Mol. Hum. Reprod. Advance Access originally published online on August 31, 2007
Molecular Human Reproduction 2007 13(11):797-806; doi:10.1093/molehr/gam063
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The expression profile of micro-RNA in endometrium and endometriosis and the influence of ovarian steroids on their expression
Department of Obstetrics/Gynecology, University of Florida, Box 100294, Gainesville, FL 32610, USA
1 Correspondence address. Tel: +1-352-273-7566; Fax: +1-352-392-6994; E-mail: cheginin{at}obgyn.ufl.edu
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Abstract |
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MicroRNAs (miRNAs), through mRNA degradation or repression, act as key regulator of gene expression. Our aim was to identify specific miRNAs that are expressed in endometrium of women with and without endometriosis. We profiled the expression of 287 miRNAs in paired eutopic and ectopic endometrium and isolated endometrial cells using microarray and validated the expression of selected miRNAs using real-time PCR. On the basis of global normalization, 65 of these miRNAs were identified to be expressed above the threshold levels set during the analysis in the endometrium of women without endometriosis with a progressive decline in expression in paired eutopic and ectopic endometrium. Statistical analysis (ANOVA) identified 48 of these miRNAs as differentially expressed among these tissues and 32 miRNAs between isolated endometrial stromal cell (ESC) and glandular epithelial cell (GEC) (P < 0.05). The expression of hsa-miR20a, hsa-miR21, hsa-miR26a, hsa-miR18a, hsa-miR206, hsa-miR181a and hsa-miR142-5p, predicted to target many genes, including TGF-ßR2, ER
, ERß and PR, respectively, was validated in these tissues and cells using real-time PCR. Treatment of ESC and GEC with 17ß-estradiol and medroxyprogesterone acetate (10–8 M) differentially regulated the expression of hsa-miR20a, hsa-miR21 and hsa-miR26a, which in part reversed following co-treatment with ICI-182780 and RU-486 (10–6 M), respectively (P < 0.05). In conclusion, we provided evidence for the expression of a number of differentially expressed miRNAs in eutopic/ectopic endometrium and isolated endometrial cells, opening up the possibility that aberrant/altered expression of some miRNAs whose expression is regulated by the ovarian steroids may influence the expression of specific target genes with central roles in normal endometrial cellular activities and pathogenesis of endometriosis. Key words: endometrium/endometriosis/miRNA/expression/regulation
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Introduction |
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Endometrium is a dynamic tissue that undergoes specific cyclic changes under the control of ovarian steroids during the reproductive years. The ovarian steroids also play a central role in pathogenesis of several uterine disorders, including endometriosis, which is characterized by the presence of endometrial tissue fragments outside the uterine cavity. Both eutopic and ectopic endometrium express ER
and ERß with predominantly higher levels of ER in ectopic lesions (Matsuzaki et al., 2000, 2001). Although eutopic endometrium expresses both PR-A and PR-B, ectopic endometrium has been reported to only express PR-A (Attia et al., 2000). In addition, altered local estrogen metabolism in ectopic endometrium has been associated with progression of endometriosis (Bulun et al., 2006). Despite the importance of ovarian steroids in pathogenesis of endometriosis, the nature of the processes that lead to establishment of endometriosis remains unknown. Conventional and recent large-scale gene expression studies have provided further evidence reflecting the molecular environments that differentiate normal endometrium from disorders affecting this tissue, including endometrial cancer, adenomyosis and endometriosis (Eyster et al., 2002; Kao et al., 2002; Riesewijk et al., 2003; Risinger et al., 2003; Matsuzaki et al., 2004; Ponnampalam et al., 2004; Hever et al., 2006; Wu et al., 2006). The product of many of these genes have been considered to contribute toward normal endometrial cyclic changes and associated endometrial disorders; however, the expression, regulation and biological significance of many of them in normal physiological and pathophysiology of endometrium remain to be established. Recent identification of a group of small non-coding RNA referred to as microRNAs (miRNAs) and their function analysis has led to the discovery of their key regulatory function in gene expression. miRNAs are expressed as 70–90 bp precursor RNA that are processed by the nuclease Drosha and transported into the cytoplasm where they are further cleaved by dicer, resulting in formation of a 17–23 bp mature miRNAs (Calin and Croce 2006a,b; Engels and Hutvagner 2006; Jovanovic and Hengartner, 2006; Zeng 2006). These mature miRNAs complex with the target sequences in a complementary manner similar to that occur during RNA interference process; however, their target sequences could be at least partially complementary to the miRNA (Bartel 2004; Pillai 2005). To date, several hundred miRNAs have been cloned and/or predicted, each with the ability to modulate the expression of their target genes through cleavage or translational regression. The expression of many of these miRNAs has been identified in a number of mammalian cells and tissues, including humans (Roldo et al., 2006; Zhao et al., 2006); however, their biological significance in many cellular processes remains to be established. Evidence indicates that the genes for many of miRNAs have been located at chromosomal fragile sites or regions of cytogenetic abnormalities associated with cancer and other disorders and their altered expression has been associated with tumorogenesis. Evidence also supports their importance in developmental processes as well as other cellular activities involving cell growth, differentiation and apoptosis (Ambros 2004; Engels and Hutvagner 2006; Jovanovic and Hengartner, 2006; Zeng 2006).
Many of the above processes are involved in cyclic endometrial changes and the establishment and progression of endometriosis, suggesting the implication of specific miRNAs in regulating the endometrial expression of genes whose products influence the outcome of this and other endometrial disorders. The aim of the present study was to profile the expression of miRNAs in eutopic and ectopic endometrium as well as in isolated endometrial stromal cell (ESC) and glandular epithelial cell (GEC) using microarray. In addition, we validated the expression of several of these miRNAs selected based on their predicted target genes, including TGF-ß, TGF-ß receptors, ER, ERß and PR, recognized to play a central role in endometriosis, in these tissues and cells using real-time PCR. We also examined the influence of ovarian steroids, 17ß-estradiol and medroxyprogesterone acetate, as well as the effect of ICI-182780 and RU-486, their respective antagonists on the expression of these miRNAs in endometrial cells.
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Materials and Methods |
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All the materials for isolation of ESC and GEC and culture media were purchased from commercial sources as previously described (Chegini et al., 1999). MirVana RNA isolation and enrichment kits and miRNA Bioarray slides for human miRNA detection were purchased from Ambion (Austin, TX, USA) and real-time PCR reagents were purchased from Applied Biosystem (Foster city, CA, USA). 17ß-estradiol (E2), medroxyprogesterone acetate (MPA) and RU486 (Mifepristone) were purchased from Sigma Chemical Co. (St Louis, MO, USA). ICI-182780 (Fulvestrant) was purchased from Tocris Cookson, Inc. (Ballwin, MO, USA) and charcoal-stripped fetal calf serum was purchased from (Hyclone, Logan, UT, USA) and utilized as previously described (Ripley et al., 2001).
Tissue collection
Portions of endometrium from women without endometriosis (n = 4; EN), paired eutopic and ectopic endometrium (n = 4; EU and EC) and ectopic endometrium (n = 4; EE, tissues without the paired eutopic endometrium) were collected from premenopausal women who were scheduled to undergo hysterectomy for indications related to symptomatic leiomyomas or endometriosis, respectively. The patients age ranged from 27 to 39 years and were not taking any medication, including hormonal therapy for 3 months prior to surgery. On the basis of their last menstrual period and endometrial histology, the tissues were from early-mid secretory phase of the menstrual cycle. The endometriosis was identified as stage III according to ASRM guideline. The tissues were collected at the University of Florida affiliated Shands Hospital under an expedited study protocol approved by the Institutional Review Board at the University of Florida without requiring written informed consent. Immediately after collection, the tissues were snapped frozen and kept in liquid nitrogen for further analysis, or used for cell isolation and culturing.
Endometrial cell isolation and culturing
A small portion of endometrial tissues was used for isolation of ESC and GEC cells as previously described (Chegini et al., 1999). The isolated cells were cultured in Dulbecco's Modified Eagle's Medium/Hams (DMEM:F12) containing antimycotic, antibiotics and 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified 5% CO2 incubator until reaching confluence. Prior to use, the cell cultures were characterized using antibodies to vimentin and cytokeratin based on immunofluoresence microscopy (Luo et al., 2003). Human endometrial epithelial cell line (HES), derived from spontaneous transformation of isolated endometrial surface epithelial cells from benign proliferative endometrium, was kindly provided by Dr D. Kniss at Ohio State University, Columbus, OH and cultured under the same condition as for the GEC (Luo et al., 2003).
miRNA expression analysis
Total RNA isolated from the above tissues and cells was subjected to mirVana miRNA isolation kit according to manufacturer's instructions (Ambion). Briefly, 30 µg of total RNA was loaded onto the top of a column filled with a denaturing acrylamide gel matrix and the miRNA fraction was obtained by mixing RNA with 2x sample buffer and flashPAGE purification using flashPAGE precast Gels and the flashPAGE Fractionator System. The RNA quality, yield and size of miRNA fractions were analyzed using Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, CA, USA).
miRNA labeling and hybridization
Purified miRNA isolated from the above samples was labeled at 3' amine-modified tails using mirVana miRNA Array Labeling Kit and fluorescently coupled with Cy5 post-Labeling Reactive Dye (Amersham, GE Healthcare Bio-Sciences, Piscataway, NJ, USA). Chemically synthesized oligoribonucleotides was used as positive control (Ambion) and labeled along with purified miRNAs. A 3x miRNA hybridization buffer (Ambion) was added to the fluorescent-labeled miRNAs and the solution was heated at 95°C for 2 min. The Cy5 labeled samples were washed three times using miRNA washing buffer, mixed in the same post-labeling cleanup kit, eluted and stored at –70°C or analyzed by hybridizing to mirVana miRNA Bioarray Slides. The slides were placed into hybridization chambers (Corning Incorporated Life Sciences, Acton, MA, USA) and 20 µl of miRNA and 10 µl of hybridization buffer were added to hybridization mixture under the Bioarray LifeSlip. The chamber was sealed and incubated at 42°C in a water bath for 12–16 h. Each probe on the mirVana miRNA Bioarray Slide is printed in duplicate with 20 positive and 100 negative controls (Ambion).
Array data processing and analysis
Following hybridization, the slides were washed, dried and scanned on a GenePix 4000B Array Scanner (Molecular Devices Corporation, Sunnyvale, CA, USA). The miRNA spots and their intensity were determined using GenePix Pro 6.0 software as recommended by the manufacturer. Background-adjusted spot intensity for each miRNA was subjected to a global variance stabilization normalization (VSN) procedure (Huber et al., 2002) recommended by Ambion (http://www.ambion.com/techlib/ resources/miRNA-array/da-bioarrays.html) and described by Davison et al. (2006). This analysis identifies differentially expressed miRNA with precision and quadratic relationship between the variance with lesser focus on absolute expression and fold-change difference. The expression values were subjected to unsupervised hierarchical clustering and Tree-View analysis (Davison et al., 2006).
Treatments
To determine whether ovarian steroids regulate the endometrial expression of miRNAs, 1 x 106 ESC and GEC cells were seeded in six-well culture dishes and incubated for 48 h as described earlier. The cells were washed and incubated under serum-free condition for 24 h and then treated with E2 (10–8M), MPA (10–8 M), ICI-182780 (10–6 M), RU486 (10–6 M), E2+ICI or MPA+RU486 added to phenol red-free medium containing 2% charcoal-stripped FBS for 24 h (Ripley et al., 2001).
Real-time polymerase chain reaction
Real-time PCR was carried out to verify the expression of hsa-miR20a, hsa-miR21, hsa-miR26a, hsa-miR18a, hsa-miR181a, hsa-miR206 and hsa-miR142-5p selected based on their predicted target genes (which include TGF-ß, TGF-ß receptor, ER, ERß and PR, respectively). Briefly, 10 ng of total RNA was reverse transcribed to cDNA with stem-loop primers for the above miRNAs and TaqMan® miRNA Reverse Transcription kit. Quantitative real-time PCR was carried out using an Applied Biosystems 7300 Real-time PCR System and a Taqman Universal PCR Master Mix at 95°C for 10 min, 95°C 15 s and 60°C for 1 min for 40 cycles. The results were analyzed using comparative method following normalization of expression values to U6 and hsa-let-7a expression as recommended by the manufacturer using Sequence Detection Software 2.2.1 (Applied Biosystems). These miRNAs were selected for verification based on their predicated target genes listed in the Sanger miRBase database (http://microrna.sanger.ac.uk/sequences, Adams et al., 2007).
Statistical analysis
All the in vitro experiments were performed three times using independent cell cultures. Where appropriate, the results are expressed as mean ± standard error (SE) and statistically analyzed using Student's t testing for comparison of two groups and ANOVA for multiple comparisons, with P < 0.05 considered significant.
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Results |
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Using miRNA Bioarray Slides containing 287 human miRNA probe sets, we first assessed their expression profile in the endometrium of women without endometriosis (EN), paired eutopic (EU) and ectopic (EC) endometrium as well as ectopic endometrium (EE; without paired eutopic tissue). We also profiled the expression of these miRNAs in ESC and GEC isolated form the same endometrium of women without endometriosis. The analysis revealed the expression of a considerable number of miRNAs in these tissues and cells with progressive decline in their numbers from EN to EU, EC and EE (figures not shown). Of these miRNAs, the expression of 65 miRNAs in these tissues was above the threshold levels set during the analysis. There was also a lower number of miRNAs expressed in GEC when compared with ESC (figures not shown). Global normalization of the miRNAs mean expression values and statistical analysis (ANOVA) resulted in the identification of 48 miRNAs as differentially expressed among these tissues with progressive decline in the level of their expression from EN to EU, EC and EE (Table 1). Figure 1 illustrates the unsupervised hierarchical clustering and Tree-View analysis of these differentially expressed miRNAs in EN, EU, EC and EE with each tissue separated into their respective subgroup some with overlapping relatedness. Using a similar analysis, we identified 32 miRNAs as differentially expressed in ESC and GEC (Table 2), a significantly lower numbers when compared with EN. These miRNAs were commonly expressed in the original endometrial tissues used for isolation of ESC and GEC.
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We selected hsa-miR20a, hsa-miR21, hsa-miR26a, hsa-miR18a, hsa-miR181a, hsa-miR206 and hsa-miR142-5p and validated their expression in the above tissues and cells using real-time PCR. The selection of these miRNAs was based on their predicted target genes (Table 3), which include TGF-ß, TGF-ß receptors, ER
, ERß and PR (http://microrna.sanger.ac.uk/sequences, Adams et al., 2007). Although these miRNAs are expressed in EN, EC, EU and EE as well as GEC and ESC, the microarray analysis indicated no significant difference in the level of their expression with the exception of hsa-miR21, hsa-miR26a and hsa-miR142-5p (Tables 1 and 2). Considering the PCR threshold cycle (Ct) values, the order of relative expression of these miRNAs in EN was hsa-miR20a> hsa-miR21>hsa-miR26a and for the remaining miRNAs was hsa-miR18a>hsa-miR181a>hsa-miR142-5P>hsa-miR206.
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Following individual normalization for comparative analysis by setting the expression value of each miRNA in EN independently as 1, the relative level of expression of hsa-miR21 and hsa-miR26a was lower in EC and EE when compared with EN and EU (Fig. 2A; P < 0.05). In contrast, the relative expression of hsa-miR20a was higher in EC and EE than in EN and EU. The level of expression of hsa-miR18a [but not consistently hsa-miR206 (ER
) and hsa-miR181a (ERß)] was higher in EU, EC and EE when compared with EN, whereas the expression of hsa-miR142-5p (PR) was higher in EU when compared with EC (Fig. 2B; P < 0.05). The level of expression of these miRNAs did not display a trend similar to their mean expression values obtained from the microarray (Table 1).
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The expression of hsa-miR20a, hsa-miR21 and hsa-miR26a was also differentially expressed in ESC and GEC (Fig. 3). Since the expression of miRNAs is altered in cell cultures and cellular differentiation when compared with their levels at tissue levels (Szafranska et al., 2007), we used HES line for comparative analysis with ESC and GEC isolated from the same EN. Setting the expression of hsa-miR20a, hsa-miR21 and hsa-miR26a in HES cells as 1, the results indicated that GEC expressed more hsa-miR20a and hsa-miR21 when compared with hsa-miR26a, whereas ESC expressed higher levels of hsa-miR26a and hsa-miR21 with a low level of hsa-miR20a when compared with HES (Fig. 3, P < 0.05).
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We next examined the effect of ovarian steroids on the expression of hsa-miR20a, hsa-miR21 and hsa-miR26a in ESC and GEC. The results indicated that E2 (10–8 M) inhibited the expression of hsa-miR20a and hsa-miR21 in ESC, and hsa-miR21 in GEC when compared with untreated control (Figs 4 and 5; P < 0.05). Although a limited difference was observed between E2- and MPA-treated cells, MPA (10–8 M) inhibited the expression of these miRNAs in ESC, while increasing the expression of hsa-miR20a and hsa-miR26a in GEC when compared with untreated control (Figs 4 and 5; P < 0.05). Treatment with ICI-182780 (10–6 M) inhibited the expression of hsa-miR20a, hsa-miR21 and hsa-miR26a in ESC as well as hsa-miR21 in GEC when compared with untreated control (Figs 4 and 5; P < 0.05). In contrast, the expression of hsa-miR26a was increased in ICI-treated GES (Fig. 5; P < 0.05). RU-486 (10–6 M) had no significant effect on hsa-miR20a and hsa-miR21 in ESC and hsa-miR20a in GEC; however, it inhibited hsa-miR26a in ESC, and hsa-miR21 in GEC, while increasing the expression of hsa-miR26a in GEC (Figs 4 and 5; P < 0.05). Co-treatment of these cells with E2+ICI or MPA+RU in most cases resulted in differential pattern of expression of these miRNAs when compared with cells treated with E2, MPA, ICI or RU486 and untreated controls, respectively (Figs 4 and 5; P < 0.05). There was no significant difference in the level of expression of these miRNAs after 12 and 24 h of treatments and the results of the 24 h treatments are presented here.
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Discussion |
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miRNAs are a novel class of regulatory molecules with the ability to control gene expression at the post-transcriptional level through degradation, repression or silencing. Since each miRNA has been predicted to have a broad range of target mRNA based on degree of sequence homology, it is estimated that the expression of
30% of genes are the potential target of miRNA regulatory function (Yu et al., 2006). As such, changes in the expression of even a single miRNA could have a significant impact on the outcome of diverse cellular activities regulated by the product of these genes. In the present study, we profiled 287 human miRNAs and identified the expression of considerable number of them in the endometrium of women without endometriosis (EN) which progressively declined in paired eutopic (EU) and ectopic (EC) endometrium. On the basis of the statistical analysis of the mean expression values, 48 of these miRNAs were identified as differentially expressed among these tissues. There was also a progressive decline in the level of expression of these miRNAs (Table 1) from EN to EU, EC and EE (ectopic endometrium without paired eutopic tissues). These miRNAs were also expressed in isolated endometrial cells with a significant reduction in numbers and level of expression (Table 2) when compared with the original tissues used for isolating these cells. As such, the results suggest that absence/altered expression of a substantial number of miRNAs in ectopic endometrium could result in expression re-programming of a large number of genes when compared with paired eutopic endometrium as well as comparing EN with EU. As a result, gene expression re-programming the endometrial fragments derived from women with endometriosis may have an altered regulatory mechanism that leads to their survival and growth at the ectopic sites when compared with tissues derived from women without endometriosis. We recognize that relatively low number of tissues (n = 16) collected from the early-mid secretory phase of the menstrual cycle as a limitation of our study. As such, further study is necessary to profile the expression of these miRNAs in the endometrium throughout the menstrual cycle and at eutopic and ectopic endometrium at various stages of endometriosis. In addition, correlation of expression of specific genes targeted by some of these miRNAs would allow establishing their biological relevance to endometrial normal physiological and pathological disorders, including endometriosis. Despite these limitations, the results of miRNA expression profiles in EN, EU and EC are consistent with studies involving other tissues and cells associating absence and/or altered expression of miRNAs with developmental and various pathological conditions such as malignant cellular transformation and cancer development. With respect to a lower number of miRNAs expressed by the isolated endometrial cells (ESC and GEC) and HES, when compared with the endometrium, studies profiling miRNAs expression in isolated cells in cultured or cell lines when compared with their corresponding benign and cancer tissues have reported similar trends (Michael et al., 2003; Lim et al., 2005; Bandres et al., 2006; Lee et al., 2006; Mineno et al., 2006; Volinia et al., 2006; Weber et al., 2006; Yu et al., 2006; Szafranska et al., 2007). In addition, we found a significantly lower level of expression of three of these miRNAs in HES, which is a spontaneously transformed endometrial epithelial cell line, when compared with GES and ESC. Although tissue variations could account for some of the difference, the biological significance of changes in the endometrial expression of specific miRNAs in vivo under normal and disease status and HES, ESC and GEC in vitro require careful investigation reflecting their function during this transition.
Using real-time PCR, we validated the expression of hsa-miR20a, hsa-miR21, hsa-miR26a, hsa-miR18a, hsa-miR181a, hsa-miR206 and hsa-miR142-5p in these tissues and observed some difference in the level of their expression when compared with microarray analysis. Although microarray profiling did not show a significant difference in the level of expression of some of these miRNAs among the tissues, they were selected because of their predicted target genes which include TGF-ß, TGF-ß receptors, ER, ERß and PR, respectively (Table 3, Adams et al., 2007). The difference between microarray and real-time PCR results could be attributed to detection of both precursor and mature forms of miRNAs by microarray and mature form by real-time PCR (Lee et al., 2006). The results provided additional evidence for differential expression of these miRNAs in the endometrium, more specifically between paired eutopic and ectopic endometrium, although correlating their expression with the expression of target genes is necessary. However, a large number of target genes for a single miRNA and multiple miRNAs targeting the expression of one gene have been recognized as a major drawback in assessment of specific target gene regulated by a given miRNA at the transcriptional or translational levels. It has been suggested that prediction of such high number of genes regulated by a single miRNA contain a significant fraction of false-positive genes and procedures have been recommended to assess the genes with a potential functional relevance in specific tissue biology (Chaudhuri and Chatterjee, 2007). Although the expression of many of the genes regulated by these miRNAs (Table 3) have been identified in the endometrium during the menstrual cycle, in ectopic endometrium and in isolated endometrial cells (Eyster et al., 2002; Kao et al., 2002; Riesewijk et al., 2003; Ponnampalam et al., 2004; Matsuzaki et al., 2004; Hever et al., 2006; Wu et al., 2006), it is essential to correlate the expression of a number of these genes with their corresponding miRNAs.
Functionally, TGF-ß and TGF-ß receptor signaling pathways have been associated with several normal cyclic endometrial activities and uterine disorders including endometriosis (Chegini and Williams 2000; Chegini et al., 2003; Luo et al., 2003, 2004). In addition, differential expression of ER, ERß and PR in ectopic endometrium is considered to reflect their response to ovarian steroid actions (Bulun et al., 2006). Despite some discrepancies, both eutopic and ectopic tissues express ER and ERß with predominantly higher levels of ER and higher relative ratio of ER:ERß in ectopic endometrium with the expression detected in both epithelial and stromal cells (Matsuzaki et al., 2000, 2001). With respect to progesterone receptors, the ectopic endometrium is reported to only express PR-A, whereas eutopic endometrium expresses both PR-A and PR-B (Attia et al., 2000). As such, altered expression of hsa-miR20a, hsa-miR21, hsa-miR26, hsa-miR18a, hsa-miR181, hsa-miR206 and hsa-miR142-5p in EC when compared with EN and EU could influence the stability of TGF-ß, TGF-ß receptors, ER, ERß and PR and other target genes, resulting in changes in their local expression and actions, and the outcome of endometriosis. Functional analysis also indicates that the expression of these miRNAs is associated with cellular apoptosis, differentiation, cell–cell communication and tumorogenesis, with their genes localized to sites of frequent chromosomal instability (Chan et al., 2005; Meng et al., 2006; Si et al., 2007).
We also provided evidence that ovarian steroids influence the expression of hsa-miR20a, hsa-miR21 and hsa-miR26a in ESC and GEC. The molecular mechanism by which ovarian steroids regulate the expression of miRNAs requires detailed investigation; however, such a regulatory function may alter the expression of their target genes and cellular activities manifested by their products. We also found that ICI-182780 and RU486, alone or in combination with E2 and MPA, in part altered the expression of these miRNAs. We believe this is the first such demonstration of regulatory action of ovarian steroids on the expression of miRNAs and alteration by ICI and RU-486. Selective estrogen and progesterone receptor modulators and ICI-182780 have been demonstrated to effectively regress the growth of endometrial implants in animal model of endometriosis, in clinical trails in human and the endometrial cell growth in vitro (DeManno et al., 2003; Chabbert-Buffet et al., 2005). Our results suggest that ICI-182780 and RU486 may act by targeting the endometrial expression of miRNAs resulting in a re-programming of their target genes expression; however, further studies are needed to examine the molecular mechanism by which these agents influence the expression of miRNAs and their target genes.
In conclusion, the results provided the first evidence for the expression of unique set of miRNAs in the endometrium and endometrial cells, as well as in paired ectopic and eutopic endometrium. Because a substantial number of miRNAs are either aberrantly and/or differentially expressed in ectopic endometrium with a selective number of them regulated by the ovarian steroids, due to expression re-programming of their target genes, various cellular activities regulated by their products may alter leading to the survival and growth of cells in ectopic site. In the future blocking or over-expression of specific miRNAs in endometrial cells may provide a method of targeting genes involved in pathogenesis of endometriosis, including ovarian steroid receptors.
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Funding |
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National Institute of Health (HD37 432).
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Submitted on July 27, 2007; accepted on August 21, 2007.
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