Overexpression of trefoil factor 3 (TFF3) contributes to the malignant progression in cervical cancer cells
Yuan et al. Cancer Cell Int
Overexpression of trefoil factor 3 ( TFF3) contributes to the malignant progression in cervical cancer cells
Zhaohu Yuan 0 2
Dandan Chen 1
Xiaojie Chen 0 2
Huikuan Yang 0 2
Yaming Wei 0 2
0 Department of Blood Transfusion, Guangzhou First People's Hospital, Guangzhou Medical University , Guangzhou 510180, Guangdong Province , China
1 Department of Radiology, Guangzhou First People's Hospital, Guangzhou Medical University , Guangzhou 510180 , China
2 Department of Blood Transfusion, Guangzhou First People's Hospital, Guang- zhou Medical University , Guangzhou 510180, Guangdong Province , China
Background: There remains a great need for effective therapies for cervical cancers, the majority of which are aggressive leaving patients with poor prognosis. Methods and results: Here, we identify a novel candidate therapeutic target, trefoil factor 3 (TFF3) which overexpressed in cervical cancer cells and was associated with reduced postoperative survival. Functional studies demonstrated that TFF3 overexpression promoted the proliferation and invasion of cervical cancer cells, and inhibited the apoptosis by inducing the mRNA changes in SiHa and Hela cell lines. Conversely, TFF3 silencing disrupted the proliferation and invasion of cervical cancer cells, and induced the apoptosis via Click-iT EdU test, flow cytometry analysis and two-dimensional Matrigel Transwell analysis. Western blot analysis showed that overexpression of TFF3 repressed E-cadherin (CDH1) expression to promote the invasion of cervical cancer cells. Furthermore, down-regulated CDH1 via overexpression of TFF3 was significantly up-regulated by virtue of inhibitor of p-STAT3. Conclusions: These results suggested that TFF3 stimulated the invasion of cervical cancer cells probably by activating the STAT3/CDH1 signaling pathway. Furthermore, overexpression of TFF3 decreased the sensitivity of cervical cancer cells to etoposide by increasing P-glycoprotein (P-gp) functional activity. Overall, our work provides a preclinical proof that TFF3 not only contributes to the malignant progression of cervical cancers and but also is a potential therapeutic target.
Trefoil factor 3 (TFF3); Malignant progression; Cervical cancer cells; Therapeutic targets
Worldwide, cervical cancer is ranked as the second most
common cancer in women and the third leading cause
of death from cancer in women [1, 2]. The incidence of
cervical cancer is very high in developing countries .
Until recently, therapeutic options for
hysterectomyresistant cervical cancers have been limited with
treatments largely palliative . Therefore, detecting or
preventing cervical cancers with progressions in early
is critical, which could help to prolong patient survival.
As we know TFF3 is a soluble peptide containing trefoil
domain and C-terminal dimerization domain which is
not only a novel prognostic marker but also a therapeutic
target in various cancers, such as mammary carcinoma,
gastric cancer and prostate carcinoma [5–8].
Upregulation of TFF3 after rectal cancer chemo-radiotherapy is
an adverse prognostic factor . Furthermore, in prostate
carcinoma cells, TFF3 reduces the sensitivity to
TFF3, behaved as an oncogene, promotes
proliferation and invasion, improves survival, and increases
oncogenicity in cancer cells, such as mammary
carcinoma, gastric cancer and prostate carcinoma [5, 11].
TFF3 promoted epithelial tumorigenesis by
inducing aberrant proliferation and inhibiting apoptosis .
TFF3 also may contribute to cancer metastasis with
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epithelial-to-mesenchymal transition (EMT)
potentially through the regulation of genes such as androgen
receptor (AR), FOXA1 and human epidermal growth
factor receptor-type 2 (HER2) [12, 13]. Moreover, TFF3,
a secreted protein, is a valuable predictive serum
biomarker in patients with metastatic colorectal cancer .
In cancer cells, TFF3 promotes cell migration, invasion
and metastasis by reducing cell–cell and cell–matrix
interactions and enhancing cell scattering in bronchiole
or other epithelia cells [14, 15]. Up-regulation of TFF3 in
cancer cells was accompanied by activation of multiple
pathways including PI3K, MAPK and JAK/STAT
pathways which were associated with cellular proliferation,
apoptosis, migration, invasion and clonogenic survival
. Despite the evidence that TFF3 could influence
various cancer cells function in vitro, the role of TFF3 in
cervical cancer cells has not been examined.
In the present study, we found that TFF3 protein
was overexpressed in cervical cancer cells and weakly
expressed in human non-tumor keratinocytes. We
detected up-regulated expression of TFF3 promoted
growth, proliferation and invasion, and inhibited
apoptosis in SiHa and Hela cells. These finding demonstrate that
TFF3 may be a potential therapeutic target in invasive
cervical cancers with multidrug resistance.
Dulbecco’s modified Eagle’s medium (DMEM) and
fetal bovine serum (FBS) were obtained from GIBCO
(Carlsbad, California, USA). Mouse anti-GPADH
polyclonal antibody (Lot#ab37168), Rabbit anti-Trefoil
Factor 3 monoclonal antibody (Lot#ab108599), Mouse
anti-E Cadherin monoclonal antibody (Lot#ab1416),
Mouse anti-Phospho-STAT3 monoclonal antibody
(Lot#ab119672), Mouse anti-Total STAT3 monoclonal
antibody (Lot#ab119672) were obtained from Abcam
(Cambridge, UK). JSI-124 was obtained from Enzo Life
Science (USA). Goat anti-Rabbit IgG IR Dye 800cw
(Lot#C30626-03) and Goat anti-Mouse IgG IR Dye
800cw (Lot#C40528-02) were from Odyssey (Licor,
USA). Click-iT Edu imaging kit and Live/Dead Bac Light
Viability Kit for microscopy were from Invitrogen
(Carlsbad, CA, USA).
Cell cultures and transfection
Human cervical cancer cell lines SiHa, CaSki, Hela,
Me180 and human non-tumor keratinocyte line HaCaT
were obtained from Nanjing KeyGen Biotech Co, Ltd
(Nanjing,China). The cells were cultured in Dulbecco’s
modified Eagle’s medium (GIBCO, Carlsbad, California,
USA) containing 10% FBS in a humidified atmosphere of
5% CO2 at 37 °C. Human TFF3 expression, TFF3 siRNA
and CDH1 siRNA plasmid constructs have been
previously described [7, 17]. Luciferase assays were performed
as previously described . Briefly, transfections were
carried out in triplicate using 1 μg of the appropriate
luciferase reporter construct and empty vector per
transfection along with 0.1 μg of Renilla luciferase construct
as control for transfection efficiency. Luciferase
activities were assayed after 24 h of transfection using the Dual
Luciferase Assay System (Promega Corp, Madison, WI,
RT‑PCR and semi‑quantitative RT‑PCR
Total RNA was isolated from cells using Trizol plus RNA
Purification system as previously described . DNase I
treatment, total RNA to complementary DNA, PCR, and
qPCR assays were performed as previously described.
Gene expression analysis was performed as previously
described  and the sequence of the primers were
described in Additional file 1: Table S1.
Total RNA was isolated using Trizol plus RNA
Purification Kit (Invitrogen, Carlsbad, CA) as previously
described . Semi-quantitative RT-PCR was
performed using a Super Script One Step RT-PCR kit
(Invitrogen, Carlsbad, CA, USA). Sequences of the
nucleotide primers for RT-PCR were: TFF3 5′-GCTG
CCAGAGCGCTCTGCATG-3′ and 5′-AAGGTGCA
TTTCTGCTTCCTGCAG-3′ (35 cycles; wild-type cell
lines); β-Actin, 5′-ATGATATCGCCGCGCTCG-3′ and
5′-CGCTCGGTGAGGATCTTCA-3′ (23 cycles).
Amplified RT-PCR products were visualized on a 1.5% agarose
In vitro invasion analysis
An in vitro invasion assay was carried out to
examine the invasion of cervical cancer cells, as previously
described . Briefly, 24-well Transwell units with
8 µm polycarbonate nucleopore filters (Corning, NY,
USA) were coated with 0.1 mL 0.8 mg/mL Engelbreth
Holm-Swarm sarcoma tumor extract (EHS Matrigel) at
room temperature for 1 h to form a genuinely
reconstituted basement membrane. Cervical cancer cells
(5 × 104 cells) were placed in the upper compartment
and 500 µL DMEM culture medium containing 10%
fetal calf serum was added to the lower compartment.
The Transwell plates were incubated at 37 °C for 36 h
in a humidified atmosphere with 5% CO2 and stained
with 10% crystal violet. Invading cells were defined as
cells that had degraded the Matrigel and moved into the
lower surface of the membrane. The non-invading cells
retained on the upper surface of the membrane were
removed by a cotton swab.
Click‑iT EdU test
We performed the Click-iT Edu test to analyze the cervical
cancer cell proliferation according to the manufacturer’s
instructions. Cervical cancer cells were incubated with
EDU for 12 h and then images were obtained to determine
percentages of EdU-labeled cervical cancer cells.
Cervical cancer cells lysates were separated by
SDSPAGE, blotted onto nitrocellulose membranes, and
probed with primary antibodies, followed by Goat
antirabbit or mouse IgG IR Dye 800 cw (1:15,000),
respectively. Images were obtained with an Odyssey Imager
(LI-COR, Lincoln, NE, USA).
Flow cytometry and analysis
Flow cytometry analysis was performed either on
FACSCAN using Cell Quest software, or on MACS quant
seven color analyzer. Data analysis was performed using
Flow Jo software.
Total cell number
1 × 105 (SiHa and Hela) cells were seeded into 35 mm2
falcon tissue culture dish in monolayers in 10% serum
media in quadruplicate. On indicated days, cells were
trypsinised and the cell number was determined using a
Graphs and statistical analysis
All graphs were generated using Prism 4 (GraphPad
Software, Inc., California, USA). Statistical significance was
assessed by using an unpaired two-tailed Student’s t test
(P < 0.05 was considered as significant) using SPSS 18.0
(SPSS, Inc., Chicago, IL, USA). Columns are the mean of
triplicate experiments; bars ± SD. *P < 0.05, **P < 0.01.
TFF3 is overexpressed in human cervical cancer cell lines
Compared with HaCaT cells, TFF3 was expressed at
higher level in all the cervical cancer cell lines tested. To
better reveal the functional roles of TFF3, SiHa and Hela
cell lines were selected for next studies, in which
expression of TFF3 was higher than the other two cervical
cancer cell lines (Fig. 1a). To generate TFF3 overexpressing
or knockdown stable cell lines, 293T cells were
co-transfected with retroviral constructs of pMSCV-TFF3 or
pSIH1-TFF3 along with the respectively package system
plasmids for 2 days. The supernatant was collected to
infect SiHa and Hela cell lines for 2 days, and then cells
were collected for semi quantitative PCR and Western
blot analysis (Fig. 1b, c).
Using RT-PCR analyses, we therefore quantified the
mRNA levels of various molecules associated with
cellular proliferation, apoptosis, migration, invasion
and clonogenic survival in SiHa and Hela cell lines with
forced expression of TFF3. SiHa-TFF3 or Hela-TFF3
cells exhibited decreased mRNA levels of BAX, TIMP2,
CDKN2A, SERPINB5 and CDH1, relative to SiHa-vector
cells or Hela-vector cells respectively. Concomitantly,
SiHa-TFF3 and Hela-TFF3 cells exhibited increased
mRNA levels of CDH2, VIM, TGFB1, TERT, SERPINE1,
TWIST, KI67, SURVIVIN, MMP2 and MMP3, relative to
SiHa-vector or Hela-vector cells (Fig. 1d, e). Considering
the rapid changes in proliferative and molecular patterns
of expression associated with the oncogenic
characteristics, which indicated TFF3 plays an important role in the
malignant progression of human cervical cancer.
Overexpression of TFF3 promotes proliferation
and survival of SiHa and Hela cells
To investigate whether TFF3 contributes to the
proliferation of cervical cancer cells in vitro, we analyzed the
proliferation upon overexpression and knockdown of TFF3
in SiHa and Hela cells. Overexpression of TFF3 increased
the total cell number of SiHa and Hela cells by 1.20 fold
and 1.26 fold respectively after 48 h. Conversely,
knockdown of TFF3 decreased the total cell number of SiHa
and Hela cells (Fig. 2a). Furthermore, TFF3
overexpressing cells (SiHa-TFF3 and Hela-TFF3 cells) showed a
statistically significant increase in proliferation, as assessed
by measuring EdU incorporation into DNA and Ki67
expression (Fig. 2b, c). In order to verify whether the
endogenous function of TFF3 is to promote proliferation,
it was followed by knocking down cervical cancer cells
expressing high level of TFF3. Specific-stranded RNA
oligonucleotides against TFF3 or negative RNA control
were transfected into SiHa and Hela cells. When
TFF3specific oligonucleotides were used, a rapid
down-regulation of TFF3 mRNA and protein was observed (Fig. 1b,
c). The decrease in TFF3 level resulted in a concomitant
decrease in proliferation. Cell proliferation assays (EdU
incorporation and Ki67 expression) showed a
statistically significant decrease in proliferation when TFF3 was
down-regulated with siRNA (Fig. 2b, c).
The number of apoptosis cells was very low in
TFF3overexpressing cells (SiHa-TFF3 and Hela-TFF3 cells),
but no significant differences from controls were
observed (P > 0.05, Fig. 3a). However, the apoptotic
proportions in SiHa-siTFF3 and Hela-siTFF3 were
significantly increased (P < 0.01, Fig. 3a). Western Blot analysis
demonstrated that forced expression of TFF3 reduced
the levels of the Bax protein, whereas the Bcl-2
protein revealed only minimal increase in SiHa and Hela
cells. The increase in Bcl-2/Bax ratio inhibits the
apoptosis of human cervical cancer cells. Conversely, siRNA
against TFF3 increased the expression of Bax protein and
(See figure on previous page.)
Fig. 1 Forced expression of TFF3 in cervical cancer cells modulates the expression of malignant progression-related gene markers. a Determination
of endogenous expression of TFF3 protein by Western blot in the cervical cancer cells lines CaSki, SiHa, Me180 and Hela and human non-tumor
keratinocyte line HaCaT. b Semi-quantitative RT-PCR analysis was used to assess the mRNA levels of TFF3 in SiHa and Hela cells with either forced or
depleted expression of TFF3 as described in “Methods” section. c Western blot analysis was used to assess the protein levels of TFF3 in SiHa and Hela
cells with either forced or depleted expression of TFF3. d, e Quantitative PCR analysis quantifying the change in expression of various genes
associated with malignant progression in SiHa-TFF3/Hela cells. Change in gene expression is expressed as fold difference, respectively. Fold change values
are representative of three independent experiments
reduced the levels of Bcl-2 protein (Fig. 3b). Decreasing
the rate of Bcl-2/Bax induces mitochondria-mediated
apoptosis in human cervical cancer cells.
CDH1 is critical for TFF3‑mediated cervical cancer cells
Transwell invasion assays frequently were utilized to
determine the invasiveness of cervical cancer cells
in vitro. We therefore examined the behavior of cervical
cancer cells with either forced or depleted expression of
TFF3 growing on two-dimensional Matrigel. SiHa-TFF3
cells exhibited increased ability to penetrate and migrate
in Matrigel-coated matrix compared with SiHa-vector
cells (100.0 ± 7.2% for control cells vs. 189.5 ± 12.4%
for TFF3-overexpressing cells; Fig. 4a). In contrast, the
ability of SiHa-sivector to penetrate and migrate in
Matrigel-coated matrix significantly decreased compared
with SiHa-vector cells (101.3 ± 8.1% for control cells vs.
39.5 ± 4.2% for TTF3-knockdown cells; Fig. 4a). Similar
results were obtained in Hela cells, as shown in Fig. 4a.
To further determine whether TFF3 could activate
CDH1 expression in cervical cancer cells,
TFF3-expressing plasmids were transiently transduced into the two
cervical cancer cell lines. Western blot analysis was
performed to examine the expression of TFF3 and CDH1.
Consistent with the results reported previously ,
overexpression of TFF3 in the two cell lines suppressed
expression of CDH1 and induced CDH1 signaling was
evidenced by increasing phosphorylation of STAT3 in
TFF3-overexpressing cells compared with control cells
(Fig. 4b). Conversely, TFF3 knockdown in two cervical
cancer cell lines increased CDH1 expression mRNA level
(Fig. 1g, h) with concomitant decrease of
phosphorylation of STAT3 (Fig. 4b). These data indicated that TFF3
could down-regulate CDH1 expression in cervical cancer
Previous studies showed that overexpression of TFF3
promoted cell migration and invasion [6, 7, 11]. We next
determined whether CDH1 mediated the effect of TFF3
on cell invasion in SiHa cells. We first utilized the
CDH1specific siRNA to knock down CDH1 expression in the
two cell lines. Western blot analysis showed that CDH1
siRNAs achieved a knockdown of CDH1 expression in
SiHa-siTFF3 cells by 70 to 80% (Fig. 4c). We performed
cell invasion assay and found that the invasive ability in
SiHa-siTFF3-siCDH1 cells was significantly stronger
than that in SiHa-siTFF3 cells (Fig. 4c). In addition,
transfection of CDH1 siRNA also increased invasive ability of
control cells and the percentage was significantly greater
compared with that in SiHa-siTFF3 cells (100.0 ± 9.1%
for control cells vs. 142.1 ± 6.2% for TFF3-overexpressing
cells; Fig. 4c). To further confirm that the STAT3/CDH1
signaling pathway is critical for TFF3-mediated cell
migration and invasion, we treated TFF3-overexpressing
or control cells with the STAT3 activity inhibitor,
JSI124. Western blots were used to confirm the blockage
of the CDH1 signaling pathway. As expected, the
phosphorylation level of STAT3 was decreased with JSI-124
treatment in TFF3-overexpressing cervical cancer cells
(Fig. 4d). Moreover, the inhibition of STAT3/CDH1
pathway significantly attenuated cell invasion ability in
TFF3overexpressing cells (Fig. 4d). This is consistent with our
hypothesis that overexpression of TFF3 could stimulate
cell invasion through activation of the STAT3/CDH1
signaling pathway. Similar results also were obtained in
TFF3 decreases the sensitivity of cervical cancer cells
to etoposide by increasing P‑gp functional activity
MDR is a common clinical problem for the treatment
of cancers in chemotherapy. Escaped cancer cells from
chemotherapy through MDR are a major reason for
(See figure on next page.)
Fig. 2 Correlation of TFF3 expression with the proliferation of SiHa and Hela cells. a Total cell number assays. Cells were seeded in both
fullserum (10%) and total cell number counted every 12 h. b EdU incorporation and c Ki67 flow cytometry expression assays showed a statistically
significant increase or decrease in proliferation when TFF3 was overexpressed or knockdown respectively in SiHa and Hela cells. Results represent
mean ± SEM. **P < 0.01. Statistical significance was assessed by using an unpaired two-tailed Student’s test (P < 0.05 was considered as significant)
using SPSS 18.0
Fig. 3 Effect of TFF3 on apoptosis of SiHa and Hela cell lines. a The flow cytometry analysis and quantification analysis of apoptotic cells. Statistical
significance was assessed by using an unpaired two-tailed Student’s t test (P < 0.05 was considered as significant) using SPSS 18.0. Columns are the
mean of triplicate experiments; bars ±SD. **P < 0.001. b Western blot analysis was used to assess the apoptosis related protein levels in SiHa and
Hela cells with either forced or depleted expression of TFF3
clinical treatment failure [22, 23]. In this study we found
forced expression of TFF3 in SiHa and Hela cells
significantly decreased the sensitivity to etoposide and
inhibited the apoptosis/death by live/dead staining.
Conversely, TFF3 knockdown increased the sensitivity of
cervical cancer cells to etoposide and promoted
apoptosis (Fig. 5a, b).
Rho123 is a fluorescence substrate that is applied
to investigate P-gp functional activity. When
functional activity of P-gp declines, the accumulation of
(See figure on previous page.)
Fig. 4 TFF3 regulates cell and invasion of cervical cancer cells through activation of the STAT3/CDH1 signaling pathway. a Capacity of SiHa and
Hela cells with either forced or depleted expression of TFF3 to penetrate to EHS Matrigel and Quantification analysis. b Western blot analysis was
used to assess the levels of CDH1, p-STAT3 andSTAT3 in cervical cancer cells with either forced or depleted expression of TFF3. c Effect of
CDH1specific siRNA on invasion of SIHA or HELA with depleted expression of TFF3. CDH1-specific siRNA increased the invasive percentage SiHa and Hela
cells with depleted expression of TFF3. d Western blot analysis was used to assess the levels of CDH1 in SiHa or Hela cells with forced expression
of TFF3on exposure to JSI-124 (0.2 μM) inhibitor and Effect of p-STAT3 on invasion of SiHa or Hela. Statistical significance was assessed by using
an unpaired two-tailed Student’s t test (P < 0.05 was considered as significant) using SPSS 18.0. Columns are the mean of triplicate experiments;
bars ±SD. **P < 0.001
the Rho123 substrate within cells increases, and vice
versa [24, 25]. Our results indicated that the amount
of Rho123 accumulation in SiHa-TFF3 and Hela-TFF3
cells was significantly higher than that in control cells
(47.06 ± 5.45% for control cells vs. 73.45 ± 8.01%
for TFF3-overexpressing cells in SiHa cell line;
59.85 ± 7.17% for control cells vs. 90.14 ± 9.45%
for TFF3-overexpressing cells in Hela; all P < 0.01;
Fig. 5c). In contrast, the amount of Rho123
accumulated in SiHa-siTFF3 and Hela-siTFF3 cells significantly
decreased compared with control cells (46.54 ± 6.32%
for control cells vs. 33.44 ± 3.53% for TTF3-knockdown
cells; 61.21 ± 6.84% for control cells vs. 41.43 ± 3.49%
for TTF3-knockdown cells; all P < 0.01; Fig. 5c).
However, western blot analysis showed that forced/deleted
expression of TFF3 didn’t alter the expression level
of P-gp. These results suggested that TFF3 increased
intracellular accumulation of Rho123 by inhibiting
P-gp pump function in SiHa and Hela cells and further
decreased the sensitivity to etoposide.
This study contributes to our understanding of the
molecular mechanism by which overexpression of TFF3
in human cervical cancers promotes tumor
progression. The present work first found that TFF3 was
overexpressed in cervical cancer cells and weakly expressed in
human non-tumor keratinocytes. Several studies
demonstrated that TFF3 overexpression strongly correlated
with poor prognosis in various tumors [26, 27], which
indicated that TFF3 could be a potentially superior
diagnostic marker or therapeutic target for cervical cancer.
In this study we demonstrated that TFF3 functionally
promoted the malignant progression of cervical cancer
cells. Forced expression of TFF3 promoted the
proliferation and invasion, and inhibited the apoptosis in SiHa
and Hela cells. Conversely, decreased expression of TFF3
inhibited the proliferation and invasion, and induced the
apoptosis in the two cell lines. Our data suggested that
TFF3 stimulated an invasive phenotype in cervical
cancer cells through STAT3 mediated repression of CDH1.
Furthermore, we found TFF3 decreased the sensitivity
of cervical cancer cells to etoposide by increasing P-gp
functional activity in the two cell lines. TFF3 silencing
increased the sensitivity of the two cell lines to etoposide
TFF3, behaved as an oncogene, promotes cancer cell
proliferation, survival, oncogenicity and invasion in
various cancers, such as mammary carcinoma, gastric
cancer and prostate carcinoma [7–9]. For the first time, we
found that TFF3 was overexpressed in cervical cancer
cells. Elevated expression level of TFF3 has also been
reported in the molecular apocrine subtype of estrogen
receptor-negative mammary carcinoma characterized
by the expression of AR, FOXA1 and a high frequency
of HER2 expression [12, 13]. In SiHa and Hela cells,
forced expression of TFF3 promoted cervical cancer
cells growth, proliferation and invasion. Overexpression
of TFF3 was caused changes in mRNA levels associated
with the cellular proliferation, apoptosis, migration,
invasion and clonogenic survival. Forced expression of TFF3
decreased mRNA expression of BAX, TIMP2, CDKN2A,
SERPINB5 and CDH1, but increased mRNA levels of
CDH2, VIM, TGFB1, TERT, SERPINE1, TWIST, KI67,
SURVIVIN, MMP2 and MMP3 which closely correlated
with increasing cell cycle progression, anti-apoptosis,
proliferation, metastasis and invasion of cervical cancer
cells [9, 10, 28, 29].
(See figure on next page.)
Fig. 5 TFF3 decreased sensitivity of cervical cancer cells to etoposide by increasing P-gp functional activity (a) Cultures were stained with the live/
dead stain. The cervical cancer cells were treated with 10 μmol/L etoposide for 24 h. Live cervical cancer cells with intact cell membranes stain
fluorescent green, whereas cells with damaged membranes and dead cells stain fluorescent red (×200). b Total cell number assays of cervical cancer
cells treated with etoposide (10 μmol/L) on 24 h. c The amount of Rho123 accumulated in cervical cancer cells was detected by flow cytometry to
reflect P-gp function and Quantification analysis of Rho 123 positive cells. d The effect of TFF3 on expression of drug resistance-related protein, P-gp.
Statistical significance was assessed by using an unpaired two-tailed Student’s t test (P < 0.05 was considered as significant) using SPSS 18.0. Columns
are the mean of triplicate experiments; bars ±SD. **P < 0.001
In the cervix cells, TFF3 expression was detected
significantly higher level in cervical cancer cells than in
human non-tumor keratinocytes. The results presented
here clearly demonstrated that TFF3 overexpression
accelerated cell cycle progression and a decrease in TFF3
levels slowed the progression of cells. In addition, TFF3
levels correlated with the proliferative potential of
cervical cancer cells as revealed by correlation between TFF3
and Ki67 levels in vivo. As an oncogene, TFF3 is
qualified with various functions that could impinge on
normal cell proliferation. It is known that TFF3 induces the
expressions of AR, FOXA1, HER2 and basic fibroblast
growth factor (bFGF) in vitro in various cancers such as
breast cancer and melanoma cells [12, 13, 30]. Our study
revealed that overexpression of TFF3 had no effect on
apoptosis in SiHa and Hela cells. But siRNA-mediated
depletion of TFF3 induced the apoptosis of cervical
cancer cells by decreasing anti-apoptotic protein, Bcl-2 and
increasing pro-apoptotic protein, Bax. The ratio of
antiapoptotic and pro-apoptotic members within the Bcl-2
family plays an important role to determine cell fate
It is well known that TFF3 is associated with invasion
and metastasis which plays very important roles in
progression of tumors. It was first discovered TFF3 might
regulate migration via a Twist-dependent pathway in
gastric cells , which was an indispensable step in the
process of cell invasion. TFF3 participated in cancer
invasion metastasis in breast cancer through repression of
CDH1 mediated by STAT3 . CDH1, as a tumor
suppressor glyco-protein, is one of the major constituents of
cell adhesion complexes and mediates
calcium-dependent cellular interactions in epithelial cells, which plays a
key role in the establishment of adherent type junctions
[34, 35]. Loss of CDH1 expression, or CDH1
dysfunction, contributes to the loss of cell–cell interaction which
stimulates cancer cells to gain an invasive cell phenotype
and metastasis [36–38]. By two-dimensional Matrigel
Transwell analysis we found TFF3 was critical for cervical
cancers to involve invasion. Moreover, we observed that
TFF3 repressed the expression of CDH1 to promote cell
invasion in cervical cancer cells. Western blot analysis
showed that siRNA against TFF3 increased the
expression of CDH1 and decreased phosphorylation of STAT3
which up-regulated the expression of CDH1.
Furthermore, up-regulated CDH1 via overexpression of TFF3
was significantly down-regulated by virtue of inhibitor
of p-STAT3, JSI-124, which was similar to those reported
by Pandey . Our results suggested that TFF3
stimulates invasion of cervical cancer cells probably by, at least
partially, activating the STAT3/CDH1 signaling pathway.
CDH1 is a downstream protein of TFF3 and may be a key
modulator of TFF3-mediated cervical cancer invasion.
Surgery combined with chemotherapy or
radiotherapy is still the optimal treatment for cervical cancer
while MDR causes the cervical cancer cells to be
resistant to chemotherapeutic drugs resulting in
chemotherapy failure [39–41]. Previously study showed that
up-regulation of TFF3 after rectal cancer
chemo-radiotherapy is an adverse prognostic factor. The
physiological role of TFF3 in restoring the mucosa during
neo-adjuvant chemotherapy could be interfering with
treatment efficacy by increase neo-adjuvant
chemotherapy resistance . In this study, we showed that
TFF3 decreased the sensitivity of cervical cancer cells
to etoposide by increasing P-gp functional activity and
had no effect on the expression of P-gp. Drug resistance
to chemotherapy is mediated mainly by the
overexpression of P-gp which is a phosphorylated
transmembrane glycoprotein pump with ATP enzyme activity
encoded by mdr-1 gene [42, 43]. P-gp exerts an impact
on drug distribution by pumping a substantial amount
of compounds from intracellular to extracellular sites,
especially hydrated cation compounds. In addition,
overexpression of TFF3 decreased the sensitivity of
cancer cells to chemotherapy by mediating Bcl-2 [44,
In this study our evidence presented that TFF3 is
expressed between human non-tumor keratinocyte line
and cervical cancer cell lines distinctively, inducing
proliferative activity and malignant progression of cervical
cancer patients, strongly supporting a role for this gene
in cervical carcinogenesis.
Additional file 1: Table S1. RT-PCR and semi-quantitative RT-PCR.
AR: androgen receptor; BAX: BCL2-associated X protein; CDH1: cadherin 1;
CDH2: cadherin 2; CDKN2A: cyclin-dependent kinase inhibitor 2A; DMEM:
Dulbecco’s modified Eagle’s medium; EMT: epithelial-to-mesenchymal
transition; FBS: fetal bovine serum; FOXA1: forkhead box A1; HER2: human
epidermal growth factor receptor-type 2; JAK: janus kinase; KI67: KI67 Antigen;
MAPK: mitogen activated protein kinase-like protein; MDR: multidrug resistant;
MMP2: matrix metallopeptidase 2; MMP3: matrix metallopeptidase 3; PI3K:
PI3K phosphatidylinositol 3-kinase; P-gp: P-glycoprotein; SERPINE1: serpin
peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1),
member 1; SERPINB5: serpin peptidase inhibitor, clade B (ovalbumin), member
5; STAT3: signal transducer and activator of transcription 3; TERT: telomerase
reverse transcriptase; TFF3: trefoil factor 3; TIMP2: TIMP metallopeptidase
inhibitor 2; TGFB1: transforming growth factor beta 1; VIM: vimentin.
ZHY, DDC and YMW designed and performed experiments and analyzed data.
DDC, XJC and HKY performed experiments and analyzed data. ZHY, XJC and
CSC reviewed all data, prepared the figures, and wrote the manuscript. All
authors read and approved the final manuscript.
This study was supported by research funds from the Guangzhou General
Science and Technology Project of Health and Family Planning (20161A011010).
Availability of data and materials
All relevant data are within the paper and its Additional file.
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