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doi:10.1111/jog.12461 J. Obstet. Gynaecol. Res. Vol. 40, No. 9: 2051–2057, September 2014

Overexpression of myosin is associated with the

development of uterine myoma

Wenchao Zhang1,2, Zhongping Cheng2, Xiaoyan Qu2, Hong Dai2, Xiaoping Ke2 and
Zijiang Chen1
1
Center for Reproductive Medicine, Provincial Hospital Affiliated to Shandong University, Jinan, and 2Department of
Obstetrics and Gynecology, Yangpu District Central Hospital, Shanghai, China

Abstract
Aim: Myosin is involved in cell contraction and motility, but it is unclear whether it is involved in cell
proliferation in uterine myoma. In this study therefore we aimed to explore the role of myosin in uterine
myoma.
Material and Methods: Immunohistochemistry and real-time polymerase chain reaction were used to deter-
mine the expression of myosin light chain (MLC), myosin heavy chain (MHC) and myosin light chain kinase
(MLCK) in patient uterine myoma and adjacent smooth muscle tissue. Human uterine fibroid cells were
isolated and cultured in vitro, myosin heavy chain 11 (MHC subtype expressed in uterine fibroid cells) was
knocked down by RNA interference to reduce the expression of myosin, then cell proliferation was determined
by the methyl thiazol tetrazolium bromide method. To explore the possible mechanism of reduced cell
proliferation after myosin heavy chain 11 knockdown, the downstream proteins collagen I, insulin-like growth
factor-1, fibronectin and proteoglycans were analyzed.
Results: Expression of MLC, MHC, MLCK and p-MLCK in uterine myoma cells was significantly higher than
in adjacent smooth muscle cells. After knockdown of MHC, smooth muscle cell proliferation decreased, and
the production of collagen I, insulin-like growth factor-1 and fibronectin was also reduced, but proteoglycans
did not show any significant change.
Conclusion: Myosin is overexpressed in uterine myoma, and the overexpression of myosin is associated with
both uterine contraction and tumor development of uterine myoma.
Key words: contraction, myosin heavy chain, myosin light chain, myosin light chain kinase, uterine myoma.

Introduction patients’ uterine cavity pressure using a pressure

Uterine fibroids are the most common benign tumors
in women, with a clinical morbidity rate of 20–40%.1
However, the pathogenesis of uterine fibroids is still
not fully understood. Previously, research on the patho-
genesis of uterine fibroids has focused on cell differen-
tiation.2 Recent studies showed that the phenomenon
of abnormal contraction was also found in myoma.3 As
early as the 1980s–1990s, some researchers monitored
sensor, and found that patients with uterine fibroids
showed abnormal uterine contraction, possibly leading
to infertility.4,5 Subsequently, by observing the ultra-
structure using electron microscopy they also found
differences in the density band of myoma cells com-
pared to normal uterine smooth muscle cells, indicat-
ing that contraction of the myoma cells is abnormal.6
As is well known, the contraction-related
pathway and its regulation are important in uterine

Received: November 26 2013.

Accepted: March 29 2014.
Reprint request to: Professor Zhongping Cheng, Department of Obstetrics and Gynecology, Yangpu District Central Hospital,
Tengyue Road 450, Shanghai, China 200090. Email: paperch@sina.com

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Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
W. Zhang et al.
pathophysiology. The thick myofilaments of the myo-
fibrils are composed of myosins, which play an im-
portant role in muscle movement. Myosins form a
structurally and functionally diverse superfamily that
consists of at least 18 distinct members.7 The classic
two-headed myosin is called conventional myosin and
represents the myosin II family; the other 17 members
are referred to as unconventional myosins.7,8 Myosin is
composed of six polypeptide chains, comprising two
myosin heavy chain (MHC) molecules and two pairs of
myosin light chains (MLC). MLC are divided into basic
light chains and regulatory light chains. The major role
of the basic light chains is to stabilize the structure of
the heavy chains, while regulatory light chains work to
regulate the activity of myosin.9
Actin and adenosine triphosphate (ATP) binding
sites are located in the head of the MHC molecule. Pairs
of spherical MHC heads join up and move rotatively, a
conformational change occurs, then the MHC head
binds to actin, hydrolyzing ATP and generating con-
traction.10 Myosin heavy chain 11 (MYH11) encodes
the smooth muscle MHC, and belongs to the family
of conventional myosins. MLC are regulated by dual
signals, being activated by myosin light chain kinase
(MLCK) signaling, which is dependent on Ca2+
/calmodulin (CaM), and by myosin phosphatase
signaling, which is dependent on the RhoA pathway.
MLCK is an important regulator of MLC through its
phosphorylation and activation.11 Thus, in this study
we analyzed both MLC and MLCK expression in
uterine myoma.
Given the important role of myosin both in cell
contraction and in proliferation, we isolated human
uterine fibroid cells and tested cell proliferation after
knockdown of myosin. We also analyzed downstream
signals to reveal the underlying mechanism.
Methods
Patients and samples
This study was approved by the medical ethics com-
mittee of Yangpu District Central Hospital, Shanghai,
China, and informed consent was obtained from each
patient. Uterine myoma and adjacent smooth muscle
tissue were collected from 30 patients in our hospital
who underwent surgical treatment in 2011. The
samples were confirmed by pathological examination
after surgery, with adjacent smooth muscle tissue
defined as tissue at a distance of 0.5 cm from myoma
tissue. The age of the patients ranged between 37 and
59 years, with a mean age of 44.5 ± 3.3 years. Patients
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Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
were all diagnosed with intramural myoma, and were
excluded if they exhibited other complications, such as
ovarian cysts or adenomyosis.
Immunohistochemistry
The immunohistochemistry was performed using the
StreptAvidin-Biotin Complex method. Briefly, after
hydration and antigen repair of tissues, the primary
antibodies were added at the dilutions indicated
(MLC, 1:200; MHC, 1:100; MLCK, 1:100; p-MLCK,
1:100; all from Abcam) and slides were incubated at
4°C overnight. The next day, the tissues were stained
with biotinylated secondary antibodies, and DAB dye
was used for color development. Images were collected
under a microscope (OLYMPUS BX-51), positive area
(brown) was abstracted by negative area (purple) and
the area size was measured by micrometer. The average
optical density (OD) was measured as follows: Unit
conversion (total area 351 000 pixels) and positive
area (1 pixel point = 0.095 μm2). Immunohistochemical
index was calculated as positive area multiplied by
OD value.
Real-time polymerase chain reaction
Total RNA was extracted using Trizol reagent
(Invitrogen). The cDNA synthesis kit and real-time
polymerase chain reaction (PCR) kit were purchased
from Takara. cDNA was synthesized as follows:
5 × reverse transcription buffer, oligo dT, dNTPs,
reverse transcriptase MMLV, DEPC water and RNA
template. Real-time PCR was then performed in a
mixture of real-time PCR mix, primers and cDNA tem-
plate in a total volume of 20 μL. PCR amplification
conditions: 95°C preliminary denaturation 5 min; 95°C
denaturation 15 s, 60°C annealing 35 s, 40 cycles. MLC
primers: forward 5′-CGGGTCCTGAAATCTTACTC-3′,
reverse 5-′AGGCTGCTTATGGCAATC-3′; MLCK
primers: forward 5′-CGCCACTTCCAGATAGACTAC-
3′, reverse 5′-ACCTTCCTCCATCGTTTCC-3′. Glycer-
aldehyde 3-phosphate dehydrogenase was used as an
internal control, using the primers: forward 5′-ATCAG
CAATGCCTCCTGCAC-3′, reverse 5′-CGTCAAAGGT
GGAGGAGTGG-3. Gene expression was calculated as
the relative gene expression to internal control (△CT).
Human uterine fibroid cells culture
The uterine fibroid tissues were isolated from patients
with uterine fibroids, the tissues were cut into small
pieces at 4°C and digested with collagenase II for 2 h at
37°C. The samples were centrifuged at 1000 rpm for
© 2014 The Authors

10 min after repeated pipetting. After this step, small
pieces of loose tissue were placed in 6-cm plastic
culture dishes and cultured in DMEM with 10% fetal
bovine serum (Gibco/Life Technologies). After 2 days,
shuttle-shaped uterine fibroid cells were observed
growing out from the pieces of tissue.
MYH11 interference
Oligonucleotide fragments were designed to target
the homo sapiens MYH11 smooth muscle 1A
(NM_002474) gene sequence, synthesized and
annealed into the double-stranded form. They were
then inserted into the expression vector pcDNA 6.2-
GW/EmGFPmiR (Invitrogen) with the BLOCK-iT
Pol II miR RNAi Expression Vector Kit. The lentiviral
vector pLENT6.3/V5-SR60_1 was constructed and
packaged, and viral titers were determined. Uterine
fibroid cells were used for transduction, and a high
transfection efficiency and interference were achieved.
Cell proliferation assay
The cell proliferation rate was tested by methyl thiazol
tetrazolium bromide (MTT) assay. The cells were plated
into 96-well plates at a concentration of 3000 cells per
well and incubated for 72 h, resulting in a final MTT
concentration of 0.5 mg/mL. MTT was added to each
well and they were returned to the incubator for a
further 4 h. The culture supernatant was then removed
from each well, 150 μL of dimethyl sulfoxide was added
and the cultures were shaken for 10 min. A wavelength
of 560 nm was used to measure the absorbance of each
well using a microplate reader, and the growth rate was
calculated relative to the control group.
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) kits for
collagen I (Genway), insulin-like growth factor (IGF)-1
(Genway), fibronectin (Abcam) and proteoglycans
(antibodies) were used to assay protein secretion in
the cell culture supernatants. After blocking non-
specific binding by adding 200 μL of 1% bovine serum
albumin, 100 μL of each culture supernatant was trans-
ferred to the wells of a microtiter plate at different
dilutions, and incubated for 2 h at 37°C. After washing,
100 μL of horseradish peroxidase-conjugated second-
ary antibody was added to the wells and they were
incubated for 1 h, then the washing step was repeated,
and finally 100 μL of tetramethylbenzidine was added
to each well and they were incubated for 30 min at
room temperature. Finally the stop solution was added
© 2014 The Authors
Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Overexpression of myosin in myoma
and the plate was read immediately on a microtiter
plate reader at 450 nm.
Statistical analysis
Data were expressed as the mean ± standard deviation
or standard error and analyzed by the Mann–Whitney
U-test or Student’s t-test using spss. A P-value <0.05
was considered statistically significant.
Results
Overexpression of myosin and MLCK in
uterine myoma
In order to explore whether crosstalk occurs between
cell contraction and proliferation in uterine myoma,
expression of two important proteins, myosin and its
upstream kinase MLCK, was determined in patient
samples of uterine myoma. As shown in Figure 1, the
expression of MLC, MHC, MLCK and p-MLCK was
detected both in uterine myoma and in adjacent
smooth muscle, with positive expression located
mainly in the cytoplasm. The expression of MLC,
MHC, MLCK, and p-MLCK in uterine myoma was
much higher than in smooth muscle tissue (P < 0.05)
(Fig. 1 and Table 1).
To verify the overexpression of MLC and MLCK
in myoma, we used real-time PCR to analyze mRNA
expression. As shown in Figure 2, the mRNA levels
of both MLC and MLCK were significantly higher in
myoma (P < 0.05).
Knockdown of MHC expression reduces
cell proliferation
In order to investigate the effect of myosin to uterine
fibroid cells, we disrupted the function of myosin by
knockdown of MYH11, which is specifically expressed
in uterine fibroid cells. Uterine fibroid cells were trans-
duced with lentiviral to knockdown the expression of
MYH11 (Fig. 3a,b). The interference effect was showed
to cause a reduction of 72% of MYH11 at the mRNA
level (Fig. 3c). In order to evaluate the effect of MHC on
cell proliferation, control cells and MYH11 knockdown
cells were examined using the MTT method to deter-
mine their growth rate. As shown in Figure 3d, the
growth rate of MYH11 knockdown cells was signifi-
cantly reduced compared to control cells (P < 0.05).
Effect of MHC downregulation on uterine
fibroid cells
The knockdown of MHC could change the structure
and distribution of the cytoskeleton. This might result
2053
W. Zhang et al.

Figure 1 Expression of myosin

light chain (MLC), myosin heavy
chain (MHC), myosin light chain
kinase (MLCK) and p-MLCK in
uterine myoma as determined
by immunohistochemistry. Rep-
resentative increased expres-
sion of MLC, MHC, MLCK and
p-MLCK in uterine myoma as
compared to smooth muscle
tissue. The expression of MLC,
MHC, MLCK and p-MLCK were
detected by their primary anti-
bodies and biotinylated second-
ary antibodies, and then DAB
dye was used for color deve-
lopment (StreptAvidin-Biotin
Complex, ×100). Strong expres-
sion is shown in brown staining,
while low expression is shown
in blue staining.

Table 1 Comparison of immunohistochemical index of MLC, MLCK, p-MLCK in

myoma and smooth muscle tissue (mean ± standard deviation)
Myoma Smooth muscle P-value*
(n = 30) (n = 30)
MLC 35 635.77 ± 8 575.76 16 161.53 ± 5 846.28 <0.05
MHC 27 724.56 ± 12 306.34 12 316.08 ± 9 058.74 <0.05
MLCK 30 313.73 ± 13 722.96 17 895.93 ± 9 819.17 <0.05
p-MLCK 38 063.57 ± 5 370.23 24 621.70 ± 6 012.04 <0.05
*Analyzed by Mann–Whitney U-test. MHC, myosin heavy chain; MLC, myosin light chain;
MLCK, myosin light chain kinase.

2054 © 2014 The Authors

Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
 Overexpression of myosin in myoma

Figure 2 mRNA expression of myosin light chain (MLC) and myosin light chain kinase (MLCK) expression in myoma as
determined by real-time polymerase chain reaction. Expression of MLC and MLCK were increased in myoma, as
compared to smooth muscle tissues. Gene expression was calculated as the relative gene expression to internal control
(△CT). The bars indicate the mean ± standard error (n = 30) for each group. Data were analyzed by Student’s t-test.
*P < 0.05, as compared to the control group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MC, myoma cell; SMC,
smooth muscle cell.

Figure 3 Knockdown of myosin

heavy chain expression in
human uterine fibroid cells.
(a) Light microscopic picture
of uterine fibroid cells that
were cultured for 72 h in vitro.
(b) Expression of pLENT6.3/
V5-SR60 (myosin heavy chain
11 [MYH11] RNAi) in uterine
fibroid cells as monitored by
EGFP fluorescence. Scale bar
50 μm. (c) mRNA expression of
MYH11 was detected by real-
time polymerase chain reaction.
(d) Cell proliferation rate was
determined by methyl thiazol
tetrazolium bromide assay. The
bars indicate the mean ± stan-
dard error (n = 3) for each group.
Data were analyzed by Student’s
t-test. *P < 0.05, as compared to
the control group.

in changes in secretion of some factors. In order of which play important roles in uterine fibroid cell
to investigate the effect of MHC downregulation on proliferation. ELISA was performed to detect the
uterine fibroid cells, we analyzed the expression of extracellular secretion of collagen I, IGF-1, fibronectin
collagen I, IGF-1, fibronectin and proteoglycans, all and proteoglycans. The results showed that collagen I

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Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
W. Zhang et al.
secretion was maintained at a stable level after MYH11
interference. In control cells, collagen I production
increased gradually (at 48 and 72 h), compared with
control group cells, so that over time the secretion of
collagen I was significantly decreased after MYH11
knockdown (P < 0.05). IGF-1 secretion was also
reduced after MYH11 knockdown compared to control
cells. In control group cells, IGF-1 remained at a high
level at 24 and 48 h, after which it decreased gradually,
but levels remained higher than those in MHY11 RNAi
cells (P < 0.05). In addition, fibronectin secretion was
also reduced after MYH11 knockdown compared to
control cells (P < 0.05). In control cells, fibronectin
production increased gradually over time. But
proteoglycans did not show any significant difference
compared to control cells (P > 0.05). Production
increased gradually in either MHY11 RNAi cells or
control group cells (Fig. 4).
Discussion
Although myomas are common, myoma research has
been slow compared with research on other non-
malignant diseases.12,13 However, with much younger
patients now presenting with this condition, and the
10-year recurrence rate after myomectomy gradually
increasing over the years,14,15 an increasing number
of studies has focused on the pathogenesis and mecha-
nisms of uterine fibroids. Most patients with uterine
fibroids have a dysfunction of contraction and endo-
crine disorders.
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Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Figure 4 Expression of collagen
I, insulin-like growth factor
(IGF)-1 and fibronectin were
decreased after myosin heavy
chain 11 (MYH11) interference
in human uterine fibroid cells, as
determined by enzyme-linked
immunosorbent assay. (a) Secre-
tion of collagen I. (b) Secretion
of IGF-1. (c) Secretion of fibro-
nectin. (d) Secretion of proteo-
glycans. The bars indicate the
mean ± standard error (n = 3) for
each group. Data were analyzed
by Student’s t-test. *P < 0.05,
as compared to the control
group. , Scramble RNAi;
, MYH11 RNAi.
Myosins are involved in various cellular functions,
including muscle contraction, cell motility, intracellu-
lar transport, endocytosis, exocytosis, and, probably,
gene expression.7,16 Myosin is a multifunctional protein
and plays an important role in muscle contraction.17 In
recent years, studies have shown that myosin is closely
involved with tumor proliferation, invasion and migra-
tion. MLC phosphorylation is necessary for tumor cell
proliferation, because cell division depends on the
activity of the cytoskeleton, and MLC phosphorylation
causes myosin and actin to interact, thus controlling
cytoskeleton activity, further influencing cell growth.18
Experiments have shown that treating liver cancer cells
with an MLCK inhibitor caused a shape change and
growth inhibition.19 Bessard showed that MLCK plays
a key role in cell cycle progression in hepatocytes, pro-
viding evidence that MLCK regulates S phase entry.20
In this study we found that the expression of myosin,
MLCK and p-MLCK were significantly increased in
uterine myoma, suggesting that an increase in the
MLCK-myosin pathway contributes to cell prolifera-
tion in the development of uterine myoma. Fibroids
are uterine smooth muscle cell-derived tumors. The
collagen components of the uterine fibroids provide for
a strong, fibrous texture.21,22 Uncontrolled secretion
of growth factors leads to this abnormal growth of
uterine smooth muscle cells.23,24 Abnormal secretion
of fibronectin can enhance cell adhesion with the sub-
strate. By sticking, fibronectin can regulate cell shape
and cytoskeleton through the cell signal transduction
pathways. As weak contractions would lead to massive
© 2014 The Authors

bleeding,25,26 we postulated that interference with the
contraction-related protein MYH11 would affect intra-
cellular protein traffic. Myosins are actin-dependent
molecular motors that use the energy of ATP hydroly-
sis to move along actin filaments.27 We knocked down
the expression of MYH11 and observed downstream
protein secretion. Our results show that collagen I,
IGF-1 and fibronectin secretion were reduced after
MYH11 knockdown, which subsequently may reduce
cell proliferation. But proteoglycans did not show any
significant change and this result suggested that the
secretion of proteoglycans may be influenced by other
factors.
In conclusion, this study shows that myosin and
MLCK are highly expressed in myoma tissues, which
suggests that they may be involved in the develop-
ment of myoma. Knockdown of myosin could reduce
the expression and secretion of collagen I, IGF-1 and
fibronectin, further supporting the important role of
myosin in myoma. However, the exact role of myosin
in cell contraction and proliferation needs further
exploration in future.
Disclosure
No author has any potential conflict of interest.
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