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. Author manuscript; available in PMC: 2018 Jan 1.
Published in final edited form as: Placenta. 2016 Nov 9;49:1–9. doi: 10.1016/j.placenta.2016.11.002

Expression Patterns of the Chromosome 21 MicroRNA Cluster (miR-99a, miR-125b and let-7c) in Chorioamniotic Membranes

Bhavi P Modi 1, Sonya Washington 2, Scott W Walsh 2, Colleen Jackson-Cook 1,2,3, Kellie J Archer 4,*, Jerome F Strauss III 1,2
PMCID: PMC5502786  NIHMSID: NIHMS829715  PMID: 28012448

Abstract

Trisomy 21 (T21) is the most common chromosome abnormality in humans and is associated with a spectrum of phenotypes, including cognitive impairment, congenital heart defects and immune system defects. In addition, T21 is also associated with abnormalities of fetal membranes including chorioamniotic separation, delayed fusion of the chorioamniotic membranes, defects in syncytiotrophoblast formation, as well as amniocyte senescence. There is evidence indicating miRNAs encoded by sequences on chromosome 21 (Chr-21) are involved in several of the cognitive and neurological phenotypes of T21, but the role of Chr- 21 derived miRNAs in fetal membrane abnormalities associated with T21 has not been investigated. In the current study, we determined the expression patterns of three miRNAs derived from a cluster on Chr-21 - hsa-miR-99a, hsa-miR-125b and hsa-let-7c in chorioamniotic membranes obtained from term pregnancies with spontaneous rupture (n = 20). Tissue and location specific expression patterns within the chorioamniotic membranes were identified. The rupture zone in the choriodecidua had distinct expression patterns compared to other fetal membrane locations. Despite the increased gene dosage associated with T21, the expression of all three miRNAs was reduced in cultured T21 amniocytes as compared to cultured euploid amniocytes. In silico analysis of experimentally validated targets of the three miRNAs suggest these Chr-21 derived miRNAs play a potential role in fetal membrane rupture and the fetal membrane defects associated with T21.1

Keywords: hsa-miR-99a, hsa-miR-125b, hsa-let-7c, trisomy-21, amnion, choriodecidua

Introduction

Trisomy 21 (T21) is associated with abnormalities of the fetal membranes, including chorioamniotic separation and delayed fusion of the amnion and chorion [1-3]. Cytotrophoblast abnormalities and early amniocyte senescence have also been reported [4,5]. Early senescence of fetal extraembryonic tissues has been proposed to contribute to preterm birth [6]. The underlying pathophysiological explanations for these abnormalities has not been elucidated. Epigenetic alterations in the chorion and amnion (differential DNA methylation) have been reported, raising the possibility that both chromosome dose, as well as epigenetic factors, contribute to dysfunction associated with cells in the fetal membranes [7].

MicroRNAs (miRNAs) are small (20-24 nucleotides) single-stranded, noncoding, regulatory RNA molecules. They are involved in post-transcriptional regulation of gene expression either by complimentary binding to the 3’ untranslated region of their target mRNA, thereby inhibiting translation or inducing mRNA degradation [8,9]. Altered expression of microRNAs, including those encoded on chromosome 21 (Chr-21), are thought to contribute to the phenotypes associated with T21 [10,11]. However, the role of these miRNAs in the fetal membrane abnormalities associated with T21 has not been studied in detail. We examined the pattern of expression of a cluster of Chr-21 microRNAs that are potential candidates for altering fetal membrane function in T21. We determined the relative levels of expression of the 3 microRNAs in this cluster in the amnion and choriodecidua, as well as differences in expression in different locations within the fetal membranes. Examination of in vitro cultured amniocytes from normal pregnancies and pregnancies hosting a T21 fetus revealed that T21 is associated with reduced expression levels of these three microRNAs. Based on the known roles of these microRNAs in the epigenetic control of gene expression, some of the fetal membrane structural defects and cellular abnormalities associated with T21 could be explained by altered expression of this microRNA cluster.

Methods

Study Population

Self-reported African-American women admitted at MCV Hospitals, Richmond, VA, with spontaneous labor at term (defined as more than 37 weeks of gestation) with rupture of membranes (n = 20) were recruited for the study. Women with multiple gestations, fetal anomalies, connective tissue diseases and pregnancy complications requiring induction of labor were excluded. Fetal membrane specimens were collected as described below from 12 male and 8 female fetal subjects delivered from 20 term pregnancies.

In order to test the effect of gene dosage on the expression of the Chr-21 localized miRNA cluster that were assessed in this study, cultured amniocytes specimens from local cell repository of cryopreserved specimens were evaluated from fetuses (race unknown) with a T21 complement (n = 7; T21 cases) or a disomic chromosomal complement (n = 6; normal controls). Fetal sex of the amniocyte cultures were as follows: T21 cases (2 males, 5 females); normal controls (4 males, 2 females).

The study was approved by the Institutional Review Board of MCV Hospitals, Richmond, VA., and written informed consent was obtained from the patients before collection of samples

Collection of samples

Human placentas and fetal membranes were obtained at the time of vaginal delivery or when C-section was performed at term. Fetal membranes specimens were collected within 1-2 hours of delivery. After visual identification of the rupture zone (Figure 1) as done previously by Nhan-Chang et al [12], the two adherent fetal membranes (amnion and choriodecidua) were separated/ teased apart from the rupture tear line. A piece of membrane tissue (~ 2cm2) spanning the rupture site was dissected sharply from both the amnion as well as the choriodecidua, constituting the rupture zone amnion (RZA) and rupture zone choriodecidua (RZC) samples, respectively. The two membranes were continuously teased apart and pieces of both the amnion and choriodecidua were dissected at the mid region, which was halfway between the rupture site and the placenta, followed by at the peri-placental region, which is adjacent to the placenta. This method yielded a set of 6 specimens from each patient: 1) amnion from the rupture zone (RZA), 2) choriodecidua from the rupture zone (RZC) (3) amnion from the mid-region (MRA), 4) choriodecidua from the mid-region (MRC), 5) amnion from the peri-placental region (PPA), 6) choriodecidua from the peri-placental region (PPC). The amniocytes from T21 cases and normal controls (defined above) were established in culture (monolayer cultures in flasks), propagated in vitro (1 – 3 passages), and cryopreserved using standard techniques [13,14]. The frozen cell aliquots were retrieved and maintained on ice prior to RNA isolation, which was accomplished as described below. The amniocyte cultures constitute a mixed population of fibroblast cells, amniotic fluid cells (derived from fetal membranes and trophoblasts) and epithelial cells (derived from epithelial fetal skin, bladder and other epithelia). The tissue culture procedure selects for growth of fibroblast-like cells, followed by amniotic fluid cells and epithelial cells with decreasing proliferative capacity in culture respectively [14].

Figure 1. Sites of tissue collection.

Figure 1

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The image of the placenta with the fetal membranes showing the representative sites of tissue collection by region i.e. gross sites for the membrane rupture called the Rupture Zone (RZ), Mid-Region (MR) and the Peri-placental (PP) regions. For RZ, MR and PP regions tissues were collected from both the amnion (A) and the choriodecidua (C) separately.

RNA isolation and miRNA qPCR

For the miRNA-qPCR assays, total RNA was extracted using miRVana miRNA isolation kit (Ambion Life Technologies) following the manufacturer’s protocol. MicroRNA qPCRs were performed using the TaqMan MicroRNA Assays (Life Technologies) following the manufacturer’s instructions. Briefly, 10ng RNA was reverse transcribed using the target (miRNA) specific stem- loop RT primer and the TaqMan MicroRNA reverse transcription kit. The cDNA was then amplified by real-time qRT-PCR using target specific TaqMan primer-probe mix. The qRT-PCR was performed in triplicates per sample (means were used for statistical analysis) and normalization was done using small RNA RNU48.

Statistical analysis

For miRNA qPCR assays in amniocyte samples from T21 cases and controls, normalized qPCR expression values for the three miRNAs (hsa-miR-99a, hsa-miR-125b and hsa-let-7c)were log2 transformed and used for student’s t- test for determining statistical significance (p < 0.05).

For miRNA expression analysis in the fetal membranes normalized expression values from qPCR for the three miRNAs (hsa-miR-99a, hsa-miR-125b and hsa-let-7c) from the 6 specimens collected for each of the 20 participating subjects were log2 transformed prior to statistical analysis. For each miRNA, a random coefficient mixed effects model predicting log2 expression was fit, including the location/tissue factor variable as a fixed effect and subject as a random effect [15]. Linear contrasts from the mixed effects model were constructed to test the following: MRA versus MRC; PPA versus PPC; RZA versus RZC; MRA versus PPA; MRA versus RZA; PPA versus RZA; MRC versus PPC; MRC versus RZC; PPC versus RZC; and Amnion versus Choriodecidua. Adjusted p-values are reported for the linear contrasts using the single-step method (p < 0.05 was considered significant) [16].

To compare male and female fetal subjects with respect to miRNA log2 qPCR expression, Welch’s t-test was performed for each miRNA (let-7c, miR-125b and miR-99a), location (MR, PP, RZ and VP) and tissue (Amnion, Choriodecidua), adjusting for possible unequal variances between the groups. A Bonferroni correction was used to control for multiple comparisons and an adjusted alpha level of 0.0071 or below was considered significant [16].

All statistical analyses were performed in the R programming environment [17].

MicroRNA target identification and pathway analysis

The miRNA target prediction filter in Qiagen’s Ingenuity® Pathway Analysis tool (IPA®, QIAGEN Redwood City, www.qiagen.com/ingenuity) was used to identify potential target genes for hsa-miR-125b, hsa-miR-99a and hsa-let-7c. Pathway analysis was performed in IPA on target genes with experimentally observed interactions with the miRNAs.

Results

Trisomy impacts expression of the Chr21Orf34 microRNA cluster

The qPCR miRNA expression assays that were completed on cultured amniocytes revealed significant differences (p < 0.05) in the expression of all three miRNAs between the T21 cases (n = 7) compared to disomic controls (n = 6), with reduced expression levels in T21 amniocytes. For each miRNA, dot-plots of log2 expression (normalized by RNU48) by case and control are presented in Figure 2.

Figure 2. MiRNA Expression in Amniocytes.

Figure 2

Figure 2

Figure 2

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Significant differences (p < 0.001) in miRNA expression were observed between T21 case (n = 7) and disomic control (n = 6) amniocyte samples for hsa-miR-let7c (A), hsa-miR-125b (B) and hsa-miR-99a (C).

Differential miRNA expression patterns are observed between the amnion and the choriodecidua and at different locations within the choriodecidua

For each miRNA, normalized qPCR expression values for all samples (n = 20) were log2 expression transformed for each of the location/ tissue types and used for statistical analysis. qPCR results are shown in Figure 3. The differences and adjusted p-values for linear contrasts from the random coefficient mixed effects model for all pairwise comparisons are reported in Table 1 and visually displayed in Figure 4. Comparisons between the amnion and the choriodecidua at different locations revealed that all three miRNAs had significant expression differences at the rupture zone. For the non-rupture sites, for miR99a and let-7c there were significant expression differences at the mid-region, whereas miR-99a and miR-125b were differentially expressed between the amnion and the choriodecidua at the peri-placental region. None of the three miRNAs had significant differences at any location within the amnion. Comparatively, the choriodecidua showed significant differences (lower abundance) between the rupture zone and non-rupture sites, with all three miRNAs showing significant lower abundances between the rupture zone and the peri-placental region, and miR-99a and let-7c showing significant differences (lower abundance) between the rupture zone and the mid-region choriodecidua as well.

Figure 3. MiRNA Expression in Fetal Membranes.

Figure 3

Figure 3

Figure 3

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qPCR results for log2 expression of hsa-let-7c (3A), hsa-miR-125b (3B) and hsa-miR-99a (3C) for each group/site of collection are shown. (PP=Periplacental, MR=MidRegion, RZ=Rupture Zone; A = Amnion/C = Choriodecidua). (Sample N = 20). Data are represented as Mean ± Standard Error.

Table 1.

Pairwise comparisons of miRNA expression by tissue and location

Comparison Let-7c Estimate (Adjusted p-value) miR-125b Estimate (Adjusted p-value) miR-99a Estimate (Adjusted p-value)
MRA vs. MRC 0.8127 (<0.001) 0.5421 (0.1222) 1.3674 (<0.001)
PPA vs. PPC 0.8161 (0.0873) 1.0005 (0.0331) 1.3056 (<0.001)
RZA vs. RZC 1.6851 (<0.001) 1.0628 (<0.001) 1.5455 (<0.001)
A vs. C 1.1046 (<0.001) 0.8685 (<0.001) 1.4062 (<0.001)
MRA vs. PPA -0.1980 (0.961) -0.70407 (0.0643) -0.1492 (0.9879)
MRA vs. RZA 0.2743 (0.287) 0.01895 (1.0000) 0.2807 (0.2066)
PPA vs. RZA 0.4722 (0.648) 0.72302 (0.3538) 0.4299 (0.5728)
MRC vs. PPC -0.1945 (0.638) -0.24567 (0.3453) -0.2110 (0.4481)
MRC vs. RZC 1.1466 (<0.001) 0.53970 (0.0757) 0.4588 (0.0027)
PPC vs. RZC 1.3412 (<0.001) 0.78537 (0.0024) 0.6698 (<0.001)
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The differences (log2 contrast estimates) and adjusted p-values for linear contrasts from the random coefficient mixed effects model for all pairwise comparisons are shown for hsa-let7c, hsa-miR-125b and hsa-miR-99a. (p < 0.05, considered significant, shown in bold)

Figure 4. Comparison of miRNA expression by tissue and location.

Figure 4

Figure 4

Figure 4

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Dot-plot of log2 expression for hsa-let7c (4A), hsa-miR-125b (4B) and hsa-miR-99a (4C) are shown for each specimen collection location, separated by amnion (left) versus choriodecidua (right). Each subject’s observations are connected with a line.

MiRNA Correlation in amnion and the choriodecidua by location

Pearson’s correlation was used to examine the strength of linear relationship (pairwise) between the log2 expression of the three miRNAs by location for amnion (Table 2) and for the choriodecidua (Table 3). All pairwise miRNA relationships in all 3 locations in the amnion as well as the choriodecidua were positively correlated, though the strength of correlation (determined by ρ) varied with specimen tissue/site. For the periplacental region, all pairwise miRNA comparisons showed significant correlations (p < 0.05) for both the amnion and the choriodecidua. Other significant pair-wise correlations in expression between miRNAs were observed in the choriodecidua at the mid-region (let-7c and miR-125b) and at the rupture zone (let-7c and miR-125b, as well as miR-99a and miR-125b) with miR-99a and miR- 125b showing a strong correlation (ρ > 0.5) at this site.

Table 2.

Pairwise Pearson’s correlation for log2 miRNA expression by location for amnion.

Comparison Periplacental ρ (p-value) MidRegion ρ (p-value) Rupture Zone ρ (p-value)
let-7c & miR-99a 0.627 (0.003) 0.255 (0.28) 0.407 (0.07)
let-7c & miR-125b 0.560 (0.01) 0.209 (0.38) 0.029 (0.90)
miR-99a & miR-125b 0.541 (0.01) 0.291 (0.21) 0.336 (0.15)
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The strength of linear relationship (pairwise) between the log2 expression of the three miRNAs in the amnion by location is shown. Values represent Pearson’s coefficient of correlation (ρ) with estimated p-values in parentheses. (p < 0.05 considered significant, shown in bold)

Table 3.

Pairwise Pearson’s correlation for log2 miRNA expression by location for the choriodecidua.

Comparison Periplacental ρ (p-value) MidRegion ρ (p-value) Rupture Zone ρ (p-value)
let-7c & miR-99a 0.469 (0.04) 0.445 (0.05) 0.383 (0.10)
let-7c & miR-125b 0.514 (0.02) 0.495 (0.03) 0.453 (0.04)
miR-99a & miR-125b 0.665 (0.001) 0.362 (0.12) 0.646 (0.002)
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The strength of linear relationship (pairwise) between the log2 expression of the three miRNAs in the choriodecidua by location is shown. Values represent Pearson’s coefficient of correlation (ρ) with estimated p-values in parentheses. (p < 0.05 considered significant, shown in bold)

Pathway analysis of genes targeted by miRNAs

MicroRNA target prediction filter in IPA identified a total of 217 experimentally observed miRNA-target gene interactions (Supplementary Information, Table S4). The 212 target genes from these interactions were used for core pathway analysis in IPA to identify pathways, diseases & bio-functions and networks that are “over-represented” in target genes of the Chr-21 microRNA cluster. The key findings from the pathway analysis are shown in Table 4. Of note, several of the functional annotations suggest involvement of cell survival, cell death and cellular development pathways (molecular mechanisms of cancer) which play a role in fetal membrane rupture. In addition, a significant number of targeted genes overlap with the categories of reproductive system disease (101 genes) and developmental disorder (45 genes), which also have a strong association with preterm birth, as well as T21. The top network (Figure 5), based on the functional annotations of the target genes, had 26 focus molecules (26 of the 212 target genes) with associated network functions involving the cell cycle, connective tissue development and function, and cancer. All of these functions have been shown to have strong correlations with membrane rupture and/ or phenotypic traits associated with Down’s syndrome.

Table 4. Ingenuity pathway analysis of the genes targeted by hsa-miR-99a, hsa-miR-125b and hsa-let-7c.

A: Top Canonical Pathways
Name p-value Overlap
Bladder Cancer Signaling 1.04E-09 12.60%
Chronic Myeloid Leukemia Signaling 5.26E-10 11.50%
Glioblastoma Multiforme signaling 4.18E-13 10.70%
Colorectal Cancer Metastasis Signaling 3.80E-09 6.50%
Molecular Mechanisms of Cancer 3.61E-12 6.10%
B: Top Diseases and Disorders
Name p-value No. of gene
Organismal Injury and Abnormalities 3.7E-07 - 3.82E-18 153
Cancer 3.7E-07 - 3.82E-18 151
Reproductive System Disease 3.7E-07 - 5.73E-15 101
Tumor Morphology 2.02E-07 - 3.85E-17 51
Developmental Disorder 3.68E-07 - 2.88E-14 45
C: Top Molecular and Cellular Functions
Name p-value No. of genes
Cellular Growth and Proliferation 3.70E-07 - 1.34E-20 125
Cellular Development 3.70E-07 - 1.34E-20 112
Cell Death and Survival 3.32E-07 - 5.40E-18 110
Cellular Movement 3.10E-07 - 3.43E-13 70
Cell Cycle 3.70E-07 - 2.93E-16 64
D: Physiological System Development and Function
Name p-value No. of genes
Organismal Development 3.31E-07 - 1.03E-17 115
Organismal Survival 5.59E-15 - 3.97E-18 90
Tissue Morphology 1.22E-07 - 4.65E-14 78
Hematological System Development and Function 3.21E-07 - 6.85E-14 67
Lymphoid Tissue Structure and Development 3.21E-07 - 2.97E-14 62
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Key findings from the pathway analysis (performed in IPA) of the 212 genes (Table S1) targeted by the three miRNAs in the Chr21 cluster are described. The Top Canonical Pathways (4A), Top Diseases and Disorders (4B), Top Molecular and Cellular Functions (4C) and Physiological System Development and Functions (4D) over-represented in the target genes are shown.

Figure 5.

Figure 5

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The top IPA network (Score = 48) with 26 of the 212 experimentally observed target genes of the 3 miRNAs. The associated network functions are cell cycle, connective tissue development and function and cancer.

Fetal gender and miRNA expression

Fetal gender is known to be associated with adverse pregnancy outcomes [18,19]. Male (n = 12) and female (n = 8) fetal subjects in the study were compared with respect to miRNA expression (log2 qPCR estimates) for each miRNA for each of the 6 groups or sampling sites. Only let-7c in mid-region choriodecidua samples was significantly different (p < 0.0071) when comparing fetal gender. (Supplemetary Information, Tables S1-S3)

Discussion

Increasing numbers of investigators are identifying miRNAs to be involved in pathologies of several diseases and disorders, including the features of T21 [10,11,20,21]. Expression patterns of miRNAs have been studied in the placenta as well as the chorioamniotic membranes and associations with human pregnancy, gestational age, and parturition and pregnancy complications (such as preeclampsia and preterm labor) have been found [22-25]. MicroRNAs are key regulators of extracellular matrix (ECM) gene expression and homeostasis, which is perturbed in preterm premature rupture of mmebranes (PPROM). Interestingly, the relationship between miRNAs and ECM is thought to be bi-directional with studies suggesting that not only can miRNAs regulate ECM composition, but the ECM can also affect miRNA expression and function [26].

Despite the presence of three chromosomes 21 (a trisomic imbalance), the T21 amniocyte specimens showed reduced expression of the microRNA cluster. This has been observed by others who quantitated expression of the same microRNAs in other tissue types and found reduced expression in T21 [11,20]. However, other studies have documented increased expression of these miRNAs in T21 as compared to euploid controls [10,21,27]. These apparent discrepancies could be attributed to differences in the tissue specimens investigated or differences in normalization techniques. Reduced expression of the microRNAs in the face of increased gene dosage may reflect epigenetic changes such as DNA methylation that dampen expression [28]. The reduced expression of the three microRNAs would presumably increase the expression of the proteins encoded by the target mRNAs.

Although all three miRNAs are known to be expressed in the placenta and chorioamniotic membranes, an in-depth expression analysis has not been performed. Prior studies investigating the roles of these miRNAs in context of other diseases reveal findings suggesting a true potential for their involvement in fetal membrane pathology. For example, miR-99a expression has been studied in several cancers and plays crucial roles in the PI3K/AKT and NF-κB signaling pathways [29,30]. Infact, the miR-99a/let-7c/miR-125b locus on Chr-21 is phylogenetically conserved and was shown to be transcribed as a polycistronic message and regulates TGFβ/Wnt signaling pathways in hematopoietic stem and progenitor cell homeostasis [31]. Members of the miR-125 family of miRNAs are involved in diverse cell functions and have been associated with several diseases. MiR-125 miRNAs target a range of genes, including transcription factors and matrix metalloproteinases (MMPs) which are involved in ECM metabolism and also have important functions in immunological pathways. Another study found that miR- 125b was upregulated in third trimester placentas compared to first trimester placentas, suggesting it has a role in gestational age [22,32]. Let-7 miRNAs are negatively regulated by the RNA-binding protein, Lin28, and both act downstream of the NF-κB signaling pathways. Like miR-125b, let-7 miRNAs, including let-7c were upregulated in third trimester human placentas compared to the first trimester [22]. In support of our results, another study found tissue-specific let-7c expression differences at term, with significantly reduced expression in choriodecidua as compared to amnion and placenta [33].

In the current study, in addition to the tissue-specific expression patterns, we also show differential expression patterns at different locations within the choriodecidua. Differences between the rupture zone and the non-rupture sites are especially biologically relevant. The rupture site of fetal membranes is characterized by unique histologic and biochemical changes. These changes are observed even in the portion of fetal membranes overlying the cervix (thought to be eventual site of spontaneous rupture) in patients at term without labor. This area is termed as the “zone of altered morphology” (ZAM) and is characterized by increased thickness of connective tissue and decreased thickness of cytotrophoblast and decidua [34-37]. These changes would require large-scale transcriptional reprogramming, which can be carried out, at least in part, by miRNA-regulation. Nhan-Chang et al [12] showed gene expression differences between the amnion and the choriodecidua at both the rupture as well as non-rupture sites, whereas within each fetal membrane individually, only the choriodecidua showed gene expression differences between the rupture and non-rupture sites. These results are in accordance with the miRNA expression patterns in our study, and support the concept that the altered gene expression in fetal membranes in the rupture zone could be due to altered miRNA expression patterns. It is important to note that the decreased thickness in the cytotrophoblast and maternal decidual cell layers at the rupture site in the chorion as compared to non-rupture sites [34-37] can influence the observed miRNA expression levels at these sites. Furthermore, the genes identified by Nhang-Chang et al (such as IL-6, IL1RN, CXCL12, ADAMTS5) were involved in inflammatory response, immunetolerance pathways as well as ECM-receptor interactions. This suggests that miRNAs from the Chr21orf34 locus could be involved in regulation of these pathways in the fetal membranes as well, which is quite plausible considering the evidence for these functional roles are already established in other tissues and disease states [26,29-32]. In silico pathway analysis of the experimentally observed gene targets of the three miRNAs revealed an over-representation of genes related to cellular growth and proliferation, cell death and cell survival pathways, all of which are crucial in ECM function during membrane rupture [38]. Notably, the top network based on functional annotation of the genes targeted by the three miRNAs included COL1A2, which is one of the major fibrillar collagens present in fetal membranes contributing to the tensile strength of the amnion and associated with preterm birth [39]. The network also included Smad which suggests involvement of the TGFβ-Smad signaling pathway which plays a role in ECM metabolism and fetal membrane rupture via transcription regulation of various collagens, MMPs and tissue inhibitors of metalloproteinases (TIMPs) [40,41].

In conclusion, we show evidence for distinct tissue and region specific miRNA expression patterns in fetal membranes from subjects with spontaneous term labor. These miRNAs are expressed at a reduced level in T21 and this alteration may promote abnormalities in fetal membranes associated with T21, including chorioamnion separation or delayed fusion of the chorion and amnion.

Supplementary Material

supplement
NIHMS829715-supplement.docx (107.4KB, docx)

Highlights.

  • Expression of Chromosome 21 miRNAs (miR-99a, miR-125b, let-7c) was investigated

  • Altered expression of these miRNAs in trisomy 21 amniocytes was established

  • Tissue/location specific expression patterns in fetal membranes were observed

  • Roles in fetal membrane abnormalities associated with trisomy 21 are suggested

Acknowledgments

This research was funded by National Institutes of Health Grants R01 HD073555 and P60 MD002256.

Footnotes

1

Abbreviations:- Trisomy 21: T21, chromosome 21: Chr-21, microRNA: miRNA, ECM: extracellular matrix

Declaration of Interest: All authors have declared that no competing interests exist.

Author Contributions:

BPM, KJA and JFS contributed to study design and performed statistical analysis; BPM and SW were involved in sample collection, performing experiments and data collection; BPM, SWW, CJC, KJA and JFS wrote and edited the manuscript. CJC also provided cultured amniocyte specimens.

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