Endocrinology 155(12): 4975-4985

PMC: PMC4239420

PMID: 25211593

C19MC MicroRNAs Regulate the Migration of Human Trophoblasts

Materials and Methods

Specimen preparation for histology and laser capture microdissection

The Institutional Review Board at the University of Pittsburgh approved the collection and analysis of de-identified specimens under an exempt protocol. We used formalin-fixed, paraffin-embedded archival placental samples from the first trimester (6–12 wk), term (37–41 wk) pregnancy, and ectopic (tubal) pregnancy. Notably, all placental slides were viewed by a perinatal pathologist, and no major abnormalities were noted. Tissue blocks were cut to generate 5-μm sections. The slides were stained with toluidine blue according to the protocol guide for laser microdissection (Leica Microsystems). Briefly, the slides were dewaxed twice with xylene for 15 seconds and rinsed twice with 100% ethanol, 95% ethanol, and distilled water for 30 seconds each. The slides were then soaked in toluidine blue solution for 3 minutes, dehydrated in 70% and 100% ethanol for 30 seconds each, and dried on a 37°C block for 2 hours. EVT regions and villi-containing regions were isolated separately, using an LMD7000 laser capture microdissection microscope (Leica). The size of the microdissected fragments ranged between 0.025 and 1 mm^.

Cell culture

First trimester cytotrophoblasts were derived from a pool of proliferating trophoblasts prepared from seven first-trimester placentas (8–10 wk). Cultures were enriched for EVTs by seeding on fibronectin for 48 hours, resulting in approximately 90% human leukocyte antigen (HLA)-G1-positive EVTs, with induction of the EVT marker alpha1 integrin and down-regulation of the proximal cell column marker alpha6 integrin (18). Enrichment of villous cytotrophoblasts (∼60%) was confirmed by expression of EGF receptor (18). The ethical committee of the Medical University of Vienna approved the protocol and the use of these de-identified tissues, and informed consent was obtained from patients.

Primary human trophoblasts (PHT cells) were prepared from normal-term placentas following a healthy pregnancy, labor, and delivery at Magee-Womens Hospital of the University of Pittsburgh Medical Center. De-identified placentas were collected under an approved exempt protocol by the Institutional Review Board of the University of Pittsburgh, and patients provided written consent for the use of de-identified, discarded tissues for research. PHT cells were dispersed using the trypsin-deoxyribonuclease (DNase)-dispase/Percoll method as previously described (19), with previously published modifications (20). PHT cells were maintained in DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (BGS, HyClone) and antibiotics, and cultured for 72 hours. HTR-8/SVneo cells were provided by C.H. Graham (Queen's University, Kingston, ON, Canada) (21) and cultured in RPMI1640 (Cellgro) supplemented with 5% bovine growth serum (HyClone) and antibiotics with weekly passages. BeWo cells were cultured in Ham's F12 nutrient mix (HyClone) supplemented with 10% bovine growth serum and antibiotics.

RNA (mRNA, miRNA) extraction, RT-qPCR, and Northern blot

Total RNA was extracted from the microdissected tissue fragments using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. Total RNA from the different cell types was extracted by PureLink RNA Mini Kit (Invitrogen) according to the manufacturer's instructions. For mRNA analysis, reverse transcription was performed using High-Capacity RNA-to-cDNA Kit (Applied Biosystems). qPCR was performed in duplicate, using Power SYBR Green PCR Master Mix and the ViiA 7 real-time PCR System (both from Applied Biosystems). The primer sequences used in this study are detailed in Supplemental Table 1. Transcript expression level was normalized to the expression level of the trophoblast housekeeping gene, YWHAZ (22, 23). The expression level of miRNA was determined by the miScript PCR system (Qiagen) and normalized to RNU6 expression. For both mRNA and miRNA, the fold change, relative to control samples, was determined by the 2^{method (}24).

For Northern blot experiments, 20 μg of total RNA was denatured at 65°C for 10 minutes and subsequently resolved using 7M urea/15% PAGE. The gel was stained with SYBR Gold (Invitrogen/Molecular Probes), and RNA was electro-transferred to a nylon Hybond N+ membrane (GE Healthcare). Hybridization was performed overnight at 37°C in 0.5M Na₂HPO₄ (pH, 7.4), 7% sodium dodecyl sulfate (SDS) and 1 mM EDTA, using DNA oligonucleotide probes, which were labeled with a ^{P-dATP (Deoxyadenosine-5′-Triphosphate, [α-32P]) using a StarFire labeling system (IDT). After washing, membranes were exposed to Kodak film for 16–24 hours for visualization of the appropriate bands.}

mRNA and miRNA expression array and pathway analysis

We used high-throughput microarray analysis to screen for transcriptional changes in cultured HTR-8/SVneo cells transfected with the C19MC BAC vs. control nontransfected cells. The quality of all RNA samples was first confirmed, using an RNA 6000 Nano Assay Kit (Agilent Technologies) in an Agilent 2100 Bioanalyzer to ensure RNA integrity and quality. mRNA labeling was performed using a One-Color Low Input Quick Amp Labeling Kit (Agilent) and prepared for hybridization on SurePrint G3 Human Gene Expression 8 × 60K slides using the Gene Expression Hybridization Kit (both from Agilent). Slides were scanned using Agilent's SureScan Microarray Scanner System, and data extracted using Agilent's Feature Extraction Software (version 11.0.1.1). A similar process was used for miRNA arrays, but the hybridization was then performed on Human miRNA V3 8× 15K slides, using the miRNA Complete Labeling and Hyb Kit (both from Agilent).

mRNA microarray data were normalized using the cyclic loess normalization method (25), as implemented in the R package affy. The R package Limma (Linear Models for Microarray Data), which implements a moderated t test, was used to identify differentially expressed mRNAs (26). Storey's q value method (27), as implemented in R package qvalue, was used to calculate the adjusted p-values to control the false discovery rate. Functional mRNA analysis was performed using DAVID Bioinformatics Resources (http://david.abcc.ncifcrf.gov) and Ingenuity Pathways Analysis (Ingenuity Systems, http://www.ingenuity.com). For microarray analysis, the robust multiarray average (28), as implemented in R package AgiMicroRna (25), was used to obtain the summarized and normalized miRNA expression level. All mRNA and miRNA array data are available on GEO (http://www.ncbi.nlm.nih.gov/geo. Accession numbers {"type":"entrez-geo","attrs":{"text":"GSE56564","term_id":"56564","extlink":"1"}}GSE56564 and {"type":"entrez-geo","attrs":{"text":"GSE56562","term_id":"56562","extlink":"1"}}GSE56562, respectively).

Western blotting

Whole-cell lysates were prepared with cold tris-buffered saline (TBS) containing 1% Triton X-100, 0.1% SDS, and 1% Halt protease inhibitor cocktail (ThermoFisher Scientific). Protein concentrations were measured using the Pierce BCA Protein Assay (ThermoFisher) according to the manufacturer's instructions. Proteins were separated on 10% Bis-Tris SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore). The membranes were blocked and incubated with primary antibodies against cleaved poly (ADP-ribose) polymerase (PARP) (dilution 1:1000, Cell Signaling), cytokeratin 18 neoepitope (M30, dilution 1:1000, Enzo Life Sciences), or actin (0.08 μg/mL final concentration, Millipore). The antimouse horseradish peroxidase-conjugated secondary antibodies (dilution 1:1000, Jackson ImmunoResearch Laboratories) were used, and the blots were developed by Pierce SuperSignal West Dura Chemiluminescent Substrate (Pierce, ThermoFisher) and exposed to film.

Plasmids, mutagenesis, and transfections

We have previously described the use of recombineering for generation of the BAC plasmid harboring the entire C19MC miRNA cluster (100 kb and an additional 60 kb of upstream flanking sequences), linked to GFP and a Zeocin-resistance gene (17). HTR-8/SVneo cells (800 000 cells per 60-mm plate) were plated 1 day before transfection. Cells were transfected with 6 μg of BAC-C19MC plasmid-using Lipofectamine LTX with Plus Reagent (Invitrogen) and selected for stable integration with 200 μg/mL Zeocin (InvivoGen) over 2 weeks. Single colonies of transfected cells (termed HTR-8-C19, distinct from wild-type HTR-8/SVneo, HTR-8-WT) were picked and expanded. HTR-8/SVneo cells were also reverse transfected (17) with miR-519d-specific mimics or with miRNA mimic control (miRIDIAN, ThermoFisher) at a final concentration of 50 nM, using DharmaFECT 1 transfection reagent (ThermoFisher) according to the manufacturer's instructions. Cells were assayed 48 hours post transfection.

The miR-519d expression vector was constructed by cloning an approximately 500-bp fragment of genomic DNA, harboring the miR-519d precursor and its flanking sequence, into a pcDNA3.1 vector (Invitrogen). The 3′UTRs of target genes were PCR-amplified and cloned into psiCHECK-2 vectors (Promega) directly downstream of a Renilla luciferase reporter gene. The mutated luciferase reporter was constructed by deleting the predicted miRNA binding sites using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Each mutation was confirmed by sequencing. The wild-type and mutated luciferase reporters were transfected into HTR-8/SVneo cells, along with the miR-519d expression vector or control vector, respectively, using polyethylenimine (29). Alternatively, the luciferase reporters were also transfected into HTR-8/SVneo cells, along with miR-519d mimic (50nM), using DharmaFECT Duo transfection reagent (ThermoFisher). Luciferase activity was measured 48 hours later using the Dual Glo luciferase assay (Promega), determined by a Veritas microplate luminometer (Turner BioSystems).

Proliferation assay using flow cytometry for BrdU incorporation

To determine the extent of cellular proliferation, the incorporation of 5-bromo-2′-deoxyuridine (BrdU) was quantified using the Apoptosis, DNA Damage, and Cell Proliferation Kit (BD biosciences) according to the manufacturer's instructions. Briefly, 1 × 10^{cells were cultured in six-well plates for 48 hours, and 10 μM BrdU was added. The cells were collected after 1 hour of BrdU incubation. Following fixation and permeabilization, DNase was added to the cells to expose the incorporated BrdU, and stained with anti-BrdU antibody. Stained cells were quantified with the multicolor flow cytometer (LSR II: BD Biosciences), using FACSDiva data analysis software (BD Biosciences).}

Trans-well migration assay

HTR-8-C19 or HTR-8-WT were seeded in the upper chamber of the 6.5-mm transwell (50 000 cells/2.5 mL of serum-free RPMI-1640 medium) with 8.0 μm pore polycarbonate membrane insert (Corning Life Sciences). The lower chambers were filled with 750 μl of RPMI-1640 media with 5% BGS. After 24 hours of culture, the inserts were washed with PBS, and the nonmigrating cells in the upper chamber were gently removed with a cotton swab. The inserts were fixed in methanol for 10 minutes at room temperature and stained with hematoxylin for 10 minutes. Cells migrating to the lower surface were photographed under a light microscope.

Scratch wound healing assay and live cell imaging

Cells were plated in six-well plates at a density of 1 × 10^{cells per well and cultured overnight. A sterile pipet tip was used to scratch down the center of each well, and the medium was replaced. HTR-8-C19 or HTR-8-WT cells were photographed every 24 hours. For video capture of the wound healing process, the same assay was performed except that the cells were plated at a density of 9 × 10}^{per well on the two-well Nunc Lab-Tek II Chambered Coverglass (ThermoFisher). Each cell culture was photographed at 30-minute intervals, for a total of 65 hours, on the A1 Confocal Microscope System (Nikon).}

Statistical analysis

Statistical analyses of the miRNA and gene expression microarray data were performed on the log₂-transformed expression levels. We used the nonparametric Wilcoxon matched-pairs signed-rank test to determine significance for comparisons performed on non-normally-distributed human samples and Student t test for other experiments, as indicated in the text. All experiments were repeated at least three times, as suggestd in the figure legends. P < 0.05 was considered significant.

Specimen preparation for histology and laser capture microdissection

Cell culture

RNA (mRNA, miRNA) extraction, RT-qPCR, and Northern blot

mRNA and miRNA expression array and pathway analysis

Western blotting

Plasmids, mutagenesis, and transfections

Proliferation assay using flow cytometry for BrdU incorporation

Trans-well migration assay

Scratch wound healing assay and live cell imaging

Statistical analysis

Results

Higher expression of C19MC in VTs vs EVTs

As a group, miRNA members from the C19MC comprise the most abundant miRNA species expressed in VTs (15). Even though the villous and EVTs are derived from a common extraembryonic trophectoderm lineage, their functions are different. We therefore sought to determine whether the levels of C19MC miRNAs in EVT differ from levels in VTs. EVTs were derived from four different sources, vs the appropriate villous trophoblast controls (Figure 1): first or third trimester EVTs in vivo, first trimester EVTs from ectopic pregnancy, cultured first trimester EVTs, and a cultured EVT cell line. Using laser capture microdissection and paraffin-embedded placental histological slides, we separated VTs from EVTs derived from the first or third trimesters of human pregnancy. Using RT-qPCR to determine C19MC miRNA expression, we found that the expression level of all representative miRNAs (miR-517–3p, miR-518b, miR519d, miR-520g, miR515–5p, and miR1323) was higher in VTs compared with EVTs in vivo (Figure 1A), with no significant difference between the first trimester and third trimester samples. We also found that placental samples from first trimester ectopic pregnancies, where trophoblasts are mainly of the extravillous type (30), have lower levels of C19MC miRNAs compared with first trimester VTs in vivo (Figure 1B). Moreover, we detected a lower expression of miR-517–3p, miR-518b, miR-519d, miR-520g, and miR-1323 in cultured primary EVTs compared with PHT cells (Figure 1C). Lastly, we used a Northern analysis (Figure 1D) to confirm the expression difference of a representative C19MC miRNA, miR-518b, in VT lines (JEG3, BeWo, or Jar) vs the EVT line HTR-8/SVneo (21). Together, these findings suggest that VTs express a higher level of C19MC miRNA than do EVTs.

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Figure 1.

C19MC miRNAs are differentially expressed in extravillous vs villous human trophoblasts. A, In-vivo expression level of miR-517a, miR-518b, miR519d, miR-520g, miR515–5p, and miR1323 in EVTs (white) and primary VTs, (black) from first or third trimester specimens. A–D, four individual patients. B, Expression level of miR-517a, miR-518b, miR519d, miR-520g, miR515–5p, and miR1323 in EVT (white) and VT (black) from tubal (ectopic) pregnancy. A–F, six individual patients. For panels A–B, laser-capture microdissection was performed as described in Methods, using paraffin-embedded placental specimens. Expression was determined by RT-qPCR in duplicate and expressed as fold change vs. a calibrator sample. For panels A–B, P < .05 for all VTs vs. EVTs (Wilcoxon paired test). The difference between miRNAs from first and third trimester placentas (panel A) was not statistically significant. C, The difference in expression of selected C19MC miRNAs, determined by RT-qPCR in duplicate, in cultured EVTs (white) and term primary human trophoblasts (PHT, black). * P < .05, ** P < .01 (paired Student t test; n = 7). D, Northern blot analysis of miR-518b expression in various placental (labeled with an arrowhead) and nonplacental cell lines compared with PHT cells. Among the placental cell lines, only HTR-8/SVneo cells are derived from EVTs.

Ectopic expression of C19MC miRNAs in HTR-8/SVneo line attenuates cell migration

Our observations raised the possibility that the higher expression level of C19MC miRNAs in VTs compared with EVTs might account for some of the phenotypic differences between the two trophoblastic lineages. To examine this possibility, we stably transfected HTR-8/SVneo cells with a BAC that harbors the entire 100-kb C19MC locus, along with its 60-kb upstream regulatory region, as we previously described (17). Among several transfected HTR-8/SVneo lines, we selected Clone C19.1 for further experiments, because its C19MC profile was similar to that of PHT cells (Supplemental Figure 1). RT-qPCR of representative C19MC miRNAs confirmed an increased expression in the range of 20–1000-fold (Supplemental Figure 2).

We used a scratch wound healing assay to evaluate the migration properties of C19MC miRNA-expressing HTR-8/SVneo cells (now termed HTR-8-C19). Compared with control cells, we found that HTR-8-C19 exhibited a longer wound closure time (Figure 2A and B, and a live cell imaging video, Supplemental Figure 3). We bolstered these observations using a transwell migration assay, which demonstrated markedly attenuated migration capacity in cells expressing HTR-8-C19 cells (Figure 2, C and D). To exclude the possibility that the reduced scratch wound healing and transwell migration was due to an altered proliferative capacity, we labeled the cells with the proliferation marker BrdU. Using flow-cytometry, we found no difference in the rate of BrdU incorporation between HTR-8-C19 and the parental HTR-8-WT cells (Figure 2E). Similarly, we detected no difference between HTR-8-C19 and HTR-8-WT in the rate of apoptosis, measured by Western analysis of the apoptotic markers cleaved-PARP or cytokeratin 18 (Figure 2F). Together, these data suggest that ectopic expression of C19MC miRNAs in the EVT line HTR-8/SVneo reduced cell migration without affecting the rate of cell proliferation or apoptosis.

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Figure 2.

Ectopic expression of C19MC attenuated the migratory capacity of HTR-8/SVneo cells without affecting cell proliferation or apoptosis. A, Photos illustrating the scratch wound healing process for HTR-8-C19 cells and HTR-8-WT cells at 0, 24, and 48 h after scratch, performed as described in Methods. Shown are representatives of three independent experiments. See also live cell imaging in Supplemental Figure 3. B, Quantification of the data in Figure 2A, showing the relative wound width *, P < .01. C, Photos illustrating the trans-well migration assay for HTR-8-C19 cells and HTR-8-WT cells. The photos were taken at 50× (upper) and 200× (lower) magnification. Shown are representatives of three independent experiments. D, Quantification of the transwell migration assay, showing the relative cell number (*, P < .01). E, Flow cytometry results indicating BrdU incorporation for HTR-8-C19 cells and HTR-8-WT cells. Shown is a typical flow cytometry graph, representing three independent experiments that were quantified in the diagram (P =NS, paired t test). F, Western immunoblot of PARP and cytokeratin-18 expression in cell lysate from HTR-8-C19 or HTR-8-WT cells. Actin was used as a loading control. Shown is a representative blot of three independent experiments.

C19MC miRNAs target pathways involved in adhesion and extracellular matrix processing

To define gene expression changes induced by ectopic expression of C19MC miRNAs, we used mRNA microarrays of HTR-8-C19 cells and compared the results to those for the parental HTR-8-WT cells. To minimize off-target effects, we analyzed data from four different clones (Supplemental Figure 1). We defined differentially expressed genes on the basis of a 2-fold cutoff and an adjusted P < .05, computed as described in Methods. Based on gene expression observed in all clones, we identified a set of up-regulated and down-regulated genes (a total of 837–1988 and 1188–1609, respectively, among the four clones). Using Ingenuity Pathway Analysis for each clone, we found that “cellular movement” was the most prevalent cellular pathway (Table 1). Considering the commonly occurring inverse correlation between the expression of a given miRNA and its putative target, we further focused on down-regulated genes in HTR-8-C19 cells and identified a total of 421 transcripts that were down-regulated in all four clones, when compared with the parental HTR-8-WT. Gene ontology analysis using DAVID identified “response to wounding” (Table 2, P < .0001) as the most common biological process associated with these 421 transcripts, thus confirming our pathway analysis.

Table 1.

Ranked Scores for Molecular and Cellular Functions and Canonical Pathways, Based on Differentially Expressed Genes in Four HTR-8-C19 Stable Cell Lines, Compared With HTR-8-WT Cells

Pathway	Score^{^a}
Cellular movement	17
Cellular growth and proliferation	15
Cellular development	11
Cell death and survival	10
Cell-to-cell signaling and interaction	3
Gene expression	2
Lipid metabolism	1
Cell cycle	1

^{A score of 5 (highest) to 1 (lowest) was based on the rank of pathways (top five) for each of the four HTR-8-C19 lines used in our study and added to generate the total score.}

Table 2.

Gene Ontology Analysis for 421 Commonly Down-Regulated Genes: Top 10 Enriched Items of Biological Process

Term	Count	%	Benjamini	FDR
Response to wounding	32	8.84	6.90E − 05	5.15E − 05
Inflammatory response	23	6.35	3.45E − 04	5.15E − 04
Cell motion	27	7.46	8.51E − 04	2.54E − 03
Leukocyte migration	10	2.76	9.49E − 04	2.12E − 03
Localization of cell	20	5.52	2.83E − 03	1.27E − 02
Cell motility	20	5.52	2.83E − 03	1.27E − 02
Cell migration	19	5.25	2.89E − 03	1.08E − 02
Regulation of programmed cell death	35	9.67	3.58E − 03	2.41E − 02
Positive regulation of protein secretion	8	2.21	3.75E − 03	1.96E − 02
Regulation of protein secretion	9	2.49	3.98E − 03	2.38E − 02

To identify direct target genes of C19MC miRNAs we combined three miRNA target algorithms, TargetScan, miRDB, and microRNA.org, and identified C19MC miRNA targets that were suggested by all predictive algorithms. From a total of 2334 predicted targets for C19MC miRNAs in our microarray analysis, we identified 87 transcripts that were down-regulated in all four HTR-8-C19 clones. Among these, we validated, using qPCR, the down-regulation of gene products that are functionally related to cellular movement, migration, or invasion, including CXCL6, FOXL2, NR4A2, NTN4, BAMBI, LOX1, IL6, CADM1, FOXF1, CCL2, PBX3, and RND3 (Figure 3) (31,–41).

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Figure 3.

The expression of C19MC miRNA targets, implicated in cell adhesion and extracellular matrix processing. RT-qPCR validation of differentially expressed genes in HTR-8-C19 cells and HTR-8-WT cells. The relative expression level is presented as fold change compared with parental HTR-8-WT cells. The experiments were performed in duplicate and repeated four independent times (*, P < .05; **, P < .01, paired Student t test).

The effect of the C19MC miRNA miR-519d on relevant targets in HTR-8-C19 cells

We sought to identify specific C19MC miRNAs that might regulate these 12 mRNA targets. We therefore constructed luciferase reporter plasmids harboring the 3′UTR of the 12 down-regulated genes. We expressed these reporter genes in HTR-8/SVneo cells, along with expression vectors for miR-519d, miR-515–5p, and miR-520g, which were predicted to target more than one of these putative mRNA targets. As shown in Figure 4A, luciferase activity of CXCL6, FOXL2, and NR4A2 reporters was attenuated by expression of miR-519d, but not by miR-515–5p or 520g miRNA expression constructs. There was an insignificant effect on the other reporters (not shown). Moreover, this effect was abrogated using transfection of reporter plasmids that harbor mutated miR-519d sites within the CXCL6, FOXL2, and NR4A2 3′UTR, downstream from luciferase (Figure 4B). To bolster these findings, we overexpressed a miR-519d mimic in the parental HTR-8/SVneo cells and found that the expression of Cxcl6, Foxl2, and NR4A2 transcripts was significantly down-regulated when compared with a scramble control mimic (Figure 4C). Together, these data confirm that CXCL6, FOXL2, and NR4A2 are direct targets for miR519d and likely contributors to the phenotypic differences in migration properties between VTs and EVTs.

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Figure 4.

miR-519d regulates the expression of discrete mRNA targets in trophoblasts. A, HTR-8/SVneo cells were transfected with the 3′-UTR reporter vectors for CXCL6, FOXL2, and NR4A2, as well as the vectors expressing miRNA precursors of miR-519d, miR-520g, miR-515–5p, or a scramble control vector. Renilla luciferase activity, expressed as relative luciferase unit and normalized to firefly luciferase activity, was determined 48 h after transfection. Each experiment was performed in duplicate and repeated three times (*, P < .05; paired Student t test). B, HTR-8/SVneo cells were transfected with either wild-type (WT) 3′UTR reporter for CXCL6, FOXL2, and NR4A2 or a reporter that harbors mutant miR-519d-responsive element. Each reporter was transfected, along with the vector expressing a miR-519d precursor or a control vector. Relative luciferase unit activity was determined in duplicate and repeated three times, as described in A. (*, P < .05; paired Student t test). C, The expression of CXCL6, FOXL2, and NR4A2 in HTR-8/SVneo cells transfected with miR-519d mimics or scrambled control mimics. The expression level of each transcript is expressed as fold change compared with control. The experiments were performed in duplicate and repeated three times independently (*, P < .05; paired Student t test).

Higher expression of C19MC in VTs vs EVTs

Figure 1.

Ectopic expression of C19MC miRNAs in HTR-8/SVneo line attenuates cell migration

Figure 2.

C19MC miRNAs target pathways involved in adhesion and extracellular matrix processing

Table 1.

Ranked Scores for Molecular and Cellular Functions and Canonical Pathways, Based on Differentially Expressed Genes in Four HTR-8-C19 Stable Cell Lines, Compared With HTR-8-WT Cells

Pathway	Score^{^a}
Cellular movement	17
Cellular growth and proliferation	15
Cellular development	11
Cell death and survival	10
Cell-to-cell signaling and interaction	3
Gene expression	2
Lipid metabolism	1
Cell cycle	1

^{A score of 5 (highest) to 1 (lowest) was based on the rank of pathways (top five) for each of the four HTR-8-C19 lines used in our study and added to generate the total score.}

Table 2.

Gene Ontology Analysis for 421 Commonly Down-Regulated Genes: Top 10 Enriched Items of Biological Process

Term	Count	%	Benjamini	FDR
Response to wounding	32	8.84	6.90E − 05	5.15E − 05
Inflammatory response	23	6.35	3.45E − 04	5.15E − 04
Cell motion	27	7.46	8.51E − 04	2.54E − 03
Leukocyte migration	10	2.76	9.49E − 04	2.12E − 03
Localization of cell	20	5.52	2.83E − 03	1.27E − 02
Cell motility	20	5.52	2.83E − 03	1.27E − 02
Cell migration	19	5.25	2.89E − 03	1.08E − 02
Regulation of programmed cell death	35	9.67	3.58E − 03	2.41E − 02
Positive regulation of protein secretion	8	2.21	3.75E − 03	1.96E − 02
Regulation of protein secretion	9	2.49	3.98E − 03	2.38E − 02

Figure 3.

The effect of the C19MC miRNA miR-519d on relevant targets in HTR-8-C19 cells

Figure 4.

Discussion

Primary human trophoblasts express high levels of C19MC miRNAs (8, 15). In this work, we further characterized the distribution of C19MC miRNAs within the human placenta. We found that the expression of C19MC miRNA was higher in VTs than in EVTs. Our findings were based on measurements in diverse types of EVTs, derived from normal pregnancies, ectopic pregnancies, or cultured EVTs, and VTs.

C19MC miRNAs are almost exclusively expressed in the human placenta. Our recent work suggested that C19MC miRNAs are packaged within trophoblast-derived exosomes and attenuate viral infection in nonplacental recipient cells by the induction of autophagy (17, 42, 43). Our findings here expand the potential roles of C19MCs in human placental trophoblasts, and may explain some of the phenotypic differences between VTs and the more invasive EVTs (44). Interestingly, in certain cancer cells, the functions of C19MC miRNAs may be oncogenic, whereas in others tumors, C19MC miRNAs exhibit tumor suppression (12). Upon stable transfection of the entire C19MC in HTR-8/SVneo, which do not naturally express C19MC miRNAs, we found that these miRNAs attenuate EVT migration without affecting cell proliferation or apoptosis. Our data therefore suggest that the expression of C19MC miRNAs in EVTs, either by enhanced synthesis in EVTs or by synthesis in VTs with subsequent delivery into EVTs, might slow EVT migration. In addition, because trophoblast invasion usually peaks during early pregnancy and decreases as pregnancy progresses, our data suggest that lingering expression of C19MC miRNAs in EVTs of the third trimester may lessen trophoblast invasion as pregnancy progresses.

The function of individual C19MC miRNA has been examined in diverse systems. With regard to cell migration and invasion, miR-520h has been shown to inhibit migration and invasion of pancreatic cells through targeting ABCG2 (45), and miR-520b has been reported to suppress the migration of breast cancer cells by targeting hepatitis B X-interacting protein and IL-8 (46). In addition, miR-520c-3p has been reported to inhibit hepatocellular carcinoma cell invasion through targeting glypican-3 (47) and to abrogate breast cancer cell metastasis by suppression of TGFBR2 (48). In contrast, miR-520c-3p may promote invasion and metastasis in breast tumor cells by suppression of CD44 (49). Our data support a general suppressive effect of C19MC miRNAs on EVT migration and highlight the potential role of several C19MC members in this process. Employing a target prediction strategy, we found that the C19MC member miR-519d might play a role in regulating trophoblast migration.

In addition to C19MCs, other miRNAs were recently shown to regulate trophoblast migration by either promoting or inhibiting this process. MiRNA-378a-5p promotes trophoblast cell migration and invasion by targeting Nodal (50). Similarly, miRNA-376c facilitates trophoblast invasion through targeting TGF-β and nodal signaling (51). In contrast, miRNA-155 silences cyclinD1 and thus inhibits migration of HTR-8/SVneo cells (52). Other migration/invasion inhibitory miRNAs include miR-210 and miR-29b (53, 54). Our new data on C19MC miRNAs further expands knowledge in this area, establishing a greater role for miRNA in regulating trophoblast migration and invasion.

Interestingly, there are conflicting data with regard to the function of miR-519d in tumorigenesis. One report suggests that miR-519d promotes cell proliferation and invasion in hepatocellular carcinoma, acting through targeting of CDKN1A/p21, PTEN, AKT3, and TIMP2 (55). In contrast, miR-519d has been shown to suppress hepatocellular carcinoma growth through targeting MKi67 (56). We found that miR-519d silenced the expression of CXCL6, NR4A2, and FOXL2, which are implicated in cell migration in other systems. Importantly, these genes harbor a miR-519d-responsive seed element in their 3′UTR sequences. CXCL6 is known to promote neutrophil migration and facilitate tumor cell invasion and metastasis (31), yet CXCL6 was recently reported to inhibit human trophoblast cell migration and invasion by suppressing MMP-2 activity in the first trimester (57). NR4A2 (NURR1) promotes mesenchymal stromal cell migration (32) and could attenuate the migration of bladder cancer cells (58, 59). FOXL2 is known for its role in ovarian development and function (60), yet the direct evidence that ties FOXL2 to regulation of cell migration is lacking. Nonetheless, FOXL2 could target some migration-related chemokines, such as IL29A and ICAM1 (33). In the placenta, FOXL2 is expressed in EVTs, but not in VTs (61, 62). Lastly, we note that comparing the robust effect of C19MC miRNAs on putative mRNA targets (Figure 3) and the effect of miR-519d on selected direct targets (Figure 4) suggests that other C19MC miRNAs may cooperate with miR-519d to silence target genes in EVTs, directly or indirectly.

In our study, we interrogated other miRNA-mRNA regulatory pairs, such as miR-520g-RND3, miR-515–5p-IL6, miR-519d-NTN4, and miR-519d-PBX3. By transfecting the 3′UTR luciferase reporter of the putative target genes along with their predicted regulating miRNA mimic we found that some constructs showed weak or minimal repression. Future work may target additional C19MC miRNAs using lentivirus-based overexpression and will allow us to better define the miRNA-based genomic network that modulates EVT invasion.

A limitation of our study is that the molecular manipulations were performed in an EVT cell line, and not in primary cells. Because the mechanisms underlying regulation of C19MC miRNAs are currently unknown, and because of the short culture time of primary trophoblasts, these approaches await further progress in the field. Moreover, C19MC miRNAs are expressed only in primates and humans, rendering genomic manipulations of C19MC miRNAs in vivo rather challenging. Lastly, our data centered on the regulation of invasion and migration, and we cannot exclude the possibility that C19MC miRNAs may affect other functions of EVTs.

In summary, our data defined the expression pattern of C19MC miRNAs in EVTs vs VTs, showing a lower expression in diverse types of EVTs. C19MC miRNAs lessen cell migration, with miR-519d targeting specific proteins that may play a role in the invasive phenotype. Our work expands the potential roles of C19MCs in human placental trophoblasts, and suggests that C19MC miRNAs contribute to the phenotypic differences between VTs and the more invasive EVTs.

Magee-Womens Research Institute (L.X., J.-F.M., T.C., W.T.P., E.S., Y.S.), Department of OBGYN and Reproductive Sciences, University of Pittsburgh, Pennsylvania 15213; Medical Systems Biology Research Center (L.X.), Department of Biomedical Engineering, Tsinghua University School of Medicine, Beijing 100084 China; Department of Obstetrics and Fetal-Maternal Medicine (M.K.), Reproductive Biology Unit, Medical University of Vienna, Vienna, A-1090 Austria; and Department of Microbiology and Molecular Genetics (Y.S.), University of Pittsburgh, Pennsylvania 15213

^{Corresponding author.}

Address all correspondence and requests for reprints to: Yoel Sadovsky, MD, Magee-Womens Research Institute, 204 Craft Avenue, Pittsburgh, Pennsylvania 15213., E-mail: ude.eegam.irwm@yksvodasy.

Received 2014 Jun 19; Accepted 2014 Sep 4.

Abstract

Early in pregnancy, trophoblast invasion into the decidua and inner myometrium is essential for establishment of proper implantation, maternal-fetal exchange, and immunological tolerance of the feto-placental allograft. Unlike villous trophoblasts (VTs), extravillous trophoblasts (EVTs) are unique in their capacity to invade the maternal decidua and myometrium. The largest human microRNA (miRNA) gene cluster, the chromosome 19 miRNA cluster (C19MC), is expressed almost exclusively in the placenta and, rarely, in certain tumors and undifferentiated cells. In the work reported here, we found that the expression of C19MC miRNAs is higher in VTs than in EVTs. Using a bacterial artificial chromosome (BAC)-mediated overexpression of C19MC miRNAs in an EVT-derived cell line, which does not naturally express these miRNAs, we found that C19MC miRNAs selectively attenuate cell migration without affecting cell proliferation or apoptosis. A microarray analysis revealed that C19MC miRNAs regulate target transcripts related to cellular movement. Our data also implicated a specific C19MC member, miR-519d, indirectly regulating the EVT invasive phenotype by targeting CXCL6, NR4A2 and FOXL2 transcripts through a 3′UTR miRNA-responsive element. Together, our data suggest a role for C19MC miRNAs in modulating the migration of EVTs.

Abstract

In the human placenta, trophoblasts largely differentiate along the villous or the extravillous trophoblast (EVT) pathways. The villous trophoblasts (VTs) form the outermost layer of the chorionic villi and play a critical role in the regulation of gas exchange, uptake of nutrients, and elimination of waste between the maternal and fetal circulations, as well as in the production of hormones and immunological protection of the fetal allograft (1). Bathed in maternal blood are the placental syncytiotrophoblasts, a layer of multinucleated, terminally differentiated cells that overlies a layer of mononuclear, less differentiated cytotrophoblasts (2). The EVTs invade the maternal decidua and myometrium during the course of implantation (3), anchoring the chorionic villi to the decidua and uterine wall. Unlike the VTs, the EVTs are characterized by their invasiveness, a process that spans cell proliferation, matrix degradation, migration, and differentiation. These components are exquisitely regulated to achieve the precise degree of invasion, formation of placental cell columns, and the respective vascular support (4, 5). Dysregulation of trophoblast invasion is associated with diverse types of placental abnormalities that affect embryonic development and, consequently, fetal growth and pregnancy health. To date, processes that govern the invasion and differentiation of EVTs are inadequately understood.

Like other cell types, trophoblasts produce diverse types of microRNAs (miRNAs), which have been implicated in placental development or physiology (6, 7). Human trophoblasts also produce uncommon miRNA species, including members of the chromosome 19 miRNA cluster (C19MC) (8). C19MC is the largest human miRNA gene cluster and consists of 46 genes encoding a total of 56 mature miRNAs (9). This cluster is only present in the primate and human genomes and expresses miRNAs almost exclusively in placenta (8), with expression detected in only a few other cell types such as embryonic stem cells and certain tumors (10,–13). C19MC miRNAs are also highly expressed in trophoblast-derived vesicles, including exosomes (14, 15). We recently showed that C19MC miRNAs are among the most abundant miRNAs in the human placenta and in the sera of pregnant women (15, 16), and that both villous syncytiotrophoblasts and cytotrophoblasts express comparable levels of C19MC miRNAs (15). Importantly, we recently showed that trophoblastic exosomes or their C19MC content confer viral resistance to recipient nonplacental cells (17).

In our quest to define the expression and function of trophoblastic miRNAs, we found that C19MC miRNAs are expressed not only in VTs, but also in EVTs, albeit at a markedly lower level. We hypothesized that C19MC miRNAs may play a role in the function of EVTs. To test this hypothesis, we used bacterial artificial chromosome (BAC)-mediated overexpression of C19MC miRNAs in an EVT-derived cell line that does not naturally express these miRNAs. We found that C19MC miRNAs selectively attenuated cell migration through interaction with a network of enzymes and proteins that regulate cell motility. Our data also implicate a specific C19MC member, miR-519d, indirectly regulating the EVT invasive phenotype.

Acknowledgments

The authors gratefully thank Dr. C.H. Graham for the HTR-8/SV-Neo cells, Judy Ziegler for technical assistance, Lori Rideout for manuscript preparation, and Bruce Campbell for editing.

This work was supported by National Institutes of Health (NIH) R01-HD06589, R21-HD071707 (both to Y.S.), and the Pennsylvania Department of Health Research Formula Funds (J.-F.M. and T.C.). The generation of the C19MC BAC, as described, was also made possible by NIH Grants UL1-RR024153 and UL1-TR000005.

Disclosure Summary: L.X., J.-F.M., T.C., W.T.P., E.S., M.K. have nothing to declare. Y.S. is a named inventor on a pending patent application describing the use of C19MC microRNAs as therapeutics.

Acknowledgments

Footnotes

Abbreviations:

BAC: bacterial artificial chromosome
BrdU: 5-bromo-2′-deoxyuridine
C19MC: chromosome 19 miRNA cluster
EVT: extravillous trophoblast
PARP: poly (ADP-ribose) polymerase
SDS: sodium dodecyl sulfate
TBS: tris-buffered saline
VT: villous trophoblast.

Footnotes

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