Effect of Different Concentrations of Leukemia Inhibitory Factor on Gene Expression of Vascular Endothelial Growth Factor-A in Trophoblast Tumor Cell Line
1
Department of Immunology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
2
Department of Immunology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran;Fertility, Infertility, and Perinatology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
3
Department of Immunology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran;Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
Background Several studies have shown that leukemia inhibitory factor (LIF) is one of the most important cytokines participating in the process of embryo implantation and pregnancy, while, the role of this factor on vascular endothelial factor-A (VEGF-A), as one of the most important angiogenic factor, has not been fully investigated yet. The aim of this study was to evaluate the effect of LIF on gene expression of VEGF in the choriocarcinoma cells (JEG-3). Materials and Methods In this experimental study, JEG-3 choriocarcinoma cells were treated with different concentrations of LIF (1, 10, and 50 ng) for 6, 12, 24, 48 and 72 hours. Expression of VEGF was analyzed by real-time PCR. Delta CTs were subjected to one-way analysis of variance (ANOVA) and a post hoc Tukey’s test by SPSS version 25.0 software for data analyzing. Results In the stimulated cells, different concentrations of LIF caused significant decrease of VEGF gene expression (P < 0.05) at 12, 24 and 48 hours. In contrast, it was increased after 72 hours (P < 0.001). Analysis of data after 6 hours also showed that level of VEGF gene expression was significantly decreased by increasing LIF concentration (P < 0.001). Conclusion Expression level of VEGF gene was decreased in trophoblast cells (except after 72 hours) under the effectof different concentrations of LIF in a time-dependent manner. So, this study showed that further studies are needed to determine the effect of LIF on other angiogenic factors in trophoblast cells.
Leukemia inhibitory factor (LIF) is a glycoprotein cytokine with a molecular weight of 38-67 kDa. That is a
member of the interleukin 6 family. LIF receptor is a heterodimer composed of two chains, gp130 and leukemia
inhibitory factor receptor-β (LIFR-β) expressing on the
surface of trophoblast cells (1, 2). LIF induce tyrosine
phosphorylation in signal transducers and transcription
factors of several trophoblast cell types, like choriocarcinoma cell line (JEG-3) (3, 4). Phosphorylation and signal
transduction lead to migration, invasion, stimulation or
suppression of various categories of genes in trophoblast
cells (5, 6). Janus kinase 1 (JAK-1) and Signal transducer
and activator of transcription 3 (STAT-3), play important
roles in the signal transduction factors and activation of
transcription in the LIF signaling (7, 8). VEGF is a homodimer glycoprotein which can stimulate angiogenesis
and vasculogenesis by two types of its receptors like Fmslike tyrosine kinase 1 (Flt1) and kinase insert domain
receptor (KDR) (9, 10). VEGF has many roles in early
pregnancy, such as oocyte maturation and development,
trophoblast proliferation, placenta angiogenesis, embryo
implantation, maternal blood vessel growth and development of the embryonic blood vessels (11). Formation of
the placenta in uterus depends on differentiation of extravillous cytotrophoblast (EVT) for invasion to the uterine stroma and forming endovascular trophoblast (12, 13).
Incorrect differentiation of EVT cells leads to disruption
of spiral artery remodeling, and this impairment in spiral
artery remodeling can lead to preeclampsia and defective
development of the fetus (13). Trophoblast invasion is a
localized and temporary process. That is the main factor
in the regulation of implantation and supply of oxygen
to the fetus. By VEGF gene inactivation, invasion and
migration of trophoblast cells are reduced (14). VEGF-A
is one of the main factors of EVT differentiation to the endovascular trophoblast (15). Anti-angiogenic factors
that reduce the amount of VEGF-A is one of the factors
inhibiting formation of spiral arteries, which eventually
associated with the creation of preeclampsia (16).
VEGFA is one of the factors encoded by VEGF gene. Studies
have shown that among all growth factors encoded by
this gene, VEGF-A is the most potent type in stimulating
angiogenesis (17). During formation of placenta, EVTs,
involving in vascular reconstruction, acquire the features
associated with epithelial cells, following the production
of VEGF and its receptor expression on the surface (12,
18). These cells migrate to decidua, followed by replacement of the endothelial cells in the spiral arteries to form
spiral arteries (19). In this study, a choriocarcinoma cell
line JEG-3 (derived from fetal trophoblast tumor) was
used as EVTs (20). This cell line has many biological and
biochemical features of EVTs (Cells lining the blood vessels of villus in the placenta) (21). This cell line is able to
produce progesterone, hCG, steroids and other hormones
in the placenta (22). In this study we aimed to evaluate
VEGF gene expression levels in trophoblast tumor cell
line (JEG-3) at different times, while these cells were
treated by different concentrations of LIF.
Materials and Methods
This experimental study approved by Ethics Committee
of Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, Iran (Ethics code: IR.AJUMS.REC.1395.577).
Cell culture and treatment
JEG-3 choriocarcinoma cells were purchased from the
Pasteur Institute of Iran (Tehran, Iran). These cells were
maintained in Dulbecco’s modified Eagle’s medium-F12
(DMEM-F12; GIBCO, Ireland) supplemented with 10%
heat-inactivated fetal bovine serum (FBS; GIBCO, Ireland) along with penicillin (BioIdea, Iran;100 units/ml)
and streptomycin (BioIdea; 100μg/ml). All JEG-3 cultures were commenced at 106 cells/175-cm2 flask and
maintained under standardized conditions (37˚C, 5% CO2,
humidified atmosphere). The cells were trypsinized twice
a week when confluence was estimated at over 75%. For
all assays, JEG-3 cells were adjusted to 105 cells/ml. The
cells (105cell/ml) were seeded in six-well plates, following the resuspension in complete growth media. Before
adding the stimuli, the cells were starved for 2 hours in
medium without FBS. The cells were cultured per well
in the presence and absence of different concentrations (1
ng/ml, 10 ng/ml, 50 ng/ml) (23, 24) of human LIF (Sigma-Aldrich, Germany), while non-stimulated cells were
included as controls. Treated and non-treated cells were
incubated for 3, 6, 12, 24, 48 and 72 hours at 37°C with
5% CO2. The cell culture supernatants were then collected
by aspiration and centrifugation at 1000 g for 5 minutes
and they were stored at -70°C until cytokine analysis.
JEG-3 cells were harvested and kept at -70°C until total
ribonucleic acid (RNA) extraction.
RNA was isolated using TRI Reagent (SinaClonCo.,
Iran). According to the manufacturer’s protocol, and the
purity of extracted RNA was determined by the A260/
A280 ratio (A260/A280 ratio was 1.8). 50-100 ng RNA
was reverse transcribed using cDNA synthesis kit (SinaClonCo.)
and relative changes in VEGF mRNA level
was quantified by real-time reverse transcription PCR
(RT-PCR). Expression level of VEGF was determined
by quantitative RT-PCR (qRT-PCR) using SYBR Green
® Premix Ex Taq (Takara, Japan) dye detection method
on ABI StepOne PCR instrument (Applied Biosystems,
USA), compared to GAPDH as an internal control. Initial
denaturation at 95°C for 10 minutes, 40 cycles of annealing at 95°C for 15 seconds and extension at 68°C for 60
seconds. Rest 2009 and Excel software were used for the
analysis of gene expression ratio. Gene-specific primers
for VEGF and GAPDH are summarized in Table1. The
fold change for target genes normalized by internal control was determined by the formula 2-ΔΔCt. All reactions
were run in duplicate.
Statistical analysis
All of the experiments were repeated in triplicates and
data were demonstrated as means ± standard error (SE).
Statistical software SPSS 25.0 and Graphpad Prism 8.0.1
were used for data analysis. Delta CTs were subjected to
one-way ANOVA and a post hoc Tukey’s test, while the
non-parametric Kruskal-Wallis test was used to compare
the results of different experimental days. P values lower
than 0.05 were considered statistically significant.
Results
Effects of different concentrations of LIF on VEGF
gene expression level This study evaluated the effects
of different concentrations of LIF on VEGF gene expression in different time periods, compared to untreated cells. The results are described (Fig .1, 2) in more
details.
Gene specific primers used for RT-PCR
Primer (accession)
Sequence (5'-3')
Tm
Amplicon size
VEGF (NM_001287044.1)
F: AGGAGGAGGGCAGAATCATCA
60
76 bp
R: CTCGATTGGATGGCAGTAGCT
GAPDH (NM_002046.5)
F: TGGGCTACACTGAGCACCAG
60
72 bp
R: CAGCGTCAAAGGTGGAGGAG
VEGF gene expression level at different time points, under treatment with different concentrations of LIF. The effect of different concentrations of
LIF (1, 10 and 50 ng) on VEGF gene expression after A. 6 hours; B. 12 hours; C. 24 hours; D. 48 hours; and E. 72 hours. Cells that did not treated by LIF were
considered at any time as control, and the VEGF gene expression was measured in treated cells relative to these untreated cells. *; P<0.05, **; P≤0.01, and
***; P≤0.001
Comparing total VEGF gene expression at different time (6, 12, 24,
48 and 72 hours) under the treatment of different concentrations of LIF.
*; P<0.05, **; P≤0.01, ***; P≤0.001) , and h; Hours.
Six hours treatment
An analysis of 6 hours data showed that by increasing
LIF concentration, level of VEGF gene expression was
decreased. In this time point, there is a significant difference (P<0.001) between the rate of VEGF gene expression in comparison with each other at different concentrations and control (Fig .1A).
Twelve hours treatment
After 12 hours, there was a significant reduction in the
VEGF gene expression in all three concentrations of LIF
treatment than control (P<0.001). The lowest VEGF gene
expression level was observed at 1 ng concentration of
LIF. The results of 10 ng concentration of LIF were almost similar to the 1 ng (the difference between 1 and 10
ng was not significant). At 50 ng concentration of LIF,
VEGF expression level was higher than the both concentrations of 1 and 10 ng (P<0.05, Fig .1B).
Twenty-four hours treatment
Twenty-four hours after cells treatment with different
concentrations of LIF, the results showed lowest expression of the VEGF gene at the concentration of 10 ng
(P<0.001). Using 10 ng (P<0.001) and 50 ng (P<0.01)
concentrations, there was a significant decrease in gene
expression compared to control, but at 1 ng concentration,
there was no significant decrease in the gene expression
(P=0.324). Comparing gene expression between different concentrations of LIF showed a significant difference
between the concentration of 1 ng and 10 ng (P=0.004,
Fig .1C).
Forty-eight hours treatment
After 48 hours, like 12 and 24 hours, VEGF gene expression was decreased by treating with different concentrations of LIF, compared to control (P<0.001), and the
lowest gene expression was observed at 1 ng in comparison with 10 ng (P<0.01) and 50 ng (P<0.05). VEGF gene
expression was more in 10 ng than the other two concentrations (1 and 50 ng) of LIF (P<0.01; Fig. 1D).
Seventy-two hours treatment
After 72 hours, effect of LIF on the VEGF gene expression
was reversed, and contrary to the previous times, in
all three concentrations of LIF, we observed a dramatic
increased expression of the VEGF gene, in comparison
with control (P<0.001; Fig.2). The maximum VEGF gene
expression was observed at 10 ng of LIF concentration,
which had significant difference in comparison with 1
ng concentration of LIF (P<0.05). But, the difference
between 50 ng and 10 ng LIF concentrations was not significant (Fig .1E).
VEGF gene expression at different time points
As shown in Figure 3, VEGF gene expression was dramatically decreased
(P<0.001) at 12, 24, and 48 hours after cell treatment with LIF,
in comparison with 6 hours
treatment. In contrast to decrease in the VEGF gene expression at
12, 24 and 48 hours, we determined a significant increase (P<0.001)
in VEGF gene expression at 72
hours compared to other time points.
Changes in VEGF gene expression at different time points of inducting LIF to the cells. *; P<0.05, **; P≤0.01, ***; P≤0.001.
Discussion
Pregnancy is a complex process that depends on many
factors. Studies have shown that cytokines, growth factors and several transcription factors play important roles
in embryo implantation. For example, production of LIF
by endometrial cells is essential for the beginning of implantation (25, 26). Data obtained from mice and humans
have shown that among the all molecules expressed in
uterus, LIF plays the most important role in embryo implantation (27). Formation of new blood vessels is called
angiogenesis and it accompanies with migration, growth
and differentiation of endothelial cells (28). Angiogenesis usually occurs during the menstrual cycle or estrus
to convert the ovulation follicles to corpus luteum which
leads to the synthesis of progesterone and restructure of
the endometrium. This culminates in maintenance of embryo implantation (29). Angiogenesis and vasculogenesis
are essential processes for increasing blood flow to the fetus and, consequently, supplying the nutrients and oxygen
needed by fetus (13, 30, 31). Several growth factors control angiogenesis and vasculogenesis during pregnancy.
Among these factors, VEGF plays a critical role in the
development of the placenta and formation of vesJahanbin sels. Carmeliet et al. (32) showed that deactivation
of onlyone VEGF allele leads to fetal death through angiogenesis disruption. Shalaby et al. (33) by disrupting
Vascular endothelial growth factor receptor 1 (VEGFR1),
Fong et al.(34) by disrupting VEGFR2 and Tsoi et al.
(35) by disruption of neuropilin-1 and -2 (all of them are
VEGF receptors) determined similar results to Carmeliet
et al. (32). Adequate blood supply to the placenta is highly
dependent on regulated invasion and trophoblast vascular
remodeling in uterus (36, 37). Extravillous trophoblast
(EVT) is a subset of trophoblasts that play the most important role in invasion (the same mechanism as cancerous cells for invasion) to the mother's uterus and vascular
remodeling. This eventually acquires the phenotype of
endothelial cells and improves artery formation (37). Previous studies have shown that EVTs have receptors for
VEGF at their surface and message through these receptors which stimulate invasion, switch phenotype to endovascular cells and tube formation in EVT cells (37, 38).
Defects in EVT invasion and angiogenesis have been observed in disorders, such as preeclampsia and intrauterine
growth restriction (IUGR) (37). Due to the vital role of
vascular formation by trophoblast cells (especially EVTs)
in pregnancy and implantation, in this study, we decided
to investigate the effect of LIF on one of the most important angiogenic factors, VEGF, in EVTs. For this purpose,
we had to select an appropriate cell line with similar features to EVT cells. According to the previous studies (20,
39), JEG-3 cell line was selected. The results of this study
showed that LIF could have a dual effect on VEGF gene
expression with respect to time. So that at 12, 24, and 48
hours, VEGF gene expression was decreased, while it was
increased at 6 and 72 hours (the increase of VEGF gene
expression at 6 hours depended on the concentration of
LIF showing a significant decrease at 50 ng concentration
of LIF in contrast to 1 and 10 ng).
Considering the mentioned roles for VEGF during pregnancy and relevant disorders, as well as the important role
of LIF during pregnancy, we decided to investigate the
effect of LIF on the level of VEGF gene expression in
trophoblast cells. Regarding to the results, it was found
that the expression of the VEGF gene in trophoblast tumor cells treated by LIF was reduced in concentrationand time-dependent manners. Although expression of the
VEGF gene was significantly increased after 72 hours, a
study has previously shown that half-life of the LIF attachment to its receptor is slightly more than 24 hours
(40). It can be concluded that after 72 hours, interactions
between LIFs and their receptors are broken-down and
the LIF signaling from their receptors are ended in trophoblast cells. As the results of this study showed. different
concentrations of LIF can reduce the rate of VEGF gene
expression depending on the time. So given the fact that
VEGF gene expression level was decreased in LIF-treated cells, assessment of the production and secretion of
VEGF protein in treated trophoblast cells is vital. Further
investigations have to be performed on the other angiogenic factors to clarify the role of LIF on angiogenesis
procedure in trophoblast cells.
Conclusion
In conclusion, recent studies have shown that both LIF
and VEGF are essential for maintaining and initiating the
pregnancy process. It has also been found that angiogenesis process is a critical procedure in embryonic trophoblast cells for a normal pregnancy. VEGF-A is one of
the most important angiogenic factors. Therefore, in this
study we investigated the effect of LIF on VEGF gene
expression in JEG-3 cell line as extravillous trophoblast
cells. According to the results of this study, LIF causes a
significant decrease in gene expression level of VEGF-A
in JEG-3 cells. Further studies are needed to determine
the action mechanism of LIF in angiogenesis of trophoblast cells.
Jahanbin, K., Ghafourian, M., & Rashno, M. (2020). Effect of Different Concentrations of Leukemia Inhibitory Factor on Gene Expression of Vascular Endothelial Growth Factor-A in Trophoblast Tumor Cell Line. International Journal of Fertility and Sterility, 14(2), 116-121. doi: 10.22074/ijfs.2020.6058
MLA
Khodakaram Jahanbin; Mehri Ghafourian; Mohammad Rashno. "Effect of Different Concentrations of Leukemia Inhibitory Factor on Gene Expression of Vascular Endothelial Growth Factor-A in Trophoblast Tumor Cell Line". International Journal of Fertility and Sterility, 14, 2, 2020, 116-121. doi: 10.22074/ijfs.2020.6058
HARVARD
Jahanbin, K., Ghafourian, M., Rashno, M. (2020). 'Effect of Different Concentrations of Leukemia Inhibitory Factor on Gene Expression of Vascular Endothelial Growth Factor-A in Trophoblast Tumor Cell Line', International Journal of Fertility and Sterility, 14(2), pp. 116-121. doi: 10.22074/ijfs.2020.6058
VANCOUVER
Jahanbin, K., Ghafourian, M., Rashno, M. Effect of Different Concentrations of Leukemia Inhibitory Factor on Gene Expression of Vascular Endothelial Growth Factor-A in Trophoblast Tumor Cell Line. International Journal of Fertility and Sterility, 2020; 14(2): 116-121. doi: 10.22074/ijfs.2020.6058
)
