Introduction
Endometriosis is a prevalent and complex
gynecological disease which affects 10% of women of reproductive
age (1), and is characterized by
the growth of endometrial glandular epithelial and stromal cells
outside the uterine cavity. Women of reproductive age with
endometriosis often suffer from painful menstrual periods, chronic
pelvic pain and infertility. This disease is multifactorial and
factors, such as hormones, immune response and the environment may
contribute to the susceptibility and progression of endometriosis
(2–4). In the majority of cases, the spread
of extrauterine endometrial tissue is due to retrograde
menstruation and lymphatic dissemination (5).
microRNAs (miRNAs or miRs) are small non-coding
RNAs, which are now known to play essential roles in the regulation
of gene expression for cell proliferation and apoptosis, cell
cycle, immune response and inflammation (6,7).
The deregulation and aberrant expression of these molecules has
been implicated in a variety of human diseases, including cancer
(8,9).
Endometriosis displays a benign morphology; however,
endometriotic cells have an invasive potential which is
characterized by malignant cells (10). To date, to the best of our
knowledge, there is no report available on the role of miR-29 in
the endometrium. In the present study, we investigated the
expression of miR-29a, miR-29b, miR-29c in endometrium samples from
women without endometriosis, as well as in paired ectopic and
eutopic endometrium samples. Furthermore, the role of miR-29c in
endometrial cell proliferation, invasion and apoptosis was examined
in vitro, and a novel target through which miR-29c exerts
its effects on endometrial cells was identified.
Materials and methods
Tissue samples
The procedures for the collection of the tissue
samples were approved by the Ethics Committee of the First
Affiliated Hospital of Xinjiang Medical University, Urumqi, China.
All patients signed an informed consent form in compliance with the
code of ethics of the World Medical Association (Declaration of
Helsinki). Paired eutopic and ectopic endometrium samples were
collected from 20 women with endometriosis who underwent
laparoscopic surgical procedures at the First Affiliated Hospital
of Xinjiang Medical University. The patients were aged 25 to 42
years, had regular menstrual cycles and had not received hormonal
therapy for at least 3 months prior to surgery. A total of 10
samples of endometrium from women with hysteromyoma were used as
the controls. The tissues were from the proliferative phase of the
menstrual cycle. After collection, the tissue samples were
immediately frozen and kept in liquid nitrogen for further
analysis.
Cell culture and transfection
The CRL-7566 endometriosis cell line was purchased
from the American Type Culture Collection (ATCC; Manassas, VA,
USA). This cell line was established from a benign ovarian cyst
obtained from a patient with endometriosis. The cells were isolated
from both the inner and outer surfaces of the cyst. The cells were
cultured in Dulbecco’s modified Eagle’s medium (Invitrogen,
Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco,
Carlsbad, CA, USA). The cells were grown at 37°C in 5%
CO2. The miR-29c mimic, miR-29c inhibitor and negative
control (NC), mutant and wild-type c-Jun 3′UTR, as well as the
c-Jun overexpression plasmid and the shRNA plasmid were transfected
into the cells using Lipofectamine 2000 reagent (Invitrogen)
following the manufacturer’s instructions and incubated at 37°C for
24 h.
Luciferase assay
The targets of miR-29c were predicted by miRanda
(http://www.microrna.org/) and c-Jun was
identified to be a target of miR-29c. The sequences of human
wild-type c-Jun 3′ untranslated region (3′UTR) and mutant c-Jun
3′UTR were then cloned into the pGL3-control luciferase reporter
vector (Promega, Madison, WI, USA). c-Jun 3′UTR reporter plasmids
were co-transfected with the miR-29c mimic or negative control (NC)
into the cells. pRL Renilla luciferase reporter vector was
used as the internal control in each assay. Following transfection,
cell lysates were collected and luciferase activity was measured
using the Dual-Luciferase Reporter assay system (Promega).
MTT assay
MTT assay was used to evaluate cell proliferation.
The cells were seeded into 96-well culture plates at a density of
1×104 cells/well. Following transfection, 10 μl
MTT solution (0.5 mg/ml; Beyotime, Shanghai, China) were added to
each well followed by incubation for 4 h. After dissolving the MTT
formazan with DMSO (Sigma, St. Louis, MO, USA), cell proliferation
was determined by reading the plates at 570 nm on a Multiskan
Ascent 354 microplate reader (Thermo Labsystems, Waltham, MA,
USA).
Cell invasion assay
Cell invasion assay was performed using
Matrigel-coated Transwell plates (Corning, New York, NY, USA).
Cells in serum-free medium at a final density of 5×104
cells/ml were added to the upper chambers and incubated at 37°C for
12 h, and cell medium containing 10% FBS was added to the lower
chambers. Following incubation, non-invaded cells were removed
using cotton swabs, and the invaded cells were fixed in 95% ethanol
and stained with hematoxylin. The number of invaded cells was
determined by counting the stained cells under an inverted
microscope (TS100; Nikon, Tokyo, Japan).
Flow cytometry (FCM)
Cell apoptosis was assessed by FCM. After washing
with phosphate-buffered saline (PBS), the cells were resuspended in
binding buffer and stained with Annexin V and propidium iodide (PI)
(Kaiji Biological Inc., Nanjing, China) following the
manufacturer’s instructions. Positive cells were detected and
quantified by FCM (BD FACSAria; BD Biosciences, Franklin Lakes, NJ,
USA).
Reverse transcription-quantitative
polymerase chain reaction (RT-qPCR)
Total RNA was isolated using TRIzol reagent
(Invitrogen) according to the manufacturer’s instructions. The
miRNAs were isolated using a miRNeasy Mini kit (Qiagen, Hilden,
Germany). Reverse transcription was performed using a RevertAid™
First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania) and
quantitative (real-time) PCR was carried out on a 7900 Real-Time
PCR System using a SYBR-Green PCR kit (both from Applied
Biosystems, Foster City, CA, USA) following the manufacturer’s
instructions. The results were analyzed using the comparative
method following normalization of the expression values to U6.
Western blot analysis
Protein samples were extracted using a total protein
extraction kit (Kaiji Biological, Inc.) and the protein
concentration was determined using the BCA kit (Beyotime). The
samples were separated in 10% SDS-PAGE and transferred onto
polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA).
The membranes were blocked in 5% non-fat milk overnight at 4°C.
After washing with PBS, the membranes were incubated with primary
antibody [mouse monoclonal to c-Jun (ab119944), mouse monoclonal to
β-actin (ab8226); Abcam, Cambridge, MA, USA] overnight at 4°C,
followed by incubation with horseradish peroxidase-conjugated
secondary antibody (BA1050; Boster, Wuhan, Hubei, China) for 1 h at
room temperature. The ECL detection kit (Pierce, Rockford, IL, USA)
was used to detect the proteins.
Statistical analysis
Data were presented as the means ± standard
deviation (SD). The two-tailed Student’s t-test was used to
statistically analyze data between 2 groups. A P-value <0.05 was
considered to indicate a statistically significant difference.
Results
Differential expression of miR-29 family
in tissue samples
The differential expression of miR-29a, miR-29b,
miR-29c in the endometrium samples from women without
endometriosis, as well as in the eutopic and ectopic endometrium
samples was determined by RT-qPCR. The results revealed that the
expression of miR-29c progressively decreased from the samples of
endometriosis to the those of the eutopic and ectopic endometrium,
and the expression of miR-29c between the eutopic and ectopic
endometrium was differed significantly (P<0.01). However, the
expression levels of miR-29a and miR-29b did not differ
significantly between the 3 groups (Fig. 1).
Differential expression of c-Jun in
tissue samples
The expression of c-Jun in the endometrium samples
from women without endometriosis, as well as in the eutopic and
ectopic endometrium samples was examined by western blot analysis.
There was no statistically significant difference in c-Jun
expression between the group without endometriosis and the eutopic
group (Fig. 2). However, the
protein expression level of c-Jun was significantly higher in the
ectopic group compared with the eutopic group (P<0.01).
c-Jun is directly regulated by
miR-29c
In order to determine whether c-Jun is a direct
target of miR-29c, wild-type c-Jun-3′UTR or mutant c-Jun-3′UTR were
co-transfected with the miR-29c mimic or NC. Luciferase assay
revealed that transfection with the miR-29c mimic significantly
inhibited the luciferase activity of wild-type c-Jun-3′UTR
(P<0.01); however, the luciferase activity of mutant c-Jun-3′UTR
did not differ statistically following transfection with the
miR-29c mimic or NC (Fig. 3).
Furthermore, the effect of miR-29c on c-Jun
expression was examined by western blot analysis. The results from
RT-qPCR revealed that miR-29c expression was significantly
upregulated in the miR-29c mimic-transfected group and
downregulated in the miR-29c inhibitor-transfected group (Fig. 4A). Western blot results revealed a
significantly decreased expression of c-Jun in the cells
transfected with the miR-29c mimic; however, the expression of
c-Jun was increased in the cells transfected with the miR-29c
inhibitor (Fig. 4B).
Expression of miR-29c and c-Jun following
transfection with the c-Jun overexpression or shRNA plasmid
The open reading frame (ORF) clone of homo c-Jun was
subcloned into the pcDNA3.1 vector to induce the overexpression of
c-Jun. We suppressed c-Jun expression using a specific shRNA
plasmid. Transfection with the c-Jun overexpression plasmid did not
alter the increase in miR-29c expression in the miR-29c
mimic-tansfected cells (Fig. 5A).
In addition, the c-Jun shRNA plasmid did not alter the decrease in
miR-29c expression in miR-29c inhibitor-tansfected cells (Fig. 5B).
Cells transfected with the miR-29c mimic and c-Jun
over-expression plasmid showed a significantly increased protein
expression of c-Jun compared with the cells transfected with the
miR-29c mimic only (Fig. 6A).
However, transfection with c-Jun shRNA significantly downregulated
the expression of c-Jun in the cells transfected with the miR-29c
inhibitor (Fig. 6B).
c-Jun reverses the miR-29c-induced
suppression of cell proliferation and invasion
In order to examine the effects of miR-29c on cell
proliferation, the miR-29c mimic or inhibitor were transfected into
the cells, and MTT assay was used to analyze cell proliferation.
Compared with the NC-transfected cells, the miR-29c
mimic-transfected cells proliferated at a significantly lower rate
(Fig. 7A). By contrast,
transfection with the miR-29c inhibitor promoted cell growth
(Fig. 7B).
Furthermore, the overexpression of c-Jun reversed
the inhibitory effects of the miR-29c mimic on cell proliferation
(Fig. 7A). Consistent with these
results, transfection with c-Jun shRNA reversed the inductive
effects of the miR-29c inhibitor on cell proliferation (Fig. 7B).
Cell invasion ability was determined by
Transwell-Matrigel assay. Transfection with the miR-29c mimic
resulted in significantly decreased cell invasion in comparison to
transfection with NC; however this effect was reversed by
transfection with the c-Jun overexpression plasmid (Fig. 8A). As expected, transfection with
the miR-29c inhibitor significantly increased cell invasion
ability, which was reversed by transfection with c-Jun shRNA
(Fig. 8B).
c-Jun reverses the miR-29c-mediated
induction of cell apoptosis
FCM was used to examine the cell apoptotic rates.
The results revealed that compared with the cells transfected with
NC, the miR-29c mimic-transfected cells showed an increased cell
apoptotic rate, while transfection with miR-29c inhibitor had the
opposite effect. The overexpression of c-Jun reversed the increase
in the cell apoptotic rate induced by the transfection with the
miR-29c mimic. Consistent with these results, transfection with
c-Jun shRNA reversed the inhibitory effects of the miR-29c
inhibitor on cell apoptosis (Fig.
9).
Discussion
Pan et al (11) provided the first evidence of the
expression of a unique set of miRNAs in the endometrium and
endometrial cells, suggesting that a substantial number of miRNAs
are aberrantly or differentially expressed in the ectopic
endometrium. Ohlsson Teague et al (12) profiled miRNAs in paired samples of
eutopic and peritoneal ectopic endometrial tissue from patients
with endometriosis using miRNA microarray analysis. Of the miRNAs
evaluated, 22 miRNAs showed a differential expression in the paired
eutopic vs. the ectopic endometrium samples. Both the studies of
Pan et al (11) and
Ohlsson Teague et al (12)
revealed that miR-29c was differentially expressed in the eutopic
and ectopic endometrium. miR-29c belongs to the miR-29 family, and
it has been suggested to be downregulated in certain types of
cancer (13–16). In the present study, the
expression levels of the miR-29 family in the endometrium samples
from women without endometriosis, as well as in paired ectopic and
eutopic endometrial tissue samples from patients with endometriosis
were examined by RT-qPCR. It was revealed that miR-29c was
differentially expressed in the paired eutopic and ectopic
endometrium samples. These findings indicate that miR-29c may be
associsated with the progression of endometriosis.
To determine the potential role of miR-29c in
endometrial cells, miR-29c mimic and miR-29c inhibitor were
transfected into CRL-7566 cells. MTT assay revealed that the cells
transfected with the miR-29c mimic proliferated at a significantly
lower rate, and transfection with miR-29c inhibitor had the
opposite effect. Endometriotic cells have an invasive potential
which may contribute to the pathogenesis of endometriosis (10). In the present study, we found that
transfection with miR-29c mimic suppressed cell invasion ability,
while transfection with the miR-29c inhibitor enhanced cell
invasion ability. FCM was used to examine the effects of miR-29c on
cell apoptosis. As expected, transfection with miR-29c mimic
increased the cell apoptotic rate, while transfection with the
miR-29c inhibitor had the opposite effect. These data indicate that
miR-29c potentially plays a negative role in the development of
endometriotic lesions. miR-29c may exert its effects on endometrial
cells by suppressing cell proliferation and invasion, as well as by
promoting cell apoptosis.
Studies on the differences in gene expression
between the ectopic and eutopic endometrium (17–22) have found that many genes related
to cell adhesion, cell migration, cell proliferation, immune
regulation and inflammation are differentially expressed in ectopic
vs. eutopic endometrial tissue. In the study by Ohlsson Teague
et al (12), it was
suggested that c-Jun mRNA expression was significantly increased in
endometriosis. c-Jun is a transcription factor which belongs to the
activating protein 1 family (23). Genetic studies have demonstrated
that c-Jun regulates a variety of genes important for diverse
cellular functions, including cell differentiation, apoptosis,
proliferation, migration and invasion (24–26). The overexpression of c-Jun has
been detected in various types of tumors (27,28). In the present study, it was
revealed that c-Jun was also differentially expressed in the
ectopic vs. eutopic endometrial tissue as determined by western
blot analysis. This result is consistent with the results of a
previous study demonstrating that c-Jun is associated with
endometriosis (12).
We predicted the targets of miR-29c by miRanda and
focused on c-Jun. Luciferase reporter assay and western blot
analysis revealed that miR-29c directly binds to the c-Jun gene and
inhibits the protein expression of c-Jun. These results indicate a
negative role of miR-29c in the regulation of c-Jun expression and
provide support for our hypothesis that miR-29c suppresses the
progression of endometriosis possibly through the downregulation of
c-Jun. In addition, c-Jun overexpression plasmid and shRNA plasmid
were transfected into the CRL-7566 cells. The results revealed that
the overexpression of c-Jun reversed the inhibitory effects of the
miR-29c mimic on cell proliferation and invasion, and the
overexpression of c-Jun reversed the increase in the cell apoptotic
rate induced by the miR-29c mimic. In addition, c-Jun shRNA
reversed the effects of the miR-29c inhibitor on cell
proliferation, invasion and apoptosis. Thus, our results suggest
that c-Jun plays an important role in the regulation of the
proliferation, invasion and apoptosis of endometrial cells by
miR-29c.
In conclusion, we identified miR-29c as a suppressor
of endometriosis, and that it is differentially expressed in paired
ectopic and eutopic endometrial tissues. miR-29c exerts its effects
on endometrial cell proliferation, apoptosis and invasion by
inhibiting the expression of c-Jun. This study may provide a novel
potential therapeutic target for the treatment of
endometriosis.
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