Invest Clin 62(3): 236 - 246, 2021 https://doi.org/10.22209/IC.v62n3a05
Corresponding Author: Qi Xing, Department of Neurological Rehabilitation, Hubei Provincial Maternal and Child
Health Hospital, Tongji Medical College, Huazhong University of Science and Technology, No.745 Wuluo Road
Hongshan District Wuhan, Hubei, China. Tel: 86018963942319. Email: 56052706@qq.com
Hepatocyte Nuclear Factor -1α stimulates
cervical cancer cells to migrate and invade
through regulating pyruvate kinase L/R.
Xiao-Ling Tao
1
, Wei-Chang Yu
1
, De-Jun Chen
1
, Li-Ming Wang
1
, Lu Liu
1
and Qi Xing
2
1
Department of Gynecology, Hubei Provincial Maternal and Child Health Hospital,
Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
China.
2
Department of Neurological Rehabilitation, Hubei Provincial Maternal and Child
Health Hospital, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, China.
Key words: cervical cancer; hepatocyte nuclear factor -1α (HNF-1α); pyruvate kinase
L/R(PKLR).
Abstract. This study was aimed to analyze the role of hepatocyte nuclear
factor -1α (HNF-1α) in regulating migrative and invasive potentials in cervical
cancer via the involvement of pyruvate kinase L/R (PKLR). The expression of
HNF-1α and PKLR in cervical cancer tissues classified by tumor size and FIGO
(Federation International of Gynecology and Obstetrics) stage were detected
by qRT-PCR. The expression correlation between HNF-1α and PKLR in cervi-
cal cancer tissues was analyzed by Pearson correlation test. After intervening
HNF-1α and PKLR levels in SiHa and Hela cells, their migratory and invasive
abilities were examined by the Transwell assay. HNF-1α was upregulated in cer-
vical cancer tissues, particularly those with large tumor size or advanced FIGO
stage. PKLR was highly expressed in cervical cancer tissues as well, presenting
a positive correlation with the HNF-1α level. Knockdown of HNF-1α attenuated
migratory and invasive abilities in SiHa cells, whereas overexpression of HNF-1α
enhanced migratory and invasive abilities in SiHa cells. PKLR was able to abol-
ish the regulatory effects of HNF-1α on cervical cancer metastasis. HNF-1α and
PKLR synergistically promote cervical cancer to migrate and invade.
HNF-1α stimulates migration and invasion in cervical cancer 237
Vol. 62(3): 236 - 246, 2021
El factor nuclear del hepatocito-1α estimula las células de
cáncer cervical para migrar e invadir a través de la regulación
de la piruvato cinasa L/R.
Invest Clin 2021; 62 (3): 236-246
Palabras clave: cáncer de cuello uterino; factor nuclear del hepatocito -1α (HNF-1α);
piruvato quinasa L/R (PKLR).
Resumen. Este estudio tuvo como objetivo analizar el papel del factor
nuclear del hepatocito -1α (HNF-1α) en la regulación de los potenciales migra-
torios e invasivos en el cáncer de cuello uterino a través de la participación de
la piruvato quinasa L/R (PKLR). Se detectó mediante qRT-PCR, la expresión de
HNF-1α y PKLR en tejidos de cáncer de cuello uterino, clasificados de acuerdo
al tamaño del tumor y su estadio FIGO (Federación Internacional de Ginecolo-
gía y Obstetricia). La correlación de expresión entre HNF-1α y PKLR en tejidos
de cáncer de cuello uterino se analizó mediante la prueba de correlación de
Pearson. Después de intervenir los niveles de HNF-1α y PKLR en las células SiHa
y Hela, se examinaron las capacidades migratorias e invasivas mediante el en-
sayo Transwell. HNF-1α se reguló positivamente en tejidos de cáncer de cuello
uterino, en particular aquellos con tumores de gran tamaño o estadio FIGO
avanzado. PKLR también se expresó en gran medida en los tejidos del cáncer de
cuello uterino, presentando una correlación positiva con el nivel de HNF-1α. La
eliminación de HNF-1α atenuó las capacidades migratorias e invasivas en las cé-
lulas SiHa, mientras que la sobreexpresión de HNF-1α aumentó las capacidades
migratorias e invasivas en las células SiHa. La PKLR pudo abolir los efectos re-
guladores del HNF-1α sobre la metástasis del cáncer de cuello uterino. HNF-1α
y PKLR promueven sinérgicamente la invasión y migración del cáncer de cuello.
Received: 02-04-2021 Accepted: 03-06-2021
INTRODUCTION
Cervical cancer is a common malignancy
of the female reproductive system (1). It is re-
ported that in 2021, the diagnostic rate and
mortality rate of cervical cancer in our coun-
try was 16.25/100,000, and 5.08/100,000,
respectively (2). It is necessary to deeply un-
derstand the molecular mechanisms of cer-
vical cancer. Seeking for effective cervical
cancer biomarkers is conductive to stop the
malignant phenotypes of cancer cells.
Hepatocyte Nuclear Factors (HNFs)
are transcription factors predominately ex-
pressed in the liver, including HNF-1, 3, 4, 6,
CCAAT / enhancer binding-proteins and D-
binding proteins. HNF-1 contains two mem-
bers, that is, HNF-1α (also known as LFB1/
TCF1) and HNF-1β. HNF-1α is a transcrip-
tion factor containing a variant homology
domain. It is located on human chromosome
12q24.2, and expressed in liver, kidney, intes-
tinal and pancreatic tissues. HNF-1α is able
to regulate expressions of multiple tissue-
specific genes (3). A previous study has iden-
tified the close relationship between HNF-1α
and hepatocellular carcinoma (HCC) pro-
gression, and HNF-1α is capable of maintain-
ing hepatocyte differentiation phenotypes
(3,4). In addition, HNF-1α mutation causes
238 Tao et al.
Investigación Clínica 62(3): 2021
maturity onset diabetes of the young type
3 (MODY3) (5). Some low-frequency muta-
tions in HNF-1α (e.g. a single rare missense
mutation) are associated with type 2 diabe-
tes (6,7). The potential influence of HNF-1α
on cervical cancer is rarely reported.
Pyruvate Kinase L/R (PKLR) is located
on human chromosome 1q22, encoding py-
ruvate kinases (PKs). PKs are involved in an-
aerobic glycolysis and provide 50% of ATP to
mature red blood cells. Dysfunctional PKLR
can lead to PKs deficiency (PKD) and ATP de-
ficiency, shortening the lifespan of red blood
cells (8). The Warburg effect (aerobic glycol-
ysis) is a well-defined metabolic change that
is relevant to cancerous phenotypes, includ-
ing the accelerated proliferative, invasive
and migratory potentials (9). Nie et al. (10)
pointed out that mineralocorticoid recep-
tors (MRs) are able to alleviate the Warburg
effect and cancer progression in HCC by
targeting the miR-338-3p/PKLR axis. There-
fore, PKLR is considered as a key regulator
of glycolytic reprogramming and tumor cell
functions. A recent research showed that
HNF-1α drives the growth and anti-apoptosis
capacity in pancreatic cancer via its target
gene PKLR (11). This study aims to eluci-
date the role of HNF-1α and PKLR in the pro-
gression of cervical cancer, and to provide a
novel guidance in clinical treatment.
MATERIALS AND METHODS
Collection of pathological tissues
A total of 50 cervical cancer tissues,
confirmed by pathological examination and
resected by surgical procedures, were col-
lected. During the same period, 50 adjacent
normal tissues that were 2 cm away from the
cancer focus were collected as well. Tissues
were immediately frozen in liquid nitrogen
for RNA extraction. All patients did not re-
ceive neoadjuvant chemotherapy or radio-
therapy before operation, and there was no
surgical contraindication. The study was ap-
proved by the Ethics Committee of Hubei
Provincial Maternal and Child Health Hos-
pital, and informed consent was signed by
patients and their families before operation.
Cell culture
HcerEpic, Hela and SiHa cell lines were
provided by Fudan University (Shanghai, Chi-
na). Cells were cultivated in Dulbecco’s modi-
fied eagle medium (DMEM) (Gibco, Rock-
ville, MD, USA) containing 10% fetal bovine
serum (FBS) (Gibco, Rockville, MD, USA) in
a humidified incubator with 5% CO
2
at 37°C.
For cell culture passages, cells were isolated
and purified by pancreatin digestion and then
were cultured and subculfured in DMEM with
20 % fetal bovine serum. Cells in the logarith-
mic growth phase were used for experiments.
Cell transfection
HNF-1α siRNAs were constructed by Am-
bion (Austin, TX, USA) based on the HNF-1α
sequences in GeneBank (Accession No. NM
000545). Primer sequences were as follows:
siHNF-1α-#1 forward primer, 5′-CCGGT-
GCTAGTGGAGGAGTGCAATTTCAAGAGA-
ATTGCACTCCTCCACTAGCTTTTTG-3′ and
reverse primer, 5′-AATTCAAAAAGCTAGT
GGAGGAGTGCAATTCTCTTGAAATTG-
CACTCCTCCACTAGCA-3′; siHNF-1α-#2
forward primer, 5′-CCGGTGCAGAAGT
ACCCTCAAGCATTCAAGAGATGCTT-
GAGGG TACTTCTGCTTTTTG-3′ and re-
verse primer, 5′-AATTCAAAAAGCAGAAGTAC
CCTCAAGCATCTCTTGAATGCTTGAGGG-
TACTTCTGCA-3′. Cells were prepared to
suspension after 0.25% trypsin digestion,
and inoculated in the 6-well plate with 1.0 ×
10
5
cells/well. Once cell confluence reached
about 70%, transfection was conducted using
Lipofectamine
TM
2000 (Invitrogen, Carlsbad,
CA, USA). Fresh medium was replaced at 6-8
h, and medium containing 2 μg/mL puromy-
cin was applied at 48 h. After 72 h cell cul-
ture, cells were passaged to a new 6-well plate
and cultivated for 1-2 weeks. Visible colonies
were picked up and inoculated in a 96-well
plate for extended culture. Their growth was
regularly observed and passaged to 6-well
plates and culture bottles two days later.
HNF-1α stimulates migration and invasion in cervical cancer 239
Vol. 62(3): 236 - 246, 2021
Quantitative real-time polymerase chain
reaction (qRT-PCR)
Total RNA isolated using TRIzol (Invi-
trogen, Carlsbad, CA, USA) was reversely
transcribed to complementary deoxyribose
nucleic acid (cDNA) using the First Chain
cDNA Synthesis Kit in a system contain-
ing 1 μL of Oligo dT Primer, 1 μL of dNTP
Mixture, 5 μL of RNA template and 3 μL
of ddH
2
O. The system was incubated at
65°C for 5 min and immediately frozen on
ice. Reverse transcription solution was pre-
pared as follows: 4 μL of 5×Prime Script II
Buffer, 0.5 μL of RNase Inhibitor, 1 μL of
Prime Script II RTase and 4.5 μL of RNase
free ddH
2
O. The reaction conditions were
45 min at 42℃, 5 min at 95℃ and cooling
on ice. PCR primers were: HNF-1α forward
primer: 5′-TCTACAGCCACAAGCCCGAG-3′
and reverse primer: 5′-GAGGTGAAGACCT-
GCTTGGT-3′; PKLR forward primer:
5′-TGGGAAAACTGGGTGGGATGGATG-3′
and reverse primer: 5′-GAAGGAAGCAGCC-
GGGGATTTGAC-3′; β-actin forward primer:
5′-TTGGCCTTAGGGTTCAGAGGGG-3′, and
reverse primer: 5′-CGTGGGCCGCCCTAG-
GCACCA-3′. PCR system in a total of 25
μL was then prepared, including 12.5 μL
of SYBR Fast qPCR Mix (2×), 1 μL of PCR
Forward Primer (10 μmol/L), 1 μL of PCR
Reverse Primer (10 μmol/L), 0.5 μL of ROX
Reference Dye (50×), 1 μL of cDNA and 8 μL
of ddH
2
O. The system was amplified at 95°C
for 30 s, 40 cycles at 95°C for 5 s and 60°C
for 32 s, followed by 95°C for 15 s, 60°C for
1 min, 95°C for 15 s and 60°C for 15 s. The
expression level was calculated by 2
-ΔΔCt
.
Western blot
Cells were digested in 0.25% trypsin,
washed in phosphate buffered saline (PBS)
for three times and lysed in radioimmu-
noprecipitation assay (RIPA) (Beyotime,
Shanghai, China). The isolated protein
samples were separated by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) and loaded on polyvinylidene
fluoride (PVDF) membranes (Millipore, Bil-
lerica, MA, USA). After blockage of non-spe-
cific antigens in 5% skim milk for 1 h, mem-
branes were reacted with primary antibodies
(1: 1000) at 4°C overnight, and horseradish
peroxidase (HRP)-labeled sheep anti-mouse
antibody (1:5000) for 1 h. Band exposure
and grey value analyses were finally conduct-
ed by Image-Pro Plus.
Transwell assay
Until cells were cultured to 75% con-
fluence, serum-free medium was replaced.
100 μL of serum-free suspension (1.0 × 10
5
cells/mL) and 600 μL of serum-containing
medium were applied to the top and bottom
Transwell chamber, respectively, and cultured
overnight. Cells in the bottom were subjected
to methanol fixation for 15 min, and crystal
violet staining for 20 min. Migratory cells
were counted in five randomly selected fields
per sample. Transwell invasion assay was con-
ducted in chambers pre-coated with 50 μL of
Matrigel diluted in serum-free medium.
Statistical analysis
Data processing was conducted by Sta-
tistical Product and Service Solutions (SPSS)
20.0 (IBM, Armonk, NY, USA). Linear relation-
ship of genes was analyzed by Pearson corre-
lation test. Differences between groups was
compared by the Student’s t-test. Compari-
son between multiple groups was done using
One-way ANOVA test followed by Post Hoc Test
(Least Significant Difference). Percentage (%)
was used to express the enumeration data and
chi-square test was used for data analysis. A sig-
nificant difference was set at p<0.05.
RESULTS
Correlation between HNF-1α and clinical
features in cervical cancer
Based on the median level of HNF-1α ex-
pression in recruited cervical cancer patients,
they were classified into high HNF-1α level
group (n=25) and low HNF-1α level group
(n=25), respectively. No significant differ-
ences in age and histology were observed be-
240 Tao et al.
Investigación Clínica 62(3): 2021
tween groups (p>0.05). Notably, tumor size,
FIGO stage and lymphatic metastasis inci-
dence were significantly different (p<0.05)
(Table I). It is indicated that HNF-1α was
closely linked to tumor size, FIGO grade and
lymphatic metastasis in cervical cancer pa-
tients, and might be a potential biomarker.
Upregulation of HNF-1α in cervical cancer
Compared with adjacent normal tissues,
HNF-1α was upregulated in cervical cancer
ones (Fig. 1A). In addition, collected cervical
cancer tissues were classified into two groups
based on the tumor size (4 cm as the cut-off
value). Higher level of HNF-1α was detected in
cervical cancer tissues with ≥ 4 cm in tumor
size compared with those < 4 cm (Fig. 1B).
In particular, we detected higher abundance
of HNF-1α in FIGO stage III-IV cervical cancer
patients than those FIGO stage I-II patients
(Fig. 1C). It is suggested that HNF-1α may
function as an oncogene driving the progres-
sion of cervical cancer.
HNF-1α overexpression and knockdown
models
HNF-1α level was first detected in cervi-
cal cancer cell lines (Fig. 2A). Based on the
differential level of HNF-1α, we constructed
HNF-1α knockdown and overexpression mod-
els in SiHa and Hela cells, respectively. Trans-
fection of either si-HNF-1α 1# or si-HNF-1α
2# could effectively decrease HNF-1α level in
SiHa cells (Fig. 2B, 2C). Besides, HNF-1α level
was markedly upregulated in Hela cells trans-
fected with pcDNA-HNF-1α (Fig. 2D, 2E).
HNF-1α promoted cervical cancer cells
to migrate and invade
Cell migration and invasion was a vital
process of tumor metastasis. Transwell as-
say was conducted to assess migratory and
TABLE I
CORRELATION BETWEEN HNF-1Α AND CLINICAL FEATURES IN CERVICAL
CANCER PATIENTS (N=50).
Clinico-pathologic
features
Number
of cases
HNF-1α expression
p
Low (n=25) High (n=25)
Age (years)
<50 31 16 15 0.7708
≥50 19 9 10
Histology
Squamous 23 12 11 0.7766
Adenocarcinoma 27 13 14
Tumor size
<4CM 26 19 7 0.0007*
≥4CM 24 6 18
FIGO stage
I~II 22 16 6 0.0044*
III~IV 28 9 19
Lymph node metastasis
No 26 17 9 0.0235*
Ye s 24 8 16
The high and low expression was classified based on the median level of HNF-1α expression in recruited cervical
cancer patients, *p<0.05.
HNF-1α stimulates migration and invasion in cervical cancer 241
Vol. 62(3): 236 - 246, 2021
Fig. 1. Upregulation of HNF-1α in cervical cancer tissues. (A) Higher level of HNF-1α was detected in cervical
cancer tissues (Tumor) (n=50) than adjacent normal ones (Peritumor) (n=50); (B) Higher level of
HNF-1α was detected in cervical cancer tissues larger than 4 cm in tumor size (≥ 4 cm) than those
smaller than 4 cm (<4 cm); (C) Higher level of HNF-1α was detected in FIGO grade III-IV(III-IV) cer-
vical cancer patients(III-IV) than those FIGO grade I-II patients(I-II). *p<0.05.
Fig. 2. HNF-1α overexpression and knockdown models. (A) HNF-1α levels in HcerEpic, Hela and SiHa cells
detected by qRT-PCR. The comparison was conducted between HcerEpic and Hela, and another com-
parison was conducted between HcerEpic and SiHa; (B) The mRNA level of HNF-1α was downregula-
ted by transfection of either si-HNF-1α 1# or si-HNF-1α 2#. The comparison was conducted between
si-NC and si-HNF-1α 1#, and another comparison was conducted between si-NC and si-HNF-1α 2#
(C) The protein level of HNF-1α was downregulated by transfection of either si-HNF-1α 1# or si-HNF-
1α 2#; The comparison was conducted between si-NC and si-HNF-1α 1#, and another comparison
was conducted between si-NC and si-HNF-1α 2# (D) The mRNA level of HNF-1α was upregulated by
transfection of pcDNA-HNF-1α in Hela cells; The comparison was conducted between pcDNA-NC and
pcDNA-HNF-1α.(E) The protein level of HNF-1α was upregulated by transfection of pcDNA-HNF-1α in
Hela cells. The comparison was conducted between pcDNA-NC and pcDNA-HNF-1α.*p<0.05.
242 Tao et al.
Investigación Clínica 62(3): 2021
invasive abilities. The number of migrated
and invading cells assessed the ability of mi-
gration and invasion. Knockdown of HNF-1α
markedly weakened the migratory and inva-
sive abilities in SiHa cells (Fig. 3A). On the
contrary, they were enhanced in Hela cells
overexpressing HNF-1α (Fig. 3B).
PKLR abolished the regulatory effects
of HNF-1α on cervical cancer metastasis
The expression of PKLR was detected by
qRT-PCR and was found upregulated in cervi-
cal cancer tissues compared with the normal
ones (Fig. 4A). Pearson correlation test iden-
tified a positive linear correlation between
HNF-1α and PKLR in cervical cancer tissues
(r
2
=0.4141, Fig. 4B). Interestingly, overex-
pression of PKLR could reverse the attenu-
ated migratory and invasive abilities in SiHa
cells with HNF-1α knockdown (Fig. 4C). It
is suggested that PKLR abolished the regu-
latory effects of HNF-1α on cervical cancer
metastasis, which might be a vital process
during cervical cancer metastasis.
Fig. 3. HNF-1α promoted cervical cancer cells to migrate and invade. (A) Knockdown of HNF-1α weakened
migration and invasion in SiHa cells; The comparison was conducted between si-NC and si-HNF-1α 1#,
and another comparison was conducted between si-NC and si-HNF-1α 2# (B) Overexpression of HNF-
1α promoted migration and invasion in Hela cells. The comparison was conducted between pcDNA-NC
and pcDNA-HNF-1α. *p<0.05.
HNF-1α stimulates migration and invasion in cervical cancer 243
Vol. 62(3): 236 - 246, 2021
DISCUSSION
Cervical cancer is one of the com-
mon malignant tumors in gynecology, and
its morbidity and mortality have remained
high, which seriously threatens the physi-
cal and mental health of women worldwide
(12). With the continuous advancement of
medical technologies, the diagnosis and
treatment strategies of cervical cancer
have greatly improved (13). However, many
patients are diagnosed in advanced stages
with distant metastases, which leads to an
unsatisfactory prognosis (13). Therefore,
it is of great significance to explore the
mechanism underlying the occurrence and
progression of cervical cancer, and to find
targets for the diagnosis and treatment.
Dysfunctional tumor-related genes
can affect tumorigenesis and tumor pro-
gression (14). Transcription factors regu-
late the initiation, extension, and termi-
nation of gene transcription (15). As a
transcription factor, HNF-1α is not only
involved in regulating the metabolism and
expressions of immune genes (16,17), but
Fig. 4. PKLR abolished the regulatory effects of HNF-1α on cervical cancer metastasis. (A) Higher level of
PKLR was detected in cervical cancer tissues (Tumor) (n=50) than adjacent normal ones (Peritumor)
(n=50); (B) Pearson correlation test identified a positive linear correlation between HNF-1α and
PKLR; (C) Overexpression of PKLR abolished the inhibited migration and invasion in SiHa cells with
HNF-1α knockdown. The comparison was conducted between control and si-HNF-1α 1#, and another
comparison was conducted between si-HNF-1α 1# and si-HNF-1α 1#+pcDNA-HNF-1α. *p<0.05.
244 Tao et al.
Investigación Clínica 62(3): 2021
also in the carcinogenesis (18,19). It is re-
ported that HNF-1α is the characteristic
of malignant gliomas (20) and childhood
T-cell acute lymphoblastic leukemia (21).
Hellerbrand et al. (22) found that HNF-1α
is downregulated in HCC cells and tissues.
Overexpression of HNF-1α induces the ex-
pression of tumor suppressor MIA2, thus
alleviating the malignant growth of HCC.
Our findings showed that HNF-1α was up-
regulated in cervical cancer tissues, and
was closely linked to tumor size, FIGO
grade and lymphatic metastasis. In cervi-
cal cancer cells, HNF-1α was able to stimu-
late the migratory and invasive abilities,
further supporting its carcinogenic role.
As the target gene of HNF-1α, PKLR
catalyzes the transphosphorylation of phos-
phoenolpyruvate to pyruvate and ATP, a
rate-limiting step in glycolysis (23). The
role of PKLR in tumor progression has been
previously reported. PKLR promotes breast
cancer progression by regulating glycolytic
reprogramming (24). By inducing gluta-
thione synthesis, PKLR triggers liver colo-
nization of colorectal cancer (25). In our
experiment, PKLR and HNF-1α level was
dysregulated in cervical cancer tissues. No-
tably, PKLR was able to abolish the regu-
latory effects of HNF-1α on cervical cancer
metastasis, which played a vital role in the
malignant progression of cervical cancer.
This result may provide a new intervention
target for the clinical treatment of cervical
cancer metastasis. We can conclude that
HNF-1α and PKLR synergistically promote
cervical cancer to migrate and invade.
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