Overexpression of myosin is associated with the development of uterine myoma
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 determine 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.
Introduction
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 pathogenesis of uterine fibroids has focused on cell differentiation.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 patients' uterine cavity pressure using a pressure sensor, and found that patients with uterine fibroids showed abnormal uterine contraction, possibly leading to infertility.4, 5 Subsequently, by observing the ultrastructure using electron microscopy they also found differences in the density band of myoma cells compared to normal uterine smooth muscle cells, indicating that contraction of the myoma cells is abnormal.6
As is well known, the contraction-related pathway and its regulation are important in uterine pathophysiology. The thick myofilaments of the myofibrils are composed of myosins, which play an important 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 contraction.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 committee 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 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 template 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′. Glyceraldehyde 3-phosphate dehydrogenase was used as an internal control, using the primers: forward 5′-ATCAGCAATGCCTCCTGCAC-3′, reverse 5′-CGTCAAAGGTGGAGGAGTGG-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 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 transferred 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 secondary 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 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).
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. Representative increased expression 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 antibodies and biotinylated secondary antibodies, and then DAB dye was used for color development (StreptAvidin-Biotin Complex, ×100). Strong expression is shown in brown staining, while low expression is shown in blue staining.
| Myoma (n = 30) | Smooth muscle (n = 30) | P-value* | |
|---|---|---|---|
| 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.
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).
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.
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 transduced 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 determine their growth rate. As shown in Figure 3d, the growth rate of MYH11 knockdown cells was significantly reduced compared to control cells (P < 0.05).
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 ± standard error (n = 3) for each group. Data were analyzed by Student's t-test. *P < 0.05, as compared to the control group.
Effect of MHC downregulation on uterine fibroid cells
The knockdown of MHC could change the structure and distribution of the cytoskeleton. This might result in changes in secretion of some factors. In order to investigate the effect of MHC downregulation on uterine fibroid cells, we analyzed the expression of collagen I, IGF-1, fibronectin and proteoglycans, all of which play important roles in uterine fibroid cell proliferation. ELISA was performed to detect the extracellular secretion of collagen I, IGF-1, fibronectin and proteoglycans. The results showed that collagen I 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).
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) Secretion of collagen I. (b) Secretion of IGF-1. (c) Secretion of fibronectin. (d) Secretion of proteoglycans. 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.
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 mechanisms of uterine fibroids. Most patients with uterine fibroids have a dysfunction of contraction and endocrine disorders.
Myosins are involved in various cellular functions, including muscle contraction, cell motility, intracellular 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 migration. 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, providing 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 proliferation 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 substrate. By sticking, fibronectin can regulate cell shape and cytoskeleton through the cell signal transduction pathways. As weak contractions would lead to massive bleeding,25, 26 we postulated that interference with the contraction-related protein MYH11 would affect intracellular protein traffic. Myosins are actin-dependent molecular motors that use the energy of ATP hydrolysis 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 development 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.
