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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2013 Dec 15;7(1):64–79.

The onset of human ectopic pregnancy demonstrates a differential expression of miRNAs and their cognate targets in the Fallopian tube

Yi Feng 1,2,3,4,*, Shien Zou 5,*, Birgitta Weijdegård 4, Jie Chen 5, Qing Cong 5, Julia Fernandez-Rodriguez 6, Lei Wang 7,8, Håkan Billig 4, Ruijin Shao 4
PMCID: PMC3885461  PMID: 24427327

Abstract

Human ectopic pregnancy (EP) is a leading cause of pregnancy-related death, but the molecular basis underlying the onset of tubal EP is largely unknown. Female Dicer1 conditional knockout mice are infertile with dysfunctional Fallopian tube and have a different miRNA expression profile compared to wild-type mice, and we speculated that Dicer-mediated regulation of miRNA expression and specific miRNA-controlled targets might contribute to the onset of tubal EP. In the present study, we used microarray analysis and quantitative RT-PCR to examine the expression of miRNAs and core miRNA regulatory components in Fallopian tube tissues from women with EP. We found that the levels of DICER1, four miRNAs (let-7i, miR-149, miR-182, and miR-424), and estrogen receptor α distinguished the tubal implantation site from the non-implantation site. Computational algorithms and screening for interactions with the estrogen and progesterone receptor signaling pathways showed that the four miRNAs were predicted to target ten genes, including NEDD4, TAF15, and SPEN. Subsequent experiments showed differences in NEDD4 mRNA and protein levels between the implantation and non-implantation sites. Finally, we revealed that increases in smooth muscle cell NEDD4 and stromal cell TAF15, in parallel with a decrease in epithelial cell SPEN, were associated with tubal implantation. Our study suggests that changes in miRNA levels by the DICER-mediated miRNA-processing machinery result in aberrant expression of cell type-specific proteins that are potentially involved in the onset of tubal EP.

Keywords: Dicer, microRNAs, target genes, tubal ectopic pregnancy, infertility

Introduction

Human ectopic pregnancy (EP) is a significant clinical problem for reproductive-aged women and their healthcare providers [1,2]. EP results from disruption of the tubal transport process and complicates up to 2% of all pregnancies in the Western world [3]. It still accounts for 4 to 10% of pregnancy-related deaths [4]. More than 98% of EPs occur in the Fallopian tube [5], and women with past tubal EP are at increased risk of infertility or tubal EP in the future [1]. Unfortunately, there are currently no effective means for the prediction, prevention, or treatment of tubal implantation [5,6]. Despite intense research efforts, the underlying cause of tubal EP remains a mystery [7-9]. This is to a large part due to our incomplete understanding of the changes in the complex molecular events behind ciliary beating, muscle contraction, and the tubal fluid microenvironment during the transport of gametes [9]. Disruption of these events results in abnormal interactions between the early embryo and tubal cells and leads to tubal implantation [5].

The process of fertilization, early embryonic development, and implantation begins in the Fallopian tube and is the result of coordinated biochemical and physiological steps [10]. Efforts have been made to identify the changes in tubal gene expression that are related to specific stages of the estrous cycle, estrogen-induced tubal transport, tubal development, and tubal secretion and contractility [11-14]. Spatiotemporal alterations in tubal transcriptome profiles are required for normal tubal functions under physiological conditions [15]. Thus it is important for us to identify the key regulators of these changes in gene expression in the Fallopian tube as a way of understanding the progression of EP. Despite decades of research, there is still a crucial need for a comprehensive understanding of the pathophysiology of tubal EP development that fully integrates gene regulation, signaling pathways, and cellular mechanisms in the Fallopian tube [16].

MicroRNAs (miRNAs) are evolutionarily conserved, small, noncoding RNAs (usually 21 to 24 nucleotides) that have emerged as regulators of overall gene expression and represent yet another layer of control over mRNA stability and translation [17]. To date, over 1,500 human miRNAs have been identified (http://www.mirbase.org/). miRNA profiling in humans shows that approximately 50% of miRNAs are expressed in a tissue-specific manner [18]. The growing body of evidence indicates that miRNA regulation is involved in human physiological and pathological processes. For example, changes in miRNA expression contribute to normal intrauterine pregnancy (IUP) [19] and to susceptibility to disease conditions such as preeclampsia, endometriosis, and endometrial cancer [20].

Dicer and Drosha, two RNase III enzymes, are key regulators that are responsible for miRNA maturation and function [17], and deletion of the Dicer1 gene results in an overall loss of miRNAs and is embryonic lethal in mice [21]. In addition, the Dicer-controlled miRNA pathway has been shown to have critical functions in the Fallopian tube. For example, adult female Dicer1 conditional knockout mice are infertile and exhibit Fallopian tubal hypotrophy and prominent tubal cysts at the isthmus near the uterotubal junction that disrupt tubal transport [22-24]. Although the regulation of human DICER1 expression in the Fallopian tubes under physiological conditions has been reported [25], very little is known about its role in tubal dysfunction and tubal EP in particular.

Mammalian miRNAs have the potential to regulate roughly 60% of all human genes [26]. In general, each miRNA can directly or indirectly regulate a diverse set of downstream target genes that are involved in all cellular processes, including those involved in disease progression [17]. It has been reported that miRNAs are differentially expressed in normal human Fallopian tubes [18,27]. Recently, the expression levels of several circulating miRNAs have been shown to be novel biomarkers for the diagnosis of tubal EP [28]. Together, these observations prompted us to investigate whether changes in key components of the miRNA biogenesis pathway contribute to widespread miRNA deregulation and to identify the specific miRNAs that target genes involved in the onset of Fallopian tube implantation.

Materials and methods

Ethics approval

This study was reviewed and approved by the Ethics Committees of the Obstetrics and Gynecology Hospital and Shanghai Medical College, Fudan University, China and in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants.

Human subject characteristics and tissue collection

All participants underwent clinical examinations at the Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China. The age, reason for surgery, gestational days, beta-human chorionic gonadotropin (β-hCG) levels, and experimental methods for all patients are provided in Supplemental Table 1. The exclusion criteria used have been described previously [29].

The present study included women in their first trimester of pregnancy (n=30) who were subdivided into the following two groups:

Group 1. This group comprised healthy pregnant women undergoing therapeutic surgical pregnancy termination as defined as decidual-endometrial specimens (n=10, mean age 29.0±4.03 years, and mean gestation 49.2±5.77 days). The gestational age was confirmed by individual menstrual cycle data and physical examination. The diagnosis of a normal IUP was made based on serum β-hCG measurements and the observation of an intrauterine gestational sac on the transvaginal or transabdominal ultrasound scans. Decidual-endometrial tissues devoid of any myometrial tissue were retrieved directly after vacuum aspiration.

Group 2. In this group, women with EP (n=20, mean age 36.30±4.26 years, mean gestation 47.83±10.68 days) were studied after tubal surgery. A full medical history was documented and a clinical examination was carried out by the attending physician. Transvaginal ultrasonography was performed, and in each case the EP was verified by the clear localization of a trophoblast in the Fallopian tube. Serum β-hCG levels were analyzed in patients at the time of their first clinical presentation. None of the women undergoing surgical management of EP presented with acute hemodynamic shock. In women with EP and with two or more children that had no plans for future childbearing, the laparoscopic surgery was combined with bilateral tubal sterilization and tubal samples were collected from both the affected EP site and the contralateral unaffected site [16].

After collection, all tissue samples were washed with ice-cold RNase-free phosphate-buffered saline (PBS) and either snap-frozen in liquid nitrogen and stored at -70°C or fixed in 4% formaldehyde and embedded in paraffin. The same sample was prepared for microarray analysis, quantitative real-time PCR (qRT-PCR), western blot analysis, and microarray analyses, qRT-PCR, western blot analysis, and immunohistochemistry; or immunofluorescence assay analysis and immunohistochemistry (Supplemental Table 1).

Total RNA isolation, miRNA microarray analysis, and array data analysis

Total RNA, including miRNAs, was isolated with the Trizol system (Invitrogen, NY) and the miRNeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturers’ instructions. The purity and quantity of the isolated RNAs were assessed by the ratio of spectrophotometric absorbance at 260 and 280 nm using a Nanodrop spectrophotometer (ND-1000, Nanodrop Technologies). A ratio of 2.0 for the sample absorbance was considered indicative of sufficient purity, and the RNA integrity was determined by gel electrophoresis. The miRNA microarray experiments were performed by KangChen Bio-tech (Shanghai, China). The samples were labeled using the miRCURY Hy3/Hy5 Power labeling kit and hybridized on the miRCURY LNA Array (v.16.0) (Exiqon, Vedbaek, Denmark). This array contains ~1223 capture probes for human miRNAs and allows for quantification of genome-wide miRNA expression. Scanning was performed with the Axon GenePix 4000B microarray scanner (Axon Instruments, Foster City, CA), and GenePix Pro V6.0 (Axon) was used to read the raw intensities of the images. miRNA expression data were normalized using the median normalization procedure, and the average values of replicate spots of each miRNA were used for statistical analysis. The following two criteria were used to identify the miRNAs with altered abundance among the different sample sets: an absolute change of ≥2-fold and a proportion of false positives <0.05. Values for gene expression (2-ΔΔCt) were reported as mean±SEM. The latter is used to control for errors in multiple tests. Hierarchical clustering was performed using MultiExperiment Viewer software (MEV, v4.6, TIGR) to show the distinguishable miRNA expression profiles among the samples.

Bioinformatics predictions

Because the various miRNA target prediction programs often produce different lists of predicted targets, we used a combination of seven databases, including TargetScan, DIANA, microRNA.org, miRBase, PITA, PicTar, and RNA22, to predict the target genes for the differentially expressed miRNAs [30]. The distribution of gene expression was unknown so only the genes identified by at least four algorithms were considered as potential target genes regulated by a given miRNA.

Quantitative real-time PCR analysis

Total RNA and miRNA were prepared as described above. Two micrograms of RNA were reverse transcribed into first-strand cDNA using SuperScript™ III Reverse Transcriptase (Invi-trogen). The reactions were performed on an ABI PRISM7900 system (Applied Biosystems, Foster City, CA) using the SYBR RT-PCR kit (Takara, Capitola, CA) according to the manufacturer’s instructions. The size and specificity of the Amplicons were confirmed by 2.5% agarose gel electrophoresis. The primer sequences and amplification products for the miRNAs and genes in this study are provided in Supplemental Table 2. All reactions were performed in duplicate and each reaction included a non-template control. The relative expression levels of the miRNAs were normalized to that of an internal control (U6, a small nuclear RNA) in all samples. The CT values for both β-actin and RPLPO (a human ribosomal protein) were not significantly different in any of the groups, and this confirmed that the loading was similar between the samples. The results for each gene of interest are expressed as the amount relative to the average value of β-actin + RPLPO in each sample. RNase-free water (QIAGEN) was included as a negative control during RNA extraction and in each qRT-PCR run.

Western blot analysis

Whole-tissue extracts for protein preparations and western blot analyses were carried out as described previously [31]. Briefly, samples were separated on 4-12% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes. The blots were probed with primary antibodies against human DICER1 and β-actin (Supplemental Table 3) overnight at 4°C. After three rinses with TBS/0.05% Tween 20, the membranes were incubated with anti-mouse IgG peroxidase-conjugated goat antibody (1:4000, A2304, Sigma-Aldrich, St. Louis, MO) for 2 h. Protein bands were visualized with SuperSignal West Dura Extended Duration Substrate (34076, Thermo Scientific, Pierce Biotechnology, Rockford, IL). Immunoblot signals were visualized with an LAS 1000 cooled CCD camera (Fujifilm). Equal protein loading was also confirmed by Coomassie staining.

Immunohistochemistry

Immunohistochemical analysis was performed as previously described [32]. Tubal tissue samples were cut into 5 μm sections and placed on Superfrost Plus slides. After deparaffinizing the sections, the slides were rinsed twice for 5 min with 0.01 M TBS. The sections were treated with 3% H2O2 to remove endogenous peroxidase activity and were blocked to prevent nonspecific binding. The primary antibodies (Supplemental Table 3) were diluted in TBST containing 0.05% NGS and incubated overnight at 4°C. After several rinses with TBST, the sections were stained using the avidin-biotin-peroxidase complex detection system (ABC kit, Vector Laboratories). Immunostaining was visualized by immersing the sections in 3-amino-9-ethylcarbazole working solution (AEC kit, Vector Laboratories) for 20-30 min. Sections were viewed on an Olympus BX60 microscope (Olympus, Shinjuku, Japan) under bright field optics, and photomicrographed with the Viewfinder program (Olympus). Negative control slides used normal rabbit serum of equivalent concentration in place of primary antibodies and were prepared identically and processed with TBST containing 0.05% NGS. Quantification of the degree and localization of the immunostaining was performed by independent observers who did not know the patient’s information or the identity of the protein studied.

Assessment of circulating β-hCG levels

Measurement of serum β-hCG levels has been described previously [29].

Statistical analysis

Results are expressed as the mean±SEM, and all statistical analysis was performed using SPSS version 16.0 for Windows (SPSS Inc., Chicago, IL). Significance was tested by two-way ANOVA followed by Bonferroni correction for multiple comparisons as necessary. P<0.05 was considered significant.

Results

Alteration of core miRNA regulatory components is associated with the onset of tubal implantation

Because it is not possible to collect Fallopian tubes from women with normal IUP, the comparison of the Fallopian tube from the implantation site to the contralateral non-implantation site of the EP women to evaluate gene/protein expression has its advantages. We used qRT-PCR to assess the mRNA expression levels of DROSHA, DGCR8, EXPORTIN5, DICER1, AGO2, and TRBP - key regulatory components of the miRNA biogenesis pathway - in Fallopian tube tissues from women with EP. We found a significantly lower level of DICER1 mRNA in the implantation site compared to the non-implantation site (Figure 1A). Because specific mRNAs and proteins are changing as normal implantation progresses [33], the decidual-endometrial tissues from women with normal IUP were used as controls. There were significant differences in EXPORTIN5 and AGO2 mRNA levels between women with tubal EP and women with IUP; however, the EXPORTIN5 and AGO2 mRNA levels were not significantly different between the implantation and non-implantation sites in women with EP. We did not find any significant differences in the expression of DROSHA, DGCR8, or TRBP mRNA levels between women with EP and IUP (Figure 1A).

Figure 1.

Figure 1

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miRNA biogenesis in women with ongoing intrauterine pregnancy (IUP) and tubal ectopic pregnancy (EP). (A) The expression of core microRNA transcripts in the decidualized endometrium (DE) of women with ongoing IUP and in the Fallopian tube of women with EP. The figure illustrates that DICER1 mRNA expression in women with tubal EP was significantly higher in the non-implantation site (NIS) compared to both the implantation site (IS) and the DE of women with ongoing IUP. (B) The expression of cell marker transcripts (cytokeratin 8 and α-smooth muscle (SM)-actin) in the DE of women with ongoing IUP and in the Fallopian tubes of women with EP. The determination of mRNA expression levels was described in the Materials and Methods. Expression of each mRNA was normalized to the average expression value of β-actin + RPLOP (a human ribosomal protein) and is shown in arbitrary units. qRT-PCR values in (A) and (B) are the mean±SEM (n=10 patients/group). Significance was tested by two-way ANOVA with Bonferroni correction for multiple comparisons when appropriate. *P<0.05; **P<0.01; ***P<0.001 vs. the respective control group. (C) Western blotting of DICER1 protein. The blot was probed with a specific antibody against human DICER1 as described in the Materials and Methods. The correct loading was evaluated by staining the gels with Coomassie blue (data not shown) and by immunoblotting with an antibody against β-actin. DGCR8, DiGeorge syndrome critical region gene 8; AGO2, argonaute RISC catalytic component 2; TRBP, trans-activation response RNA binding protein.

To ensure that changes in the DICER1 mRNA level were not influenced by differences in the population of tubal cell types, we also examined the mRNA levels of the cell markers cytokeratin 8 and α-smooth muscle (α-SM) actin in the Fallopian tube tissues from women with EP and in the decidual-endometrial tissues from women with IUP (Figure 1B). We found a significant difference in α-SM actin mRNA levels between the Fallopian tube tissues from women with EP and the decidual-endometrial tissues from women with IUP, but the mRNA levels for cytokeratin 8 and α-SM actin were similar between the implantation and non-implantation sites in women with EP, suggesting that tubal EP did not influence the population of cell types.

The levels of DICER1 protein were also examined by western blot analysis (Figure 1C). Eight out of ten tubal tissue samples from women with EP showed lower levels of DICER1 protein in the implantation site compared to the non-implantation site, consistent with the DICER1 mRNA expression.

Differential expression of miRNAs at the onset of tubal implantation

We hypothesized that changes in DICER1 expression result in the widespread miRNA deregulation that is associated with the onset of tubal implantation (Figure 2A). To test this hypothesis, we used microarray analysis to identify the globally expressed miRNAs in the Fallopian tubes from the implantation and non-implantation sites of women with EP. We compared the miRNA expression profiles between the tubal implantation site and the non-implantation site, between the tubal implantation site and the decidualized endometrium from women with an IUP, and between the tubal non-implantation site and the decidualized endometrium. We focused our attention on miRNA expression that met our criteria of P<0.05 and a greater than 2-fold difference in expression (Supplemental Tables 4, 5 and 6).

Figure 2.

Figure 2

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Comparisons of miRNAs that are differentially expressed in women with ongoing intrauterine pregnancy and tubal ectopic pregnancy. A: Flowchart of this investigation. B: Hierarchical clustering analysis of differentially expressed miRNAs using miRNA microarray analysis. Differentially expressed miRNAs are defined as those with at least a 2-fold change in expression between the decidualized endometrium and the tubal implantation site or between the decidualized endometrium and the tubal non-implantation site as determined by ANOVA using all 20 participants. Red blocks represent higher miRNA expression, green blocks represent lower miRNA expression, and black blocks represent no significant difference. C: Scatter plot of miRNA expression in paired IS vs. NIS Fallopian tubes from women with ectopic pregnancy. The vertical lines correspond to a 2-fold increase or decrease, and the horizontal line represents a p-value of 0.05. Red spots in the plot represent miRNAs with significantly different expression levels. D: A Venn diagram showing the numbers of differentially expressed miRNAs identified in the current study. The differentially expressed miRNAs are listed below the diagram. Red indicates increased miRNA expression and blue indicates decreased miRNA expression. DE, decidualized endometrium; IS, implantation site; NIS, non-implantation site.

The microarray results revealed that a specific miRNA expression significantly discriminated between women with tubal EP and women with IUP. Interestingly, 10 miRNAs were up-regulated and 14 miRNAs were down-regulated in the tubal implantation site compared with the tubal non-implantation site (Figure 2C, Supplemental Table 4). Furthermore, we found that a total of 47 miRNAs, 19 up-regulated and 28 down-regulated, were differentially expressed in the tubal implantation site compared with those in the tubal non-implantation site after comparison to the expression of miRNAs in the decidualized endometrium (Figure 2D). A total of 25 miRNAs were up-regulated and 34 miRNAs were down-regulated in the tissue from the tubal EP implantation site compared to the decidualized endometrium from women with IUP (Figure 2B, Supplemental Table 5). A total of 46 miRNAs were up-regulated and 47 miRNAs were down-regulated in the tubal EP non-implantation site compared to the decidualized endometrium control tissue (Figure 2B, Supplemental Table 6).

Experimental verification of miRNA expression

To verify the accuracy of the microarray results, we used qRT-PCR to measure the expression levels of let-7i, miR-100, miR-125b, miR-142-5p, miR-143, miR-145, miR-149, miR-182, miR-183, miR-200c, miR-22, miR-224, miR-25, miR-298, miR-29c, miR-424, miR-513b, miR-518f, miR-618, miR-96, and miR-9*. Of the 20 differentially regulated miRNAs, four miRNAs (let-7i, miR-149, miR-182, and miR-424) had significantly different expression levels between the implantation and non-implantation sites in women with EP (Figure 3).

Figure 3.

Figure 3

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Identification of the miRNAs that are differentially expressed in women with ongoing intrauterine pregnancy compared to those with tubal ectopic pregnancy. The figure (red star) shows that the expression of the let-7i, miR-149, miR-182, and miR-424 miRNAs was significantly higher in the implantation site (IS) of women with tubal ectopic pregnancy compared to both the non-implantation site (NIS) and the decidualized epithelium (DE) of women with ongoing intrauterine pregnancy. Determination of selected miRNA expression levels is described in the Materials and Methods. Expression of each miRNA was normalized to U6 and shown in arbitrary units. qRT-PCR values are shown as the mean±SEM (n=10 patients/group). Significance was tested by two-way ANOVA with Bonferroni correction for multiple comparisons when appropriate. *P<0.05; **P<0.01; ***P<0.001 vs. the respective control group.

Target prediction of differentially and selectively expressed miRNAs

Because the steroid hormones 17β-estradiol (E2) and progesterone (P4) regulate critical tubal functions during implantation and pregnancy [34], we focused on the expression of the two human estrogen receptor (ER) subtypes (ERα and ERβ1/2) and the two progesterone receptor (PR) isoforms (PRA and PRB). These receptors act as transcription factors in response to E2 and P4 stimulation in the Fallopian tube [35]. In line with the data available in the literature [36,37], the qRT-PCR analysis revealed that the ERα mRNA level was significantly lower in the tubal implantation site than in the tubal non-implantation site in women with EP (Figure 4A). We also found that the ERβ1 mRNA level was significantly lower in the tubal implantation site than in the decidualized endometrium from women with IUP and that the PRB mRNA level was significantly lower in the tubal non-implantation site than in the decidualized endometrium from women with IUP. This suggests that different ER subtypes and PR isoforms are involved in early pregnancy processes in a tissue-specific manner.

Figure 4.

Figure 4

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Identification of steroid hormone receptor-related and miRNA-regulated target genes in women with ongoing intrauterine pregnancy and tubal ectopic pregnancy. (A) The expression of estrogen receptor (ER) subtype and progesterone receptor (PR) isoform transcripts in the decidualized endometrium (DE) of women with ongoing intrauterine pregnancy and in the Fallopian tube of women with ectopic pregnancy (EP). (B) The target genes predicted to be regulated by let-7i, miR-149, miR-182, and/or miR-424. (C) The expression of predicted target genes in the DE of women with ongoing intrauterine pregnancy and in the Fallopian tube of women with EP. Determination of mRNA expression levels was described in the Materials and Methods. Expression of each mRNA was normalized to the average expression value of β-actin + RPLOP and is shown in arbitrary units. qRT-PCR values in (A) and (C) are the mean±SEM (n=10 patients/group). Significance was tested by two-way ANOVA with Bonferroni correction for multiple comparisons when appropriate. *P<0.05; **P<0.01; ***P<0.001 vs. the respective control group. IS, implantation site; NIS, non-implantation site.

A number of miRNAs have been reported to be involved in the regulation of ER and PR at either the transcriptional or post-transcriptional levels [38,39]. The combination of several target prediction algorithms, as we have used in the current study, provides fewer false miRNA target genes than the individual algorithms [30]. This computational analysis was followed by screening for interaction with the ER and PR signaling pathways. We found that four miRNAs (let-7i, miR-149, miR-182, and miR-424), either alone or in combination, were predicted to target several different genes (Figure 4B).

Identification of miRNA co-targeted genes at the onset of tubal implantation

We used qRT-PCR to compare the expression levels of KRAS, RBFOX2, NEDD4, KLF9, UBR5, SOS1, TAF15, MED1, PHB, and SPEN in Fallopian tube tissues from the tubal implantation and non-implantation sites of women with EP (Figure 4C). Decidual-endometrial tissues from women with IUP were used as controls. The qRT-PCR analysis revealed that the expression of NEDD4, UBR5, TAF15, MED1, and SPEN mRNAs was significantly lower in women with EP than in women with IUP. We did observe a significant difference in NEDD4 mRNA between the tubal implantation and non-implantation sites in women with EP (Figure 4C), but we did not find any significant differences in the levels of UBR5, TAF15, MED1, or SPEN mRNAs between the two sites. We did not find any significant differences in the levels of KRAS, RBFOX2, KLF9, SOS1, or PHB mRNA between any of the groups.

Tubal cell-specific regulation of selected miRNA targets at the onset of tubal implantation

To further determine whether alterations of NEDD4, UBR5, TAF15, MED1, and SPEN protein levels were associated with changes in their mRNA levels, we determined the cellular localization and expression level of these proteins in the Fallopian tube tissue from women with EP (Figures 5 and 6). Consistent with changes in NEDD4 mRNA levels, we found a sharp induction of NEDD4 protein expression in the α-SM cells of the tubal implantation site (Figure 5A) compared to the non-implantation site (Figure 5B). In contrast, the protein levels of NEDD4 in the epithelial cells of the tubal implantation site (Figure 5A) were comparable to those of the non-implantation site (Figure 5B). We also showed that the protein levels of TAF15 were decreased in the epithelial cells but increased in the stromal cells of the tubal implantation site (Figure 5C) compared to the non-implantation site (Figure 5D). Specific UBR5 and MED1 immunohistochemical staining was observed, but we observed no differences between the epithelial cells of the tubal implantation (Figures 5E and 6A) and non-implantation (Figures 5F and 6B) sites. Finally, the immunoreactivity for SPEN was lower in the epithelial and stromal cells of the tubal implantation site (Figure 6C) than in these cell types on the non-implantation site (Figure 6D).

Figure 5.

Figure 5

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Distribution of NEDD4, TAF15, and UBR5 proteins in the Fallopian tube tissue from women with tubal ectopic pregnancy. NEDD4 (A and B), TAF15 (C and D), and UBR5 (E and F) expression was determined by immunoperoxidase staining with antibodies against these proteins. Representative images (n=5-10 patients/group) from two independent experiments are shown. Epi, epithelial cells; Str, stromal cells; SM, smooth muscle cells. Scale bar = 100 μm.

Figure 6.

Figure 6

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Distribution of MED1 and SPEN proteins in the Fallopian tubes of women with tubal ectopic pregnancy. MED1 (A and B) and SPEN (C and D) expression was determined by immunoperoxidase staining with antibodies to MED1 and SPEN. MED1-positive cells among the epithelial cells are shown in the inset at higher magnification. A negative immunological control (normal rabbit serum) did not show any staining in the tubal epithelial or stromal cells (E). Representative images (n=5-10 patients/group) from two independent experiments are shown. Epi, epithelial cells. Scale bar = 100 μm.

Discussion

The biogenesis of miRNAs requires the action of Dicer [17], and female Dicer1 conditional knockout mice have highlighted the importance of miRNAs in regulating the functions of the Fallopian tube [22-24]. To our knowledge, the potential involvement of miRNAs in the onset of implantation in human Fallopian tube has not previously been investigated. Our data reveal that the expression levels of the miRNA processing enzyme DICER1 in tissues from the tubal implantation site from women with EP and from the decidual-endometrial tissues from women with IUP are lower than in tissues from the tubal non-implantation site from women with EP. Using microarray technology, qRT-PCR, and computational algorithms, we determined the miRNA expression profiles of the Fallopian tube tissues from women with EP and identified specific miRNAs (let-7i, miR-149, miR-182 and miR-424) that are linked to the ER and PR signaling pathways. Furthermore, we observed that cell-specific regulation of the miRNA targets NEDD4, TAF15, and SPEN occurred in parallel to changes in the ultrastructural morphology of the tubal cells after the onset of tubal EP.

Although the molecular events leading to tubal EP are poorly understood, abnormally elevated E2 levels have been suggested to increase the risk for tubal EP [35]. The cellular responses to E2 are mediated by two nuclear ERs [40]. Both ERα and ERβ1/2 are expressed in the healthy Fallopian tube, and ERα expression levels do not change during the menstrual cycle [25,37]. In our previous study we showed that the temporal variation of human DICER1 expression was correlated with ERα and ERβ2 expression in the Fallopian tubes throughout the menstrual cycle [25]. E2 stimulation induces uterine receptivity [41] and the hatching and activation of the blastocyst [42] during the establishment of an IUP, and in the current study we found that DICER1 and ERα expression levels were significantly decreased in the cells of the tubal implantation site compared to the non-implantation site in women with EP. These results led us to ask whether the E2-ERα interaction contributes directly to the regulation of DICER1 expression during aberrant implantation of the early embryo in the Fallopian tube.

It has been reported that DICER1 has an ERα-binding site and that its expression is enhanced by E2 stimulation in human breast cancer cells in vitro [43]. Such regulation, however, has not been observed in the mouse uterus in vivo [44]. In vitro treatment of Fallopian tubal tissues with E2 or the ER antagonist ICI-182,780 failed to change DICER1 expression in cells from either the tubal implantation or non-implantation site of women with EP (unpublished data). This indicates that an apparent feedback loop between E2, ERα, and DICER1 seems unlikely to exist during the onset of tubal EP. In addition, previous experiments provide evidence that several miRNAs target genes involved in ER expression and regulation in the human endometrium [45]. It is more probable that as yet unidentified upstream triggers of DICER1 dysregulation and DICER1-mediated processing of specific miRNAs play a dominant role in the diminished ERα expression during implantation in the human Fallopian tube.

The Fallopian tube is normally unable to initiate implantation [10] and the tubal microenvironment is normally hostile to the full development of the embryo [15]. Both of these factors act as barriers to prevent the early embryo from interacting with the tubal epithelium [7,46], and the implantation of the blastocyst in the Fallopian tube is probably the result of an intricate succession of cellular and molecular interactions [15]. In the present study, the observation of altered DICER1-mediated processing of miRNAs in the tubal implantation site compared to the non-implantation site provides new hope for unraveling the complex relationship between miRNA processing and onset of tubal EP. Interestingly, we have identified four miRNAs (let-7i, miR-149, miR-182, and miR-424) that are differentially expressed in the implantation site compared to the non-implantation site. However, it should be noted that our data suggest an association but not necessarily causality between the expression of specific miRNAs and the progression of tubal implantation.

To increase our understanding to predict which women will be more likely to suffer a tubal EP is limited by the lack of animal models of tubal EP [5,6]. This also makes it difficult to determine which miRNAs might be critical regulators of the development of tubal EP. The current understanding of the molecular events that occur during intrauterine implantation stems primarily from research into normal pregnancies in rodents. The expression levels of several miRNAs have been shown to be significantly different in the mouse uterus between the implantation site and non-implantation site. For example, let-7i [47,48], miR-182, and miR-424 [49] are very important candidate miRNAs for embryo implantation in the mouse uterus, and this is consistent with our findings that let-7i and miR-182 had higher expression levels in the tubal implantation site of women with EP. However, miR-424 expression in women with EP is down-regulated in the tubal implantation site in contrast to its up-regulation in the uterine implantation site in mice. This divergent result likely reflects differences between the two species, tissue-specific effects, and/or differences between ectopic and intrauterine implantation. Nevertheless, the potential role of these miRNAs in the Fallopian tube will need to be evaluated once better approaches, such as tubal-specific conditional knockout animal models and well-defined tubal cells, are developed.

miRNAs function as regulatory RNA transcripts that lead to protein translation inhibition or messenger RNA degradation [17]. Up to now, however, any role for miRNAs in the regulation of expression of the genes and proteins involved in the pathogenesis of tubal EP was still unknown. Using a computational approach, we show here that the let-7i, miR-149, miR-182, and miR-424 miRNAs have several putative target genes in the pathways involved in tubal EP. This implies a broad regulatory potential for these miRNAs in the Fallopian tube under pathophysiological conditions. It would be interesting to test whether a gain or loss of function of one miRNA regulates one or multiple targets in the Fallopian tube. In addition to miRNA expression analysis, the potential biological significance of the four miRNAs identified in the Fallopian tubes from women with EP was demonstrated by studying the regulation of their targets NEDD4, TAF15, and SPEN. We found differences in both the mRNA and protein levels of NEDD4 between the tubal implantation and non-implantation sites, but we only saw significant differences in the mRNA expression of TAF15 and SPEN. Because miRNAs can either retard or accelerate mRNA degradation [17], we speculate that the rate of TAF15 and SPEN mRNA decay triggered by the miRNAs might not exceed the rate of their transcription. A more intriguing possibility is that miRNAs are direct regulators of the TAF15 and SPEN gene expression at the level of translation control during tubal implantation.

Although the localization of Nedd4 in the uterus is unclear, the levels of Nedd4 mRNA have been shown to be regulated in the mouse uterus at the implantation site [50]. Knockout of Nedd4 in mice is embryonically lethal and mutant embryos exhibit a reduction in skeletal muscle fiber size [51]. We report here the novel observation of up-regulation of NEDD4 in the smooth muscle cells from the tubal implantation site in women with EP. These results suggest that NEDD4 may be involved in tubal functions specific to muscle cells such as tubal contractility.

TAF15 is an RNA binding protein that has been shown to be a coactivator of basal transcription [52]. In human endometrium, the immunoreactivity of TAF15 is detected in glandular epithelial and stromal cells and probably varies in the stromal cells between the proliferative and secretory stages [53]. We observed that protein levels of TAF15 are decreased in the epithelial cells in the tubal implantation site, which is the opposite of what was observed in the stromal cells. The initial intercelluar interaction between the uterine epithelial cells and the blastocyst are essential for successful uterine implantation [41,42]. The shift in high levels of TAF15 expression from epithelial cells to stromal cells may suggest a role for TAF15 in making the Fallopian tube more receptive to implantation.

SPEN is an E2-inducible gene that functions as a transcriptional repressor [54], and our immunohistochemical analysis demonstrates that SPEN expression levels are reduced in the epithelial and stromal cells from the tubal implantation site in women with EP. At present, the mechanisms underlying the E2-mediated down-regulation of SPEN expression in the Fallopian tube remain unclear. Even though the full biological functions of NEDD4, TAF15, and SPEN in the Fallopian tube have yet to be completely elucidated, our study provides new insights into the candidate molecules that might be participating in Fallopian tube dysfunction.

In summary, our investigation constitutes the first detailed analysis to show that a significant decrease in DICER1 expression is accompanied by significant dysregulation of numerous miRNAs, including let-7i, miR-149, miR-182, and miR-424, in Fallopian tube tissues from the tubal implantation site of women with EP. Our findings not only provide a global miRNA expression profile but also identify four miRNAs that are specifically misexpressed in women with EP. We also show how aberrant regulation of the specific and distinctive miRNA targets NEDD4, TAF15, and SPEN might contribute to the onset of tubal EP. Extracellular miRNAs are very stable and abundant in the circulation [55] and they can be used as potential biomarkers for human diseases, including tubal EP [28]. Although several potential risk factors for tubal EP have been proposed [5,7-9,35], future prospective studies using relatively large cohorts are still needed to determine whether the alteration of miRNA biogenesis and subsequent changes in the regulation of miRNA targets presented in this study are associated with women at risk for EP.

Acknowledgements

The authors thank Dr. Andor Pivarcsi (Department of Medicine, Center for Molecular Medicine, Karolinska Institute, Sweden) for bioinformatics analysis. This work was supported by grants from the Swedish Medical Research Council (Grant 5859 and 10380), the Swedish federal government under the LUA/ALF agreement (ALFGBG-147791), the Sahlgrenska Academy Research Council, Göteborgs Läkaresällskap, Jane and Dan Olsson’s Foundation, the Hjalmar Svensson Foundation, Anna Cederberg’s Foundation, Åke Wiberg’s Foundation, Wilhelm-Martina Lundgren’s Foundation, the Wennergren Foundation, Clas Groschinskys Minnesfond and the Royal Society of Arts and Sciences in Gothenburg, as well as National Nature Science Foundation of China (81001544 and 81102668) and “ZuoXue” Foundation of Fudan University, China, to YF. The funding sources of this study were not involved in the study design, the collection, analysis, or interpretation of the data, the writing of the report, or in the decision to submit the paper for publication. Part of the study was presented in a preliminary form at the 24th European Congress of Pathology: Pathology - Science for Patients, Czech Republic, September 8-12, 2012.

Disclosure of conflict of interest

None.

Supporting Information

ijcep0007-0064-f7.pdf (159.2KB, pdf)

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