activity was measured at 24 h after transfection using
the dual-luciferase reporter assay system (Promega, WI,
USA) as previously reported [
Results are presented as mean ± s.e.m. from at least
three independent experiments performed in triplicate.
2-tailed Student’s t-test was used for statistical analysis.
P-values < 0.05 were defined as significant.
Benign and tumor tissues
50 pairs of primary EC and adjacent non-tumor
endometrial tissues, 60 epithelial OC tissues and 20 normal
epithelial ovarian tissues were collected in the China-Japan Union
Hospital of Jilin University, China. This study was approved
by the Ethics Committee of China-Japan Union Hospital of
Jilin University and conducted according to the principles
of the Declaration of Helsinki. The patients signed a written
inform consent for the procedures in the study. Samples
were snap-frozen immediately at –80 °C, and total RNA
was isolated using TRIzol reagents.
MiR-548c is downregulated in endometrial and ovarian cancer tissues and decreased miR-548c levels associates with worse prognosis in patients with endometrial cancer
To examine the levels of miR-548c in EC tissues, we
measured the endogenous miR-548c expression by
realtime quantitative RT-PCR (qPCR) in 50 EC samples and
their adjacent normal tissues. Intriguingly, miR-548c was
significantly decreased in ECs compared with adjacent
normal tissues (Fig. 1a). We also analyzed miR-548c
expression by qPCR in clinical samples of 60 OC tissues
(Table 1) compared to 20 normal ovarian tissues.
Decreased miR-548c expression was detected in OC
tissues relative to normal ovarian tissues, and the
expressions of miR-548c were significantly lower in serous,
advanced stage and grade 3 tumor samples compared
with non-serous, early stage and grade 1/2 tumor
samples (Fig. 1b), indicating that the deregulation of
miR548c may play a critical role in EC or OC development.
To investigate whether the downregulation of
miR548c is related with prognosis of EC or OC patients, we
used the SurvMicro web tool [
] to investigate the
relationship between miR-548c levels and prognosis of
cancer patients. The analysis of published data on EC and
OC from The Cancer Genome Atlas (TCGA) revealed
that the low expression levels of miR-548c were
associated with very poor overall survival in EC patients
compared with those patients whose ECs express a high level
of miR-548c (Fig. 1e and f ). Although our meta-analysis
of OCs using TCGA data did not show a clear
separation between high-risk OCs and low-risk OCs based on
miR-548c expression (P = 0.425), OC patients whose
tumors display low miR-548c expression tend to have
shorter survival (Fig. 1g and h). Thus, these data uncover
an association between the loss of miR-548c expression
and poor survival of EC patients, and indicate that
miR548c repression might be important for EC/OC growth
MiR-548c inhibits proliferation, migration and invasion of
EC and OC cells
To select appropriate model for functional studies, we
first evaluated miR-548c expression in two EC cell lines
(RL95-2 and HEC-1) and OC cell line SKOV-3 using
qPCRs. We found that invasive HEC-1 and SKOV-3
cells express much lower levels of miR-548c, compared
with less invasive RL95-2 and OVCAR3 cells (Fig. 2a).
The downregulation of miR-548c in cancer tissues and
invasive cancer cells implicated that it might have the
tumor suppressive roles in the tumorigenesis and
metastasis of EC and OC. To test this, we transiently transfected
anti-miRNA inhibitor against miR-548c (anti-548c) or
control inhibitor (anti-ctr) into RL95-2 or OVCAR3 cells,
and then validated the depletion of miR-548c expression
by qPCR assays (Fig. 2b). Furthermore, the ectopic
overexpression of miR-548c in HEC-1 and SKOV-3 cells was
achieved by transient transfection of pre-miR-548c mimic
(pre-548c) (Fig. 2c). Then, cell proliferation was measured
by the Cell Counting Kit-8 assay. The downregulation of
miR-548c in RL95-2 and OVCAR3 cells resulted in
greater proliferation rate, whereas forced miR-548c
expression significantly reduced proliferation of HEC-1 and
SKOV-3 cells compared with the control mimic (pre-ctr)
(Fig. 2d). To determine the effect of miR-548c on cell
apoptosis, we examined apoptosis-associated DNA
fragmentation by using a colorimetric TUNEL staining assay.
MiR-548c knockdown decreased RL95-2 or OVCAR3 cell
death, however miR-548c overexpression caused apoptotic
cell death of HEC-1 and SKOV-3 cells (Fig. 2e). These
results suggest that miR-548c inhibits EC and OC cell
proliferation and triggers cell apoptosis.
To verify whether miR-548c affect the progression and
metastasis of EC and OC, we evaluated the effect of
either knockdown or overexpression of miR-548c on the
migratory and invasive abilities of these cells. Transwell
migration and invasion assays demonstrated that
downregulation of miR-548c expression significantly
promoted migration and invasion of RL95-2 and OVCAR3
cells (Fig. 2f and g). In contrast, transient overexpression
of miR-548c in HEC-1 and SKOV-3 cells significantly
impaired in vitro cell migration and invasion (Fig. 2f, g
and h). Together, these data support that miR-548c acts
as a tumor suppressors to inhibit tumor growth,
migration and invasion in both EC and OC cells.
Twist is a direct target of MiR-548c in EC and OC cells
To explore gene targets and molecular pathways that
could be mediated by miR-548c, we performed
computational target prediction using TargetScan and
conducted in silico enrichment analysis of miR-548c-target
genes in KEGG pathways using miRSystem [
note, miR-548c-regulated genes can potentially modulate
numerous well-known pathways associated with tumor
growth and metastasis, including pathways in cancer,
mTOR pathway, adherens junction, cell cycle, acute
myeloid leukemia, pancreatic cancer, notch signaling
pathway and jak-stat signaling pathway (Fig. 3a),
indicating the possibility that miR-548c has broad influence
over diverse oncogenic pathways. These findings are
consistent with our results (Fig. 2), supporting the roles
of miR-548c to suppress multiple malignant phenotypes
of EC and OC cells including proliferation, migration
Because Twist was identified as a potential target of
miR-548c (Fig. 3b), we speculated that Twist serves as a
direct target of miR-548c. Our western blot analysis
suggested that the introduction of anti-548c led to an
increase in Twist protein level in RL95-2 cells, while
pre-548c resulted in the opposite effects in HEC-1 and
SKOV-3 cells (Fig. 3c). To test whether miR-548c
repression is mediated through the predicted binding site,
the reporter plasmid containing putative miR-548c
responsive site was transfected with anti-548c or anti-ctr
into RL95-2 cells. The downregulation of miR-548c
enhanced Twist 3’-UTR luciferase reporter activity
compared with the negative control (Fig. 3d, left), suggesting
that miR-548c suppresses translation of Twist. To verify
that miR-548c directly represses Twist on its 3’-UTR, we
cotransfected pre-548c or pre-ctr with the Twist 3’-UTR
reporter into HEC-1 and SKOV-3 cells. The ectopic
expression of pre-548c caused a repression on luciferase
activity as compared with pre-ctr (Fig. 3d, middle and
right). To further evaluate the interaction between
miR548c and Twist 3’-UTR, we generated Twist 3’-UTR
mutated for miR-548c binding site and performed the
luciferase assays upon either miR-548c overexpression
or knockdown. Mutant 3’-UTR reporter activity was
resistant to the addition of anti-548c or pre-548c
(Fig. 3d), confirming that Twist is a direct target of
miR548c, and binding of miR-548c to the 3’-UTR of Twist
mRNA is sufficient to reduce Twist protein levels.
Twist binds the E-cadherin promoter and represses
E-cadherin transcription, whereas directly activating
N-cadherin expression [
]. In addition, the EMT
induction has been linked to the emergence of a
cancer stem cell (CSC)-like phenotype .
Consistent with this notion, sorted CD133+ EC cells have
been shown to demonstrate greater tumor initiating
capacity than CD133− cells [
], and CSC-like cells
obtained from SKOV-3 cells express higher levels of
]. Matrix metalloproteinase 9 (MMP-9) is a
downstream target of Twist in tumor cells [
test whether miR-548c regulates these genes in cancer
cells used in this study. By using qPCR, we found a
significant decrease in the expression of E-cadherin
and an increased expression in N-cadherin, CD133
and MMP-9 levels in anti-548c-transfected RL95-2
cells (Fig. 3e). In contrast, miR-548c overexpression
in HEC-1 and SKOV-3 cells significantly induced
Ecadherin levels, but inhibited the expression of
N-cadherin, CD133 and MMP-9 (Fig. 3e). Furthermore, we
used qPCR to examine the mRNA expression of
Twist in EC and OC tissues. As expected, Twist levels
was markedly elevated in EC samples when compared
to adjacent normal tissues (Fig. 1c). We also observed
significantly increased Twist expression in OCs
relative to normal ovarian tissues, and found higher levels
of Twist in serous, advanced, high-grade ovarian
cancers (Fig. 1d). Altogether, these data are consistent
with our hypothesis that increased Twist expression
in EC and OC is a functional consequence of
miR548c repression (at least in part).
Twist mediates miR-548c-reduced cancer cell migration and invasion
To further study whether miR-548c can attenuate
oncogenic phenotypes in EC and OC cells through
downregulation of Twist, we performed cell proliferation,
migration and invasion assays using EC or OC cells
transduced with anti-548c or pre-548c, along with either
Twist siRNA or Twist cDNA expression plasmid
without 3’-UTR sequence, which renders the escape from
miR-548c regulation. Anti-548c-induced RL95-2 cell
proliferation, migration and invasion were significantly
abrogated by Twist siRNA (Fig. 4a, c and e). On the
other hand, overexpression of Twist cDNA largely
rescued pre-548c-suppressed proliferation, migration and
invasion in HEC-1 cells (Fig. 4b, d and f, left). Although
expression of Twist cDNA restored SKOV-3 cell
migration and invasion that were reduced by pre-548c (Fig. 4d
and f, right), cell proliferative phenotype was not
significantly restored by Twist overexpression in SKOV-3 cells
(Fig. 4b, right), consistent with a previous report
showing that Twist confers migratory and invasive potential,
but does not affect proliferation of OC cells [
eO (% 200
leaR lvau 100
Collectively, these data support the mechanistic roles for
miR-548c in impairing migration and invasion of EC
and OC cells by targeting Twist.
Targeting of metastasis-related miRNAs in human
tumors may offer potential therapeutic strategies for these
]. Recent findings have identified
miR548c family members as universe miRNA, and illustrated
the possibility that these miRNAs exert broad effects on
many features of tumor cells through a broad range of
targets and pathways . Although one such miRNA,
miR-548c, has been shown to inhibit cancer cell growth
], its cellular functions, underlying mechanisms and
clinical significance have not been comprehensively
studied in gynecological cancers. Here, we found that
miR-548c was under-expressed in both EC and OC
samples, compared to their non-tumor counterparts. At the
molecular level, miR-548c represses migration and
invasion of EC and OC cells partially by targeting Twist. We
demonstrate that miR-548c expression was inversely
correlated with Twist level in EC and OC tissues, and
reduced miR-548c expression was associated with poor
prognosis in EC patients. These findings reveal for the
first time that in EC and OC cells, miR-548c is a
tumorsuppressive miRNA that inhibits multiple malignant
features of the metastatic cascade.
Twist has been known as a novel oncogene
overexpressed in diverse tumors [
], and activation of Twist
inhibits apoptosis and promotes the induction of EMT,
cancer stemness, proliferation, angiogenesis and
vasculogenic mimicry in human tumor cells, all of which
contributes to metastasis [
]. Several upstream regulators
and downstream effector of Twist have been reported
, however our knowledge of the detailed molecular
mechanisms involved in Twist overexpression, especially
the epigenetic regulation of Twist expression, is still
incomplete. Here, we reported a previously unidentified
mechanism whereby loss of miR-548c expression is
partially responsible for increased Twist levels in EC and OC,
two of the most common gynecologic cancers.
Twist has been shown to upregulate the expression of
some miRNAs, such as miR-10b [
] and miR-424 [
Twist promotes the expression of miR-10b by directly
binding to the promoter of miR-10b, which initiates
tumor invasion and metastasis in breast cancer [
Furthermore, miR-424, another miRNA that can be
induced by Twist, drives EMT-like phenotypes in breast
]. The impact of Twist on miR-548c
expression in EC and OC cells warrants further investigation.
MiR-548c was reported to be an independent prognostic
factor for breast cancer. Patients with a good prognosis
presented higher intratumoral expression of miR-548c
]. Consistently, our meta-analysis suggested that high
expression of miR-548c in EC and OC appears to correlate
with better survival in patients with EC and OC, implying
that miR-548c may be a new promising prognostic factor
in these cancers. Since miR-548c directly regulates Twist
in EC and OC cells, this miRNA might also represent an
attractive target for the treatment of Twist-overexpressing
Our findings provide a novel mechanism by which the
repression of miR-548c increases Twist levels to
stimulate cancer cell migration and invasion of both EC and
OC cells. Our results indicate that miR-548c is a
potential therapeutic target in EC and OC.
The authors declare no competing financial interests.
MC designed experiments. XS, AZ and LT conducted all experiments. MC
and XS wrote the manuscript. AZ, LT, KW, KL, XW, ZS and HZ contributed to
data analysis and discussed the results. All authors read and approved the
We are grateful to the staff and patients at the Department of Obstetrics and
Gynecology, China-Japan Union Hospital of Jilin University for providing the
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1. Dong P , Kaneuchi M , Konno Y , Watari H , Sudo S , Sakuragi N. Emerging therapeutic biomarkers in endometrial cancer . Biomed Res Int . 2013 ; 2013 : 130362 .
2. Kim A , Ueda Y , Naka T , Enomoto T. Therapeutic strategies in epithelial ovarian cancer . J Exp Clin Cancer Res . 2012 ; 31 : 14 .
3. Bast Jr RC , Hennessy B , Mills GB . The biology of ovarian cancer: new opportunities for translation . Nat Rev Cancer . 2009 ; 9 : 415 - 28 .
4. Mehlen P , Puisieux A . Metastasis: a question of life or death . Nat Rev Cancer . 2006 ; 6 : 449 - 58 .
5. Vergara D , Merlot B , Lucot JP , Collinet P , Vinatier D , Fournier I , et al. Epithelial-mesenchymal transition in ovarian cancer . Cancer Lett . 2010 ; 291 : 59 - 66 .
6. Kaufhold S , Bonavida B . Central role of Snail1 in the regulation of EMT and resistance in cancer: a target for therapeutic intervention . J Exp Clin Cancer Res . 2014 ; 33 : 62 .
7. Wang WS , Yu SL , Yang XS , Chang SD , Hou JQ . Expression and significance of twist and E-cadherin in ovarian cancer tissues . Asian Pac J Cancer Prev . 2013 ; 14 : 669 - 72 .
8. Wang WS , Yang XS , Xia M , Jiang HY , Hou JQ . Silencing of twist expression by RNA interference suppresses epithelial-mesenchymal transition, invasion, and metastasis of ovarian cancer . Asian Pac J Cancer Prev . 2012 ; 13 : 4435 - 9 .
9. Kyo S , Sakaguchi J , Ohno S , Mizumoto Y , Maida Y , Hashimoto M , et al. High Twist expression is involved in infiltrative endometrial cancer and affects patient survival . Hum Pathol . 2006 ; 37 : 431 - 8 .
10. Yoshida J , Horiuchi A , Kikuchi N , Hayashi A , Osada R , Ohira S , et al. Changes in the expression of E-cadherin repressors, Snail, Slug, SIP1, and Twist, in the development and progression of ovarian carcinoma: the important role of Snail in ovarian tumorigenesis and progression . Med Mol Morphol . 2009 ; 42 : 82 - 91 .
11. Hosono S , Kajiyama H , Terauchi M , Shibata K , Ino K , Nawa A , et al. Expression of Twist increases the risk for recurrence and for poor survival in epithelial ovarian carcinoma patients . Br J Cancer . 2007 ; 96 : 314 - 20 .
12. Pencheva N , Tavazoie SF . Control of metastatic progression by microRNA regulatory networks . Nat Cell Biol . 2013 ; 15 : 546 - 54 .
13. Liu X , Chen X , Yu X , Tao Y , Bode AM , Dong Z , et al. Regulation of microRNAs by epigenetics and their interplay involved in cancer . J Exp Clin Cancer Res . 2013 ; 32 : 96 .
14. Katz B , Tropé CG , Reich R , Davidson B. MicroRNAs in ovarian cancer . Hum Pathol . 2015 ; 46 : 1245 - 56 .
15. Dong P , Kaneuchi M , Watari H , Sudo S , Sakuragi N. MicroRNA-106b modulates epithelial-mesenchymal transition by targeting TWIST1 in invasive endometrial cancer cell lines . Mol Carcinog . 2014 ; 53 : 349 - 59 .
16. Bing L , Hong C , Li-Xin S , Wei G . MicroRNA -543 suppresses endometrial cancer oncogenicity via targeting FAK and TWIST1 expression . Arch Gynecol Obstet . 2014 ; 290 : 533 - 41 .
17. Shirdel EA , Xie W , Mak TW , Jurisica I. NAViGaTing the micronome-using multiple microRNA prediction databases to identify signalling pathwayassociated microRNAs . PLoS ONE . 2011 ; 6 : e17429 .
18. Piriyapongsa J , Jordan IK . A family of human microRNA genes from miniature inverted-repeat transposable elements . PLoS ONE . 2007 ; 2 , e203 .
19. Hu B , Ying X , Wang J , Piriyapongsa J , Jordan IK , Sheng J , et al. Identification of a tumor-suppressive human-specific microRNA within the FHIT tumorsuppressor gene . Cancer Res . 2014 ; 74 : 2283 - 94 .
20. Odero-Marah VA , Wang R , Chu G , Zayzafoon M , Xu J , Shi C , et al. Receptor activator of NF-kappaB Ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells . Cell Res . 2008 ; 18 : 858 - 70 .
21. Chiou SH , Wang ML , Chou YT , Chen CJ , Hong CF , Hsieh WJ , et al. Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelialmesenchymal transdifferentiation . Cancer Res . 2010 ; 70 : 10433 - 44 .
22. Figueira RC , Gomes LR , Neto JS , Silva FC , Silva ID , Sogayar MC . Correlation between MMPs and their inhibitors in breast cancer tumor tissue specimens and in cell lines with different metastatic potential . BMC Cancer . 2009 ; 9 : 20 .
23. Dong P , Kaneuchi M , Watari H , Hamada J , Sudo S , Ju J , et al. MicroRNA -194 inhibits epithelial to mesenchymal transition of endometrial cancer cells by targeting oncogene BMI-1 . Mol Cancer . 2011 ; 10 : 99 .
24. Dong P , Karaayvaz M , Jia N , Kaneuchi M , Hamada J , Watari H , et al. Mutant p53 gain-of-function induces epithelial-mesenchymal transition through modulation of the miR-130b-ZEB1 axis . Oncogene . 2013 ; 32 : 3286 - 95 .
25. Dong PX , Jia N , Xu ZJ , Liu YT , Li DJ , Feng YJ . Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro . J Exp Clin Cancer Res . 2008 ; 27 : 7 .
26. Aguirre-Gamboa R , Trevino V. SurvMicro: assessment of miRNA based prognostic signatures for cancer clinical outcomes by multivariate survival analysis . Bioinformatics . 2014 ; 30 : 1630 - 2 .
27. Lu TP , Lee CY , Tsai MH , Chiu YC , Hsiao CK , Lai LC , et al. miRSystem: an integrated system for characterizing enriched functions and pathways of microRNA targets . PLoS ONE . 2012 ; 7 : e42390 .
28. Vesuna F , van Diest P , Chen JH , Raman V . Twist is a transcriptional repressor of E-cadherin gene expression in breast cancer . Biochem Biophys Res Commun . 2008 ; 367 : 235 - 41 .
29. Alexander NR , Tran NL , Rekapally H , Summers CE , Glackin C , Heimark RLAlexander NR , et al. N-cadherin Gene Expression in Prostate Carcinoma Is Modulated by Integrin-Dependent Nuclear Translocation of Twist1 . Cancer Res . 2006 ; 66 : 3365 - 9 .
30. Singh A , Settleman J. EMT , cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer . Oncogene . 2010 ; 29 : 4741 - 51 .
31. Nakamura M , Kyo S , Zhang B , Zhang X , Mizumoto Y , Takakura M , et al. Prognostic impact of CD133 expression as a tumor-initiating cell marker in endometrial cancer . Hum Pathol . 2010 ; 41 : 1516 - 29 .
32. Ma L , Lai D , Liu T , Cheng W , Guo L. Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3 . Acta Biochim Biophys Sin (Shanghai) . 2010 ; 42 : 593 - 602 .
33. Zhao XL , Sun T , Che N , Sun D , Zhao N , Dong XY , et al. Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial-mesenchymal transition regulator Twist1 . J Cell Mol Med . 2011 ; 15 : 691 - 700 .
34. Brunetti O , Russo A , Scarpa A , Santini D , Reni M , Bittoni A , et al. MicroRNA in pancreatic adenocarcinoma: predictive/prognostic biomarkers or therapeutic targets? Oncotarget . 2015 ; 6 : 23323 - 41 .
35. Luo G , Chao YL , Tang B , Li BS , Xiao YF , Xie R , et al. miR -149 represses metastasis of hepatocellular carcinoma by targeting actin-regulatory proteins PPM1F . Oncotarget . 2015 ; 6 : 37808 - 23 .
36. Kang Y , Massagué J . Epithelial-mesenchymal transitions: twist in development and metastasis . Cell . 2004 ; 118 : 277 - 9 .
37. Khan MA , Chen HC , Zhang D , Fu J . Twist: a molecular target in cancer therapeutics . Tumour Biol . 2013 ; 34 : 2497 - 506 .
38. Jung HY , Yang J . Unraveling the TWIST between EMT and Cancer Stemness . Cell Stem Cell . 2015 ; 16 : 1 - 2 .
39. Ma L , Teruya-Feldstein J , Weinberg RA . Tumour invasion and metastasis initiated by microRNA-10b in breast cancer . Nature . 2007 ; 449 ( 7163 ): 682 - 8 .
40. Drasin DJ , Guarnieri AL , Neelakantan D , Kim J , Cabrera JH , Wang CA , et al. TWIST1 -Induced miR-424 Reversibly Drives Mesenchymal Programming while Inhibiting Tumor Initiation. Cancer Res . 2015 ; 75 ( 9 ): 1908 - 21 .
41. Boukerroucha M , Josse C , ElGuendi S , Boujemla B , Frères P , Marée R , et al. Evaluation of BRCA1-related molecular features and microRNAs as prognostic factors for triple negative breast cancers . BMC Cancer . 2015 ; 15 : 755 .