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. 2009 Oct 6;94(11):4533–4539. doi: 10.1210/jc.2009-1286

Progesterone and Mifepristone Regulate L-Type Amino Acid Transporter 2 and 4F2 Heavy Chain Expression in Uterine Leiomyoma Cells

Xia Luo 1, Ping Yin 1, Scott Reierstad 1, Hiroshi Ishikawa 1, Zhihong Lin 1, Mary Ellen Pavone 1, Hong Zhao 1, Erica E Marsh 1, Serdar E Bulun 1
PMCID: PMC2775649  PMID: 19808856

Abstract

Context: Progesterone and its receptor (PR) play key roles in uterine leiomyoma growth. Previously, using chromatin immunoprecipitation-based cloning, we uncovered L-type amino acid transporter 2 (LAT2) as a novel PR target gene. LAT2 forms heterodimeric complexes with 4F2 heavy chain (4F2hc), a single transmembrane domain protein essential for LAT2 to exert its function in the plasma membrane. Until now, little is known about the roles of LAT2/4F2hc in the regulation of the growth of human uterine leiomyoma.

Objective: The aim of the study is to investigate the regulation of LAT2 and 4F2hc by progesterone and the antiprogestin mifepristone and their functions in primary human uterine leiomyoma smooth muscle (LSM) cells and tissues from 39 premenopausal women.

Results: In primary LSM cells, progesterone significantly induced LAT2 mRNA levels, and this was blocked by cotreatment with mifepristone. Progesterone did not alter 4F2hc mRNA levels, whereas mifepristone significantly induced 4F2hc mRNA expression. Small interfering RNA knockdown of LAT2 or 4F2hc markedly increased LSM cell proliferation. LAT2, PR-B, and PR-A levels were significantly higher in freshly isolated LSM cells vs. adjacent myometrial cells. In vivo, mRNA levels of LAT2 and PR but not 4F2hc were significantly higher in leiomyoma tissues compared with matched myometrial tissues.

Conclusion: We present evidence that progesterone and its antagonist mifepristone regulate the amino acid transporter system LAT2/4F2hc in leiomyoma tissues and cells. Our findings suggest that products of the LAT2/4F2hc genes may play important roles in leiomyoma cell proliferation. We speculate that critical ratios of LAT2 to 4F2hc regulate leiomyoma growth.

Progesterone and its antagonist mifepristone regulate the amino acid transporter system LAT2/4F2hc in leiomyoma tissues and cells.

Uterine leiomyomata (fibroids) are the most common benign tumors in women, originating from uterine smooth muscle cells. Symptomatic leiomyoma occurs in as many as 30% of women over 35 yr of age (1). They are a frequent cause of abnormal uterine bleeding and are involved in recurrent pregnancy loss. Uterine leiomyomata are the single most common underlying indication for hysterectomy (2). Uterine leiomyomata are responsible for at least one third of approximately 600,000 hysterectomies performed annually in the United States (3).

Although the etiology of uterine fibroids is unknown, the growth of these tumors has been known to be dependent on ovarian steroid hormones (4). Accumulating data support the concept that progesterone plays an important role in regulating the growth of uterine leiomyoma (4,5). The role of progesterone and its receptor (PR) in the pathogenesis of fibroids is highlighted by the following set of findings. First, PR has been observed to be higher in fibroid tissue compared with the myometrial tissue by several groups (6,7). Secondly, the presence of PR in fibroid tissue was also shown to be positively correlated with their growth (8). Most importantly, progestins are able to reverse the reduction of fibroid size that is induced by GnRH agonist therapy when administered as add-back treatment (9,10).

The in vivo effects of progesterone on leiomyoma growth, however, are still not clearly understood. For example, the average leiomyoma size during the first trimester of pregnancy is significantly higher compared with that before the onset of pregnancy (11). On the other hand, leiomyoma size remains stable or can decrease slightly when the first and third trimesters of pregnancy are compared (12,13,14). Thus, progesterone may play dual roles regarding leiomyoma growth under various in vivo conditions.

Previously, we and others have described functions of progesterone-responsive genes that promote fibroid growth (5,15,16). We recently performed a genome-wide chromatin immunoprecipitation-cloning procedure to identify novel binding sites of PR in chromatin isolated from leiomyoma smooth muscle (LSM) cells. We noted that PR was recruited to intron 12 of the L-type amino acid transporter 2 (LAT2) gene (our unpublished observations). Here, we define novel roles of LAT2 and its functional partner, a heavy chain of 4F2 antigen (4F2hc) in LSM cell fate.

LAT2 has a 12-membrane-spanning domain that mediates Na+-independent amino acid exchange. It requires an additional single-membrane-spanning domain protein, 4F2hc, to exert its function in the plasma membrane. LAT2 and 4F2hc form a heterodimeric complex via a disulfide bond (17,18). The mRNAs of LAT2 and 4F2hc are expressed in most embryonic and adult tissues (18,19). LAT2 transports large neutral amino acids, as well as small neutral amino acids (18,19). LAT2 is involved mostly in the basolateral efflux step of transepithelial amino acid transport in the kidney and intestine (19). However, the exact localization of LAT2 on cell membranes is not fully known, and the tissue distribution data are sometimes conflicting (20). The expression and functional properties of amino acid transporters for supplying organic nutrition to cells have not been entirely clarified. Furthermore, nothing is known regarding the expression and function of LAT2/4F2hc in human uterine leiomyoma.

Materials and Methods

Tissue collection and primary cell culture

Human uterine leiomyoma and matched myometrial tissues were obtained at surgery from 39 women (mean age, 40 yr; range, 33–48) undergoing hysterectomy for symptomatic leiomyoma, following a protocol approved by the Institutional Review Board for Human Research of Northwestern University (Chicago, IL). The size of the tumors ranged from 3.5 to 15 cm in diameter. The subjects had not received any hormonal treatment for at least three menstrual cycles before surgery. Each specimen was evaluated histologically by a pathologist. The cycle phase was estimated by the last menstrual period; this was confirmed by endometrial histology. Twenty-two samples were obtained during the follicular phase, 12 during the luteal phase, and five during menstruation. Of these 39 samples, 16 were used for cell culture experiments, whereas 29 were used for the tissue experiments. We used tissues from six women to prepare cells and also for in vivo studies. We isolated LSM cells from the peripheral portions approximately 1 cm from the outer capsule of the leiomyoma, and cultured them as previously described with minor modifications (21). Immunocytochemistry using an antibody against smooth muscle α-actin confirmed purity of the cells (data not shown). Primary cells were used only up to the second passage to avoid changes in phenotype and gene expression. LSM cells were cultured in DMEM/F12 1:1 (GIBCO/BRL, Grand Island, NY) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA). The monolayer cultures at about 70% confluency were starved in serum-free medium overnight and treated with vehicle (ethyl alcohol 1:1000; Sigma-Aldrich, St. Louis, MO), progesterone (3 × 10−7 m; Sigma-Aldrich), or mifepristone (10−8-10−4 m; Sigma-Aldrich).

All cell culture-based experiments were repeated using cells from at least four subjects. One representative experiment was illustrated. Each cell culture-based experiment was carried out in triplicate replicates using cells in first or second passage.

RNA preparation and real-time quantitative PCR

Total RNA from LSM cells was extracted using Tri-reagent (Sigma-Aldrich). cDNA was prepared with qScript cDNA SuperMix (Quanta BioSciences, Inc., Gaithersburg, MD) from 2 μg of RNA. Primers against LAT2, 4F2hc, and the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as described in previous reports (22,23). For LAT2, the forward and reverse primers were 5′-AATGCATTTGAGAATTTCCAGGA-3′ and 5′-GAGCCCTGAAGGAAAGCCA-3′, respectively (22). For 4F2hc, the forward and reverse primers were 5′-CTCAGGCAAGGCTCCTGACT-3′ and 5′-GGCAGGGTGAAGAGCATCA-3′, respectively (22). For GAPDH, the forward and the reverse primers were 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′, respectively (23). Primer specificity was confirmed by the demonstration of single peaks using dissociation curves after amplification of cDNA and a lack of amplification of genomic DNA.

Real-time quantitative PCR was performed to determine the relative amounts of each transcript using the DNA-binding dye SYBR green (Applied Biosystems, Foster City, CA) and the ABI Prism 7900HT Detection System (Applied Biosystems). Cycling conditions started at 50 C for 2 min, followed by 95 C for 10 min, then 40 cycles of 95 C for 15 sec and 60 C for 1 min. The cycle threshold (Ct) was placed at a set level where the exponential increase in PCR amplification was approximately parallel between all samples. Relative fold change was calculated by comparing Ct values between the target gene and GAPDH as the reference guide. The 2−ΔΔCt method was used to analyze these relative changes in gene expression (24).

Small interfering RNA (siRNA)

RNA oligonucleotides directed against LAT2, 4F2hc, and a mismatch negative control siRNA were purchased from Dharmacon (Lafayette, CO). LSM cells were plated at a density of 1.0 × 106 cells per 10-cm dish in phenol free DMEM/F12 containing 10% charcoal stripped fetal bovine serum, but lacking gentamycin/amphotericin B, 1 d before transfection to achieve approximately 50% confluence at the time of transfection. On the day of transfection, the RNAiMAX lipofectamine-based reagent was combined in conjunction with 200 nm siRNA duplexes diluted in Opti-Mem I (Invitrogen) and applied to the cells according to the manufacturer’s instructions. Five hours after the start of the transfection, the medium was changed to growth medium without antibiotics and incubated for 24 h for mRNA and 72 h for Western blot.

Cell viability assay

The effects of LAT2 siRNA or 4F2hc siRNA on the viability of LSM cells was also evaluated by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay (Invitrogen). LSM cells were plated at a density of 4.0 × 104 cells per well in 24-well culture plate 1 d before transfection to achieve approximately 50% confluence at the time of transfection. The transfection was done as previously described and incubated for 72 h. The MTT assay was performed as described by the manufacturer’s protocol with minor modifications. Briefly, at 72 h from start of transfection, 40 μl MTT solution was added to each well, and cells were incubated at 37 C for 4 h. Then 400 μl of the sodium dodecyl sulfate-HCl solution were added to each well and mixed thoroughly. The plate was then incubated at 37 C for another 16–18 h, and samples were transferred to a 96-well plate and read at an absorbance of 570 nm.

Protein isolation and immunoblotting

Cultured LSM and myometrial cells were lysed using Mammalian Protein Extraction Reagent (M-PER; Pierce, Rockford, IL) and 1 × protease inhibitor (Sigma-Aldrich). Protein concentrations were determined by colorimetric BCA protein Assay (Pierce), and equal concentrations of total protein were loaded in each well. Samples were subjected to PAGE (Bio-Rad, Hercules, CA) and transferred onto nitrocellulose membranes (Invitrogen). Membranes were probed using antibodies against β-actin (Sigma-Aldrich), proliferation cell nuclear antigen (PCNA; Millipore, Burlington, MA), LAT2 (Santa Cruz Biotechnology, Santa Cruz, CA) or PR (PGR; courtesy of Dr. Dean Edwards, Baylor College of Medicine, Houston, TX). Antimouse and antigoat IgG conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA) were used as secondary antibodies. Immunoreactive bands were visualized using the ECL-detection system (GE Healthcare, Piscataway, NJ). Quantification of chemiluminescence signal intensity was performed after completion of all autoradiographic studies with ImageJ software (National Institutes of Health, Bethesda, MD).

Statistical analysis

Statistical significance was determined by Student’s t test and one-way ANOVA followed by Fisher’s protected least significance difference test. Significance was accepted at P < 0.05.

Results

Regulation of LAT2 and 4F2hc mRNA levels by progesterone or mifepristone in LSM cells

Our preliminary dose-response (10−10-10−6 m) experiment showed that progesterone regulated LAT2 expression in LSM cells at 10−8, 10−7, and 10−6 m concentrations; peak regulation occurred at 10−6 m dose after 4 h of treatment (data not shown). We chose to use the 3 × 10−7 m concentration of progesterone to further examine its effects on LAT2 and 4F2hc gene expression.

Real-time quantitative PCR analyses were performed after LSM cells were incubated with progesterone (3 × 10−7 m) for 2 and 4 h. LAT2 mRNA levels were significantly induced by progesterone at both 2 and 4 h (Fig. 1A). However, progesterone did not affect 4F2hc mRNA levels (Fig. 1B). Treatment at the same concentration for 72 h also significantly up-regulated LAT2 protein levels (Fig. 1C).

Figure 1.

Figure 1

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Regulation of LAT2 and 4F2hc expression by progesterone or mifepristone. Primary cultured LSM cells were starved in phenol red-free and serum-free DMEM/F12 (1:1) medium overnight and then treated with vehicle (ethanol), progesterone, or mifepristone for different times. LAT2 (A, D, E) and 4F2hc (B, D, F) mRNA levels were measured by real-time PCR and normalized to GAPDH mRNA levels. LAT2 protein levels were evaluated by Western blot using a LAT2 peptide antibody (C). Blots were reprobed with a β-actin antibody for loading control. Results for A, B, D, E and F were reported as a fold change compared with cells treated with vehicle only and represented as the mean ± sem. All graphs were derived from one representative experiment, and all experiments were repeated in triplicate in four subjects. *, P < 0.05, compared with vehicle treatment.

To determine whether progesterone antagonist mifepristone affects LAT2 or 4F2hc expression differentially, we treated LSM cells with various concentrations (10−8-10−4 m) of mifepristone for 4 h. In contrast to the regulation of these two genes by progesterone, mifepristone induced 4F2hc mRNA levels in a dose-dependent manner, but it did not influence LAT2 mRNA level at any concentration (Fig. 1D).

To elucidate whether mifepristone could block the action of progesterone, we treated LSM cells with progesterone alone, mifepristone alone, or their combination for 4 h. Consistently, mifepristone alone did not affect LAT2 expression, but it abolished progesterone-induced LAT2 mRNA level (Fig. 1E). On the other hand, progesterone did not influence 4F2hc mRNA levels, whereas mifepristone significantly induced them (Fig. 1F).

Knockdown of LAT2 is associated with increased LSM cell proliferation

We performed knockdown of endogenous LAT2 to investigate how it may be involved in the regulation of LSM cell proliferation by examining PCNA protein levels and MTT assays. Real-time PCR demonstrated that siRNA against LAT2 significantly reduced its endogenous mean mRNA level by over 90% (Fig. 2A). Western blot analysis confirmed that LAT2 protein level was also markedly decreased by LAT2 siRNA (Fig. 2, B and C). Knockdown of LAT2 increased the proliferation marker PCNA (Fig. 2, B and C), suggesting that LAT2 inhibited proliferation of LSM cells. The effect of siRNA LAT2 knockdown on the proliferation of LSM cells was confirmed by MTT assay, which reflects the total number of viable cells. As shown in Fig. 2D, knockdown of LAT2 significantly increased the number of viable cells. Knockdown of LAT2 also increased the proliferation of myometrial cells from matched tissues (data not shown).

Figure 2.

Figure 2

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The effects of knocking down LAT2 on LSM cell proliferation. LSM cells were transfected with control siRNA or LAT2 siRNA for 72 h. Knockdown efficiency and specificity of the LAT2 gene were examined by both real-time PCR (A) and Western blot (B). Cell proliferation was analyzed by measuring PCNA protein level with mouse antihuman PCNA antibody (B). Blots were reprobed with a β-actin antibody for loading control. Western blot densities were quantified with ImageJ software (C). The effect of knocking down LAT2 on LSM cell proliferation was confirmed with MTT assay (D). Data for A (represented as mean ± sem) and B were from one representative experiment and were repeated in four subjects. Data for C and D show the mean ± sem from four subjects. Each experiment was done in triplicate. *, P < 0.05, compared with control siRNA.

Knockdown of 4F2hc is also involved in increased LSM cell proliferation

We performed knockdown of endogenous 4F2hc to investigate its effects on proliferation of LSM cells. Real-time PCR analysis showed that 4F2hc siRNA reduced endogenous 4F2hc mRNA expression by nearly 80% (Fig. 3A). 4F2hc protein expression could not be demonstrated because a commercial antibody was not available. 4F2hc knockdown by siRNA significantly increased PCNA level (Fig. 3, B and C). Alterations in LSM cell proliferation were also verified by the MTT assay. Figure 3D illustrates that 4F2hc knockdown by siRNA modestly but significantly increased the number of viable cells. These results suggest that 4F2hc may also be involved in inhibition of LSM cell growth. Similar to its effects on LSM cell proliferation, knockdown of 4F2hc stimulated myometrial cell proliferation (data not shown).

Figure 3.

Figure 3

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The effects of knocking down 4F2hc on LSM cell proliferation. LSM cells were transfected with control siRNA or 4F2hc siRNA for 72 h. The cells were then harvested for analysis. Knockdown efficiency and specificity of the 4F2hc gene were examined by real-time PCR (A). LSM cells were harvested for determination of cell proliferation by measuring of PCNA protein level with mouse antihuman PCNA antibody (B). Western blot densities were quantified with ImageJ software (C). The effect of knockdown 4F2hc on LSM cell proliferation was confirmed with MTT assay (D). Blots were reprobed with a β-actin antibody, which was used as a control. 4F2hc mRNA measured by real-time PCR was normalized to GAPDH mRNA. Data for A (represented as mean ± sem) and B were from one representative experiment and were repeated in four subjects. Data for C and D are shown as the mean ± sem from four subjects. Each experiment was done in triplicate. *, P < 0.05, compared with control siRNA.

Expression of LAT2 and PR in cultured LSM cells and matched myometrial cells

The effects of progesterone on target tissues are mediated by PR. Previous studies have demonstrated that PR levels are higher in LSM cells than in myometrial cells (6). To determine whether cell-specific differences in LAT2 levels are accompanied by comparable differences in PR, LAT2 and PR protein expression in LSM cells and matched myometrial cells was examined using Western blot analysis. Compared with myometrial cells, both PR and LAT2 protein expression was higher in cultured LSM cells (Fig. 4, A and B).

Figure 4.

Figure 4

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Comparison of LAT2 and PR protein levels in cultured LSM cells with matched myometrial cells. Cell lysates were prepared for immunoblotting with anti-LAT2, anti-PR and β-actin (loading control) antibodies (A) and quantified with ImageJ software (B). Data in A from one representative result were reproduced in cells from four patients. Data in B represent the mean ± sem from four subjects. *, P < 0.05, compared with myometrial cells.

Gene expression of LAT2, 4F2hc, and PR in leiomyoma and matched myometrial tissues

To understand the in vivo relevance of progesterone-regulated LAT2 and 4F2hc expression in leiomyoma, we analyzed mRNA levels of PR, LAT2, and 4F2hc genes in human leiomyoma and matched myometrial tissues from 29 subjects. To allow comparisons of data obtained from samples of different subjects, the mRNA levels of LAT2, 4F2hc, and PR were normalized to GAPDH, and then the mRNA levels in leiomyoma tissues were expressed as a multiple of that value in matched myometrial tissues, allowing the detection of relative mRNA differences between the two tissues.

As shown in Fig. 5, mean LAT2 and PR mRNA levels were significantly higher in leiomyoma than myometrial tissues. However, there was no significant difference in mRNA levels of 4F2hc between leiomyoma and matched myometrial tissues.

Figure 5.

Figure 5

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LAT2, 4F2hc, and PR mRNA levels in human leiomyoma and matched myometrial tissues. Overall, 58 samples from 29 patients were analyzed; 29 samples were obtained from leiomyoma and 29 from adjacent myometrial tissues. To allow comparisons of data obtained from samples from different patients, mRNA levels in the myometrial tissues were normalized to 1. The data were shown as the mean ± sem. *, P < 0.01, compared with myometrial tissues.

Discussion

In this study, we have demonstrated that progesterone and its antagonist mifepristone regulated LAT2 and 4F2hc expression. LAT2 knockdown studies suggested that LAT2 inhibits LSM cell growth, although its product is considered to be an amino acid transporter. Thus, the role of LAT2 in leiomyoma pathology may seem paradoxical at first sight. The functional properties of LAT2 for supplying organic nutrition to cells have not been entirely clarified. It has been reported that LAT2 operates as an amino acid exchanger (19,25). There is also evidence to suggest that amino acid efflux via LAT2 is not dependent upon extracellular amino acids (18,26). The net direction of the transport of large neutral amino acids by LAT2 is believed to depend on the unidirectional transporters that are coexpressed in the cells. Thus, the role of LAT2 is most likely an equilibration of the amino acid distribution across the two membranes, whereas other transporters determine the actual net amino acid flux (27). Knockdown of LAT2 in LSM cells might have changed the equilibration of the amino acid distribution across the cell membrane, which could then alter the net flux of amino acid determined by other transporters. The final outcome, therefore, may be the increase of cell growth.

We also found that knockdown of 4F2hc increased the proliferation of LSM cells, suggesting that this gene may inhibit leiomyoma growth. Our result is different from what was demonstrated in a previous study (28), which showed that a monoclonal antibody against 4F2hc antigen inhibited lymphocyte activation and cell proliferation. This difference may be due to the different cell systems and methods used. To our knowledge, the present study is the first to demonstrate the function of LAT2/4F2hc through knockdown of these genes, and the first to report the expression and function of LAT2/4F2hc in leiomyoma.

It should be noted that LAT2 requires 4F2hc to function normally in the plasma membrane (17,18). Knocking down 4F2hc could lead to a loss of function of LAT2. Thus, our observation that knockdown of 4F2hc stimulated leiomyoma cell proliferation is consistent with the effect of LAT2 knockdown on the same cell type. In vivo, however, LAT2 but not 4F2hc expression in leiomyoma tissue was higher. It is likely that critical ratios of LAT2 to 4F2hc determine the net in vivo effects of this L-type amino acid transporter system in leiomyoma tissue. To add a further twist, treatment with progesterone increases, whereas mifepristone decreases, the LAT2 to 4F2hc ratio. Evidence from a number of laboratories suggests that progesterone stimulates, whereas mifepristone inhibits leiomyoma growth (4,5,29). The sum of these observations is consistent with a model that a higher in vivo LAT2 to 4F2hc ratio in leiomyoma tissue may favor its growth. Further studies are needed to clearly understand this complex mechanism.

We find that progesterone or mifepristone regulates LAT2 or 4F2hc expression via PR. PR exists as two isoforms, PR-A and PR-B (30). In humans, PR-B and PR-A may have distinct or similar functions depending on the promoter context and cell type (31,32). Thus, it seems justified to further evaluate the effects of each isoform on LAT2 or 4F2hc expression in LSM cells.

Information on the hormonal mechanisms regulating system-L transporter activity is still scarce (33,34,35). Steroid hormones may act via multiple mechanisms. They affect not only the transcriptional modulation of target genes via intracellular receptors within minutes to hours but also elicit rapid nongenomic effects within seconds to minutes. Steroid hormones have been shown to enhance or diminish amino acid transport depending on the nature of the hormone. The stimulation may involve either increased carrier synthesis or rapid nongenomic effects at the cell membrane (36,37), whereas inhibition may involve synthesis of a labile protein that either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system (38).

In summary, we demonstrated the functions of two genes involved in amino acid transport through the cell membrane in leiomyoma cell growth. The products of these genes heterodimerize to regulate amino acid transport and also regulate leiomyoma proliferation; moreover, progesterone or an antiprogestin regulates their expression. We speculate that critical ratios of LAT2 to 4F2hc may regulate leiomyoma growth.

Acknowledgments

We thank Dr. Dean Edwards (Baylor College of Medicine, Houston, TX) for the PR antibody.

Footnotes

This work was supported by National Institutes of Health Grant HD46260.

Disclosure Summary: X.L., P.Y., S.R., H.I., Z.L., M.E.P., H.Z., and E.E.M. have nothing to declare. S.E.B. serves as a consultant for the pharmaceutical companies Repros, Meditrina, and Novartis.

First Published Online October 6, 2009

Abbreviations: Ct, Cycle threshold; 4F2hc, 4F2 heavy chain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LAT2, L-type amino acid transporter 2; LSM, leiomyoma smooth muscle; PCNA, proliferation cell nuclear antigen; PR, progesterone receptor; siRNA, small interfering RNA.

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