International Journal of Endocrinology

Volume 2014 (2014), Article ID 393707, 15 pages

http://dx.doi.org/10.1155/2014/393707

Research Article

Gene Expression Profiling of the Paracrine Effects of Uterine Natural Killer Cells on Human Endometrial Epithelial Cells

Xin Gong,¹ Zhenzhen Chen,² Yanxia Liu,¹ Qiudan Lu,¹ and Zhe Jin¹

¹Reproductive Endocrinology Centre, Dongfang Hospital of Beijing University of Chinese Medicine, No. 6 Fangxingyuan 1 Qu, Fengtai District, Beijing 100078, China

²School of Chinese Materia Medica, Beijing University of Chinese Medicine, No. 6 Zhonghuan South Road, Wangjing, Chaoyang District, Beijing 100102, China

Received 19 December 2013; Accepted 18 February 2014; Published 26 March 2014

Academic Editor: F. Xavier Donadeu

Copyright © 2014 Xin Gong et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The endometrium contains a population of immune cells that undergo changes during implantation and pregnancy. The majority of these cells are uterine natural killer (uNK) cells; however, it is unclear how these cells interact with endometrial epithelial cells. Therefore, we investigated the paracrine effects of the uNK cell-secretion medium on the gene expression profile of endometrial epithelial cells in vitro through microarray analysis. Our results, which were verified by qRT-PCR and western blot, revealed that soluble factors from uNK cells alter the gene expression profiles of epithelial cells. The upregulated genes included interleukin-15 (IL-15) and interleukin-15 receptor alpha (IL-15RA), which result in a loop that stimulates uNK cell proliferation. In addition, vascular endothelial growth factor C (VEGF-C) and chemokine (C-X-C motif) ligand 10 (CXCL-10) were also determined to be upregulated in epithelial cells, which suggests that uNK cells work synergistically with epithelial cells to support implantation and pregnancy. In addition, oriental herbal medicines have been used to treat infertility since ancient times; however, we failed to find that Zi Dan Yin can regulate these endometrial paracrine effects.

1. Introduction

In the human endometrium, uterine leucocytes undergo cyclic changes during the menstrual cycle. Uterine adaptation to pregnancy includes highly regulated immune cell trafficking [1]. According to recent studies, uterine natural killer (uNK) cells are an in vivo factor that contributes to the pathological elongation of the window of endometrial receptivity [2]. The genetic deficiency of uNK cells results in a lack of remodeling of the uterine vasculature, leading to hypertrophied vascular media, swollen endothelial cells, and narrow vessel lumens [3]. Abnormal activation and numbers of uNK cells have been implicated in pregnancy complications [4].

uNK cells are the most abundant leucocytes during the implantation window and early pregnancy [5]. This sharp increase is likely due to the proliferation of existing uNK cells [6–8]. Hormonal effects may control this proliferation. However, to date, progesterone receptors have not been localized on these cells [9]; therefore, it has been proposed that progesterone may exert its effects on uNK cells indirectly via cytokines, such as interleukin-15 (IL-15), and other soluble factors [8, 10]. A previous study indicated that the paracrine communication between uterine leucocytes (>85% uNK) and uterine stromal cells can upregulate IL-15 and interleukin-15 receptor alpha (IL-15RA) to induce the proliferation of uNK cells and contributes to the support of trophoblast migration during implantation [11].

uNK cells are resident throughout the endometrium at the time of conception and throughout early pregnancy. Although uNK cells predominantly localize to the uterine stroma and the surrounding areas of the spiral arteries, Matrigel-supported cocultures of endothelial cells and uNK cells have shown that vascular endothelial growth factor C (VEGFC) producing uNK cells induce transporter 1 (TAP-1) expression in endothelial cells [12]. Furthermore, the endometrium undergoes precisely defined morphological and biochemical changes during implantation and early pregnancy; thus, there is no doubt that the preparation of epithelial cells is directed towards these process. We hypothesize that there similar paracrine communications occur between uNK cells and uterine epithelial cells. Our results revealed that soluble factors from uNK cells have substantial effects on endometrial epithelial gene expression. uNK cells were found to upregulate transcript levels in epithelial cells that are known to promote uNK cell proliferation, and these upregulated transcripts may contribute to the preparation for implantation and trophoblast migration and invasion.

In China, oriental herbal medicines have been used to treat infertility since ancient times [13]. Zi Dan Yin (ZDY), a compound of nine herbs (Table 1), was found to improve the morphology of the endometrium in mice [14]. Although the treatment effects of ZDY on mice have been confirmed, it is unclear whether ZDY contributes to the local immune responses. However, our results do not support the hypothesis that ZDY can improve the paracrine effects between uNK and endometrial epithelial cells.

Table 1: Composition of Zi Dan Yin (ZDY).

Components
Ratio

(1) Sheng Di (Rehmannia glutinosa (Gaertn.) Libosch., root)
15

(2) Dan Shen (Salviae Miltiorrhizae Bge., root)
10

(3) Dang gui (Angelica sinensis (Oliv.) Diels., root)
12

(4) Chuan Duan (Dipsacus asperoides C. Y. Cheng et T.M. Ai., root)
15

(5) Du Zhong (Eucommia ulmoides Oliv., cortex)
12

(6) Shan Yao (Dioscorea opposita Thunb., rhizome)
15

(7) Mei Gui-hua (Rosa rugosa Thunb., flower)
6

(8) Chuan Xiong (Ligusticum Chuanxiong Hort., rhizome)
6

(9) Yi Yi-ren (Coix lacryma-jobi L. var. ma-yuen (Roman.) Stapf., seed)
12

2. Materials and Methods

2.1. Ethics Statement

All of the subjects understood and signed the informed consent form before participation. The experimental protocols were approved by the Ethics Committee of the Dongfang Hospital Human Ethics Committee (no. 2011090201).

2.2. Collection of Material

Decidual tissues were obtained from healthy women undergoing elective termination of a normal pregnancy between 7 and 8 weeks of gestation. The endometrial tissues were collected from endometrial biopsies taken during the proliferative phase of the cycle from women undergoing laparoscopy (Dongfang Hospital of Beijing University of Chinese Medicine, China) for benign disease. The average age was 30 1.3 years, and the body mass index (BMI) was 22.1 0.8 kg/m². All of the nine women were Chinese of Han ethnicity. The exclusion criteria were hormonal stimulation, cancerous lesions, and irregular menstrual bleeding.

2.3. Isolation of the Uterine NK (uNK) Cells

The uNK cells were isolated as previously described [15, 16]. Briefly, the decidual tissues were extensively washed with Ca²⁺- and Mg2⁺-free Hank's balanced salt solution (HBSS) containing antibiotics, minced thoroughly between two scalpels into fragments of 1-2 mm³, and digested for 1 h at 37°C with gentle agitation in HBSS with 0.1% (w/v) collagenase I (Gibco, USA). The cell suspensions were layered over Ficoll-Hypaque medium (General Electric, USA) and centrifuged at 800 ×g and room temperature for 25 min. The cells at the interface were washed twice with Roswell Park Memorial Institute (RPMI)-1640 media with 10% fetal calf serum (FCS) and antibiotics. After incubation for 20 min at 4°C with anti-CD56 microbeads (Miltenyi Biotec, Ltd., Germany), the cells were washed with washing buffer (PBS, 2 mM EDTA, and 0.5%BSA (w/v)) and then loaded onto a MiniMACS Separator (MS) column in a MiniMACS magnet (Miltenyi Biotec, Ltd., Germany). After flushing the MS column three times, the CD56⁺ cells were flushed as indicated by the manufacturer. The purity of the uNK cells was >90% CD56⁺CD3⁻ according to the flow cytometric analysis. The uNK cells were cultured in RPMI 1640 media with 1% FCS and IL-15 (10 ng/mL) (R&D systems, USA), in which to maintain the viability of the purified uNK cells [17].

2.4. Production of uNK Cell-Secretion Medium

The uNK cell-secretion medium was prepared using 200 μL of RPMI 1640 media with 1% FCS and IL-15 (10 ng/mL) containing of the purified uNK cells. The cell suspension was placed in the upper chamber of a 0.4 μm pore hanging cell culture insert (Millipore, USA) in a 24-well tissue plate, and 1300 μL of the same media without cells was placed in the lower chamber. As a result, only the soluble molecules from the uNK cells can pass through the filter into the lower chamber. The control medium was 1500 μL of the RPMI 1640 media with 1% FCS and IL-15 (10 ng/mL), and this control medium was also used in the subsequent experiments. After incubation for 24 h at 37°C, the uNK cell-secretion medium (in the lower chamber) and the control medium were collected and frozen at −80°C. The cells in the upper chamber were collected, and the cell viability was measured using a live/dead viability kit (Invitrogen, USA). Only the conditional medium from uNK populations with less than 35% dead cells after overnight incubation was used in the subsequent experiments.

2.5. Human Endometrial Epithelial Cell Culture

Human endometrial tissue was cut using scissors and dissociated into single-cell suspensions using 0.1% (w/v) collagenase I for 50–60 min. Every 15 min, the digests were pipetted, and their dissociation was monitored by microscopy. The cell suspensions were filtered using a 40-mesh screen to separate the single cells from undigested tissue. To remove the erythrocytes, the cells were resuspended in 4 mL of Dulbecco’s modified Eagle’s medium and Ham’s nutrient F12 (DMEM/F12) with 1% FCS, layered over Ficoll-Paque PLUS, and centrifuged for 25 min at 800 ×g. The leukocytes were removed with CD45-coated Dynabeads (Dynal Biotech, USA). The purified epithelial cell suspensions were then obtained through a further round of magnetic bead sorting using Collection Epithelial Dynabeads (Dynal Biotech, USA). The epithelial cell preparations were >95% pure.

The endometrial epithelial cells were cultured in serum-free bronchial epithelial growth medium (BEGM, Clonetics Crop) containing 2 mL of bovine pituitary extract (BPE), 0.5 mL of insulin, 0.5 mL of hydrocortisone (HC), 0.5 mL of gentamicin sulfate and amphotericin-B (GA-1000), 0.5 mL of retinoic acid, 0.5 mL of transferrin, 0.5 mL of triiodothyronine, 0.5 mL of epinephrine, and 0.5 mL of recombinant human epidermal growth factor (hEGF) (all supplied by Clonetics Corp., USA). The media was changed every five days. After two weeks, the epithelial cells were passed from six-well plates into 25 cm² cell culture flasks.

2.6. ZDY Preparation

All of the crude drugs of ZDY were obtained from the Pharmacy Department of Dongfang Hospital of Beijing University of Chinese Medicine (Beijing Province, China). The quality of the raw herbs was controlled according to the requirement of the Pharmacopoeia of China. The aqueous extract of ZDY was prepared as follows. In brief, nine medicinal materials were mixed at the appropriate proportion, macerated for 1 h with eight volumes of distilled water and then decocted for 2 h. The cooled extract was then filtered. This extraction procedure was repeated twice. The extracts were then combined and concentrated by boiling to a final volume of 100 mL (10.3 g/mL). The ZDY extract concentration of 2 mg/mL was further diluted with the epithelial cell culture medium and passed through 0.2 μm sterilization filters for use in subsequent studies. The ZDY extract was stored at 2–8°C until further use. The concentration of ZDY was used according to previous analyses using a cell counting kit-8 (CCK-8).

2.7. Treatment of Epithelial Cells with uNK Cell-Secretion Medium

The endometrial epithelial cells were allocated into three groups: control, uNK, and ZDY groups. The cells in the control and uNK groups were treated with DMEM medium with 10% FBS for 24 h, and the cells in the ZDY group were treated with 2 mg/mL ZDY for 24 h. All of the cells were then washed twice with PBS and placed in serum-free DMEM for 24 h prior to the subsequent experiments. The uNK cell-secretion medium was pooled from different batches to decrease the variability. The DMEM in the ZDY and uNK groups was then replaced with 80% uNK cell-secretion medium and 20% serum-free DMEM. The control group was treated with 80% control medium and 20% serum-free DMEM. After 6 h of incubation, the cells from the three groups were collected.

2.8. Microarray Experiments

The total RNA from endometrial epithelial cells from the control, uNK, and ZDY groups was extracted using Trizol (Invitrogen, USA). The RNA was purified using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions, quantified by spectrophotometry. The microarray analysis was performed using the GeneChip 3′ IVT Express Kit (Affymetrix). Briefly, the total RNA was subjected to reverse transcription, first-strand cDNA synthesis, double-strand DNA, in vitro transcription, and fragmentation. The samples were hybridized onto the GeneChip PrimeView Human Gene Expression Array (Affymetrix, USA), which covers more than 36,000 transcripts and variants. After 16 h of hybridization at 45°C, the arrays were washed on a Fluidics Station 450 (Affymetrix, USA) and then scanned with a Scanner 3000 (Affymetrix, USA) to obtain the quantitative gene expression levels. The endometrial epithelial cells from the control, uNK, and ZDY groups were processed simultaneously throughout the experimental process, and three chips were used for each group.

2.9. Quantitative Real-Time PCR (qRT-PCR) Analysis

To verify the results obtained from the microarray experiments, qRT-PCR verification was performed for five genes: chemokine (C-X-C motif) ligand 10 (CXCL-10), interleukin-15 (IL-15), intercellular adhesion molecule 1 (ICAM-1), NLR family CARD domain containing 5 (NLRC-5), and interferon regulatory factor 1 (IRF-1). The cells from the control, uNK, and ZDY groups were washed twice with PBS, and the total RNA was then extracted using Trizol. The RNA was quantified by spectrophotometry. Reverse transcription was performed with 8 μL of total RNA in a 20 µL reaction volume using a standard cDNA Synthesis Kit (Takara BIO, Japan). The qRT-PCR primer sequences for the target genes were self-designed, and the PCR primers were ordered from Invitrogen. The primer sequences for target genes are shown in Table 2.

Table 2: Sequences of primers used in the quantitative real-time PCR analysis.

Gene
Forward primer
Reverse primer
Amplicon

CXCL-10
CTTTCTGACTCTAAGTGGCATTC
CACCCTTCTTTTTCATTGTAGCAA
176 bp

IL-15
TGGCTGCTGGAAACCC
CACAAGTAGCACTGGATGGAAAT
123 bp

ICAM-1
GAGGAAGGAGCAAGACTCAA
AGCATACCCAATAGGCAGCAAG
141 bp

NLRC-5
AAACTTGATGACTCCTCCCTTACTT
TTAGACCTGGCTTTGTCCCTTAC
120 bp

IRF-1
CCAGAAAAGCATAACACCAATCC
CCACTTTCCTTCACATTTCACTG
144 bp

GAPDH
GAGCCAAAAGGGTCATCATCT
AGGGGCCATCCACAGTCTTC
231 bp

For each qRT-PCR reaction, the typical thermal cycling conditions included an initial activation step of 95°C for 5 min and 45 cycles of 95°C each for 30 sec, 65°C for 35 sec, and 72°C for 5 min. The PCR reactions were performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems, USA). For comparison of the target mRNA levels, the cDNA concentration was normalized with that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR product. The target mRNA expression was analyzed using the 2-ΔΔCt algorithm.

2.10. Western Blot Analysis of ICAM-1

The cells from the control, uNK, and ZDY groups were lysed with RIPA lysis buffer (Applygen, China) supplemented with a protease inhibitor cocktail (Applygen, China). The protein concentrations were quantified using bicinchoninic acid (BCA; Applygen, China). The proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose. The blots were blocked overnight in Tris-buffered-saline with Tween (TBST) containing 5% dried milk, washed three times for 5 min each in TBST, and then incubated overnight in a rocker at 4°C with a cocktail containing ICAM-1 (Abcam, UK; at a dilution of 1 : 1000) and 2% dried milk in TBST. The blots were then washed three times for 10 min each in TBST and incubated with the secondary antibody (Applygen, China; at a dilution of 1 : 10000) for 1 h at room temperature in a rocker. The blots were washed three times for 10 min each in TBST and then visualized with the Super ECL Plus Detection Reagent (Applygen, China). The ECL signals were detected with the Quantity One software (Bio-Rad, USA). GAPDH (Abcam, UK) was used as the internal control to validate the amount of protein.

2.11. Statistical Analysis

All of the variables were tested in three different culture experiments. The data were analyzed by ANOVA. In the microarray experiments, the significance level was determined by ANOVA with Benjamini-Hochberg correction (). We selected the up- and downregulated genes that showed a median fold change greater than 2 and a . The data from the qRT-PCR and western blot analyses are presented as means SEM after normalization with the average value of the housekeeping gene obtained for each group, and the significance level was . The graphs of the data were produced using the Microsoft Excel software.

3. Results

3.1. Microarray Experiments

Gene expression profiling using a microarray was used to compare the transcript expression levels in epithelial cells treated with control medium, uNK cell-secretion medium, and ZDY+ uNK cell-secretion medium.

This analysis identified 169 transcripts that exhibited a statistically significant change in their median expression level (either upregulated or downregulated) in response to uNK cell-secretion medium compared with their control level. These transcripts are listed in Table 3. A total of 40 genes were found upregulated, and 129 genes were downregulated. The largest group of upregulated genes included immunomodulatory gene regulators, cytokines, such as interleukin-15 (IL-15), chemokine (C-X-C motif) ligand 10 (CXCL-10), and vascular endothelial growth factor C (VEGF-C), and cytokine receptors, such as interleukin-15 receptor alpha (IL-15RA). Additionally, the upregulated transcripts encode proteins associated with transport, structuration, and transcription. The level of adhesion molecule intercellular adhesion molecule 1 (ICAM-1) was also altered. ICAM-1 binds to the leucocyte integrins LFA-1 and Mac-1 [18, 19], which are expressed on uNK cells. Thus, this change in the level of ICAM-1 should increase the localized interactions between uNK cells and epithelial cells. A number of transcripts regulating enzyme activity, such as the deltex 3-like (Drosophila) (DYX3L) and the proteasome (prosome, macropain) subunit beta type 9 (large multifunctional peptidase 2) (PSMB9), and several transcripts regulating nucleotide metabolism genes, such as guanylate binding protein 5 (GBP5) and guanylate binding protein 1 interferon-inducible (GBP1), were also upregulated. In addition, genes with various functions, such as the icon binding complement component 1S subcomponent (C1S), SP110 nuclear body protein (SP110), tryptophan metabolism indoleamine 2,3-dioxygenase 1 (IDO1), and tryptophanyl-tRNA synthetase (WARS), and several genes with unknown function were found to be upregulated. The largest group of downregulated genes encodes transcription factors, such as B double prime 1 subunit of RNA polymerase III transcription initiation factor IIIB (BDP1) and thyroid hormone receptor interactor 11 (TRIP11). The levels of immunomodulatory gene regulators, including interleukin-36 gamma (IL-36G) and interleukin-33 (IL-33), were also altered. The levels of transcripts encoding molecules responsible for signaling, transport, structuration, and cell-cell adhesion were also downregulated. In addition, genes with various functions, such as ion binding, kinase, enzyme activity, and nucleotide metabolism, were found to be downregulated.

Table 3: Transcripts that were altered by more than twofold in endometrial epithelial cells by stimulation with the uNK cell-secretion medium (). The transcript levels were obtained using a GeneChip PrimeView Human Gene Expression Array.

Gene symbol
Fold

change
Gene

ID
Description

Upregulated genes: 40 transcripts

Cytokines/membrane

proteins/immunological factors

    NLRC5
14.1
84166
NLR family, CARD domain containing 5

    IL15
5.8
3600
Interleukin-15

    IFIT3
5.8
3437
Interferon-induced protein with tetratricopeptide repeats 3

    CXCL10
3.1
3627
Chemokine (C-X-C motif) ligand 10

    IFIH1
2.9
64135
Interferon induced with helicase C domain 1

    IFIT5
2.8
24138
Interferon-induced protein with tetratricopeptide repeats 5

    IL-15RA
2.7
3601
Interleukin-15 receptor, alpha

    VEGFC
2.5
7424
Vascular endothelial growth factor C

    PDCD1LG2
2.5
80380
Programmed cell death 1 ligand 2

Transporters

    TAP1
3.4
6890
Transporter 1, ATP-binding cassette, sub-family B (MDR/TAP)

    TAP2
2.6
6891
Transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)

    APOL3
2.1
80833
Apolipoprotein L, 3

    VLDLR
2.1
7436
Very low density lipoprotein receptor

    APOL2
2.1
23780
Apolipoprotein L, 2

Structural factors

    LMNB1
4.3
4001
Lamin B1

Transcription

    IRF1
11
3659
Interferon regulatory factor 1

    IRF9
4.9
10379
Interferon regulatory factor 9

    STAT1
4.5
6772
Signal transducer and activator of transcription 1, 91 kda

    TAF15
2.1
8148
TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68 kda

Cell-cell adhesion

    ICAM1
5.1
3383
Intercellular adhesion molecule 1

Ion binding

    C1S
3.2
716
Complement component 1, s subcomponent

    SP110
2.9
3431
SP110 nuclear body protein

    PARP12
2.3
64761
Poly (ADP-ribose) polymerase family, member 12

    ZC3HAV1
2.3
56829
Zinc finger CCCH-type, antiviral 1

Tryptophan metabolism

    IDO1
6.2
3620
Indoleamine 2,3-dioxygenase 1

    WARS
4.2
7453
Tryptophanyl-trna synthetase

Enzyme activity

    DTX3L
4.6
151636
Deltex 3-like (Drosophila)

    PSMB9
3.3
5698
Proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional peptidase 2)

    MSRB1
2.5
51734
Methionine sulfoxide reductase B1

    CTSZ
2.2
1522
Cathepsin Z

Nucleotide metabolism

    GBP5
10.5
115362
Guanylate binding protein 5

    GBP1
5.8
2633
Guanylate binding protein 1, interferon-inducible

    PARP14
5.2
54625
Poly (ADP-ribose) polymerase family, member 14

    GBP2
3.2
2634
Guanylate binding protein 2, interferon-inducible

    MX1
2.5
4599
Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse)

    RAD23B
2.3
5887
RAD23 homolog B (S. Cerevisiae)

    GBP7
2
388646
Guanylate binding protein 7

Others

    FAM117B
3.6
150864
Family with sequence similarity 117, member B

    C19orf66
2.6
55337
Chromosome 19 open reading frame 66

    NUB1
2.5
51667
Negative regulator of ubiquitin-like proteins 1

Downregulated genes: 129 transcripts

Cytokines/membrane proteins/immunological factors

    IL36G
−3.4
56300
Interleukin-36, gamma

    IL33
−3.0
90865
Interleukin-33

    IL1RN
−2.5
3557
Interleukin-1 receptor antagonist

    DNAJB14
−2.5
79982
Dnaj (Hsp40) homolog, subfamily B, member 14

    KITLG
−2.3
4254
KIT ligand

    LARP4
−2.3
113251
La ribonucleoprotein domain family, member 4

    CLDN4
−2.2
1364
Claudin 4

    CUL5
−2.2
8065
Cullin 5

Intracellular/signalling factors

    ITGB8
−3.5
3696
Integrin, beta 8

    RICTOR
−2.8
253260
RPTOR independent companion of MTOR, complex 2

    IL6ST
−2.6
3572
Interleukin-6 signal transducer (gp130, oncostatin M receptor)

    SOCS4
−2.5
122809
Suppressor of cytokine signaling 4

    NRIP1
−2.5
8204
Nuclear receptor interacting protein 1

    SEMA3A
−2.5
10371
Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A

    PLCB4
−2.2
5332
Phospholipase C, beta 4

    COPS2
−2.2
9318
COP9 constitutive photomorphogenic homolog subunit 2 (Arabidopsis)

    BMPR2
−2
659
Bone morphogenetic protein receptor, type II (serine/threonine kinase)

Transporters

    TPR
−3.4
7175
Translocated promoter region, nuclear basket protein

    KIAA1033
−2.5
23325
Kiaa1033

    BAZ2B
−2.5
29994
Bromodomain adjacent to zinc finger domain, 2B

    TMED5
−2.2
50999
Transmembrane emp24 protein transport domain containing 5

    SLC4A7
−2
9497
Solute carrier family 4, sodium bicarbonate cotransporter, member 7

Structural factors

    ROCK2
−2.9
9475
Rho-associated, coiled-coil containing protein kinase 2

Transcription

    BDP1
−3.6
55814
B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB

    TRIP11
−3.4
9321
Thyroid hormone receptor interactor 11

    ZNF644
−3.3
84146
Zinc finger protein 644

    CHD9
−3.2
80205
Chromodomain helicase DNA binding protein 9

    ZNF480
−3.1
147657
Zinc finger protein 480

    ZNF292
−3
23036
Zinc finger protein 292

    ZNF148
−2.8
7707
Zinc finger protein 148

    ZNF267
−2.8
10308
Zinc finger protein 267

    ZBTB20
−2.7
26137
Zinc finger and BTB domain containing 20

    CREB1
−2.7
1385
Camp responsive element binding protein 1

    ZNF638
−2.6
27332
Zinc finger protein 638

    ZNF518A
−2.6
9849
Zinc finger protein 518A

    ESF1
−2.6
51575
ESF1, nucleolar pre-rrna processing protein, homolog (S. Cerevisiae)

    FAR1
−2.6
84188
Fatty acyl coa reductase 1

    ZNF91
−2.5
7644
Zinc finger protein 91

    CENPF
−2.5
1063
Centromere protein F, 350/400 kda (mitosin)

    FOXN2
−2.4
3344
Forkhead box N2

    DNTTIP2
−2.2
30836
Deoxynucleotidyl transferase, terminal, interacting protein 2

    TMF1
−2.2
7110
TATA element modulatory factor 1

    BCLAF1
−2.2
9774
BCL2-associated transcription factor 1

    CEBPZ
−2.2
10153
CCAAT/enhancer binding protein (C/EBP), zeta

    ZNF146
−2.1
7705
Zinc finger protein 146

    HIF1A
−2
3091
Hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)

Cell-cell adhesion

    DST
−2.9
667
Dystonin

Ion binding

    ATRX
−4.6
546
Alpha thalassemia/mental retardation syndrome X-linked

    EEA1
−3.4
8411
Early endosome antigen 1

    RSF1
−2.6
51773
Remodeling and spacing factor 1

    NIN
−2.6
51199
Ninein (GSK3B interacting protein)

    SCAF11
−2.5
9169
SR-related CTD-associated factor 11

    MIB1
−2
57534
Mind bomb E3 ubiquitin protein ligase 1

    TRMT13
−2
54482
Trna methyltransferase 13 homolog (S. Cerevisiae)

Kinase

    CCDC88A
−3.1
55704
Coiled-coil domain containing 88A

    FER
−3.1
2241
Fer (fps/fes related) tyrosine kinase

    KTN1
−3
3895
Kinectin 1 (kinesin receptor)

    SCYL2
−2.6
55681
SCY1-like 2 (S. Cerevisiae)

    AKAP11
−2.5
11215
A kinase (PRKA) anchor protein 11

    PIK3C2A
−2.5
5286
Phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2 alpha

    CDK6
−2.3
1021
Cyclin-dependent kinase 6

    NEK1
−2.3
4750
NIMA (never in mitosis gene a)-related kinase 1

    SLK
−2.2
9748
STE20-like kinase

    UTRN
−2.2
7402
Utrophin

    RB1CC1
−2
9821
RB1-inducible coiled-coil 1

    YES1
−2
7525
V-yes-1 Yamaguchi sarcoma viral oncogene homolog 1

Enzyme activity

    RANBP2
−2.9
5903
RAN binding protein 2

    JMJD1C
−2.7
221037
Jumonji domain containing 1C

    RNF6
−2.7
6049
Ring finger protein (C3H2C3 type) 6

    LTN1
−2.6
26046
Listerin E3 ubiquitin protein ligase 1

    RBBP6
−2.5
5930
Retinoblastoma binding protein 6

    PPIG
−2.5
9360
Peptidylprolyl isomerase G (cyclophilin G)

    PTAR1
−2.4
375743
Protein prenyltransferase alpha subunit repeat containing 1

    PYROXD1
−2.3
79912
Pyridine nucleotide-disulphide oxidoreductase domain 1

    LIG4
−2.3
3981
Ligase IV, DNA, ATP-dependent

    RAD50
−2.1
10111
RAD50 homolog (S. Cerevisiae)

    DBF4
−2
10926
DBF4 homolog (S. Cerevisiae)

Nucleotide metabolism

    CHD1
−2.6
1105
Chromodomain helicase DNA binding protein 1

    CHML
−2.5
1122
Choroideremia-like (Rab escort protein 2)

    ELMOD2
−2.1
255520
ELMO/CED-12 domain containing 2

    NAA15
−2
80155
N(alpha)-acetyltransferase 15, nata auxiliary subunit

    KIF20B
−2
9585
Kinesin family member 20B

Others

    GOLGA4
−3.9
2803
Golgin A4

    ASPM
−3.4
259266
Asp (abnormal spindle) homolog, microcephaly associated (Drosophila)

    CEP350
−3.3
9857
Centrosomal protein 350 kda

    C10orf118
−3.2
55088
Chromosome 10 open reading frame 118

    GALNT5
−3.2
11227
UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 5 (galnac-T5)

    BOD1L1
−3.1
259282
Biorientation of chromosomes in cell division 1-like 1

    ANKRD12
−3.1
23253
Ankyrin repeat domain 12

    RIF1
−3
55183
RAP1 interacting factor homolog (yeast)

    KIAA2026
−3
158358
Kiaa2026

    FAM111B
−2.9
374393
Family with sequence similarity 111, member B

    EIF5B
−2.8
9669
Eukaryotic translation initiation factor 5B

    OSBPL8
−2.8
114882
Oxysterol binding protein-like 8

    GOLGB1
−2.8
2804
Golgin B1

    MALAT1
−2.7
378938
Metastasis associated lung adenocarcinoma transcript 1 (non-protein coding)

    PRPF40A
−2.7
55660
PRP40 pre-mrna processing factor 40 homolog A (S. Cerevisiae)

    QSER1
−2.6
79832
Glutamine and serine rich 1

    THOC2
−2.6
57187
THO complex 2

    RASSF6
−2.6
166824
Ras association (ralgds/AF-6) domain family member 6

    STAG2
−2.6
10735
Stromal antigen 2

    NUFIP2
−2.5
57532
Nuclear fragile X mental retardation protein interacting protein 2

    SMC2
−2.5
10592
Structural maintenance of chromosomes 2

    SLC30A1
−2.5
7779
Solute carrier family 30 (zinc transporter), member 1

    ZNF268
−2.4
10795
Zinc finger protein 268

    KIAA1109
−2.4
84162
Kiaa1109

    NIPBL
−2.4
25836
Nipped-B homolog (Drosophila)

    ANKRD30BP2
−2.4
149992
Ankyrin repeat domain 30B pseudogene 2

    KIF5B
−2.4
3799
Kinesin family member 5B

    SMC4
−2.3
10051
Structural maintenance of chromosomes 4

    SMC5
−2.3
23137
Structural maintenance of chromosomes 5

    NEMF
−2.3
9147
Nuclear export mediator factor

    GCC2
−2.3
9648
GRIP and coiled-coil domain containing 2

    EXOC5
−2.3
10640
Exocyst complex component 5

    FCHO2
−2.3
115548
FCH domain only 2

    SMC6
−2.3
79677
Structural maintenance of chromosomes 6

    HOOK3
−2.2
84376
Hook homolog 3 (Drosophila)

    LGALSL
−2.1
29094
Lectin, galactoside-binding-like

    SEMA3C
−2.1
10512
Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C

    TMEM106B
−2.1
54664
Transmembrane protein 106B

    PCM1
−2.1
5108
Pericentriolar material 1

    KRTAP2-3
−2.1
730755
Keratin associated protein 2-3

    MACC1
−2.1
346389
Metastasis associated in colon cancer 1

    CCDC82
−2.1
79780
Coiled-coil domain containing 82

    TTC37
−2.1
9652
Tetratricopeptide repeat domain 37

    UACA
−2.1
55075
Uveal autoantigen with coiled-coil domains and ankyrin repeats

    CEP290
−2
80184
Centrosomal protein 290 kda

    USP53
−2
54532
Ubiquitin specific peptidase 53

    METRNL
−2
284207
Meteorin, glial cell differentiation regulator-like

The microarray analysis identified 51 transcripts in epithelial cells that exhibited a statistically significant change in their median expression level (either upregulated or downregulated) in response to pretreatment with ZDY and treatment with the uNK cell-secretion medium compared with their level in response to treatment with uNK cell-secretion medium (Table 4). Of these genes, 33 were downregulated, and 18 were upregulated. The largest group of upregulated genes includes the following: enzyme asparagine synthetase (glutamine-hydrolyzing) (ASNS) and aldo-keto reductase family 1 member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase) (AKR1C1). The transcripts encoding molecules responsible for signal transduction, namely, aldo-keto reductase family 1 member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase, type III) (AKR1C2) and structuration, such as talin 1 (TLN 1) and keratin 75 (KRT75), were also upregulated. The levels of transcripts encoding proteins that regulate transcription and ion binding, such as nuclear protein transcriptional regulator 1 (NUPR1) and S100 calcium binding protein A8 (S100A8), were also altered, and several genes with unknown function were found to be upregulated. The downregulated transcripts included only two genes of known function that were downregulated by more than 4fold: lamin B1 (LMNB1, a structural factor) and tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2 (TNKS2, an enzyme activator). The genes that were downregulated by more than 2fold included the genes encoding the cytokines chemokine (C-X-C motif) ligand 3 (CXCL3) and caprin family member 2 (CAPRIN2), the transporter component of the oligomeric golgi complex 5 (COG5), and various genes associated with transcription, kinase, ion binding, and nucleotide metabolism.

Table 4: Transcripts that were altered by more than twofold in endometrial epithelial cells by stimulation with ZDY and uNK cell-secretion medium (). The transcript levels were obtained using a GeneChip PrimeView Human Gene Expression Array.

Gene symbol
Fold change
Gene ID
Description

Upregulated genes: 18 transcripts

Intracellular/signalling factors

    AKR1C2
2.7
1646
Aldo-keto reductase family 1, member C2 (dihydrodiol dehydrogenase 2; bile acid binding protein; 3-alpha hydroxysteroid dehydrogenase, type III)

Structural factors

    TLN1
2.7
7094
Talin 1

    KRT75
2.0
9119
Keratin 75

Transcription

    NUPR1
2.6
26471
Nuclear protein, transcriptional regulator, 1

Ion binding

    S100A8
2.1
6279
S100 calcium binding protein A8

Enzyme activity

    ASNS
5.2
440
Asparagine synthetase (glutamine-hydrolyzing)

    AKR1C1
3.3
1645
Aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase)

    NQO1
2.9
1728
NAD(P)H dehydrogenase, quinone 1

    CBS
2.5
875
Cystathionine-beta-synthase

    AKR1C3
2.3
8644
Aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroid dehydrogenase, type II)

    PSAT1
2.2
29968
Phosphoserine aminotransferase 1

    HMOX1
2.2
3162
Heme oxygenase (decycling) 1

    GPX2
2.2
2877
Glutathione peroxidase 2 (gastrointestinal)

    DHRS3
2.1
9249
Dehydrogenase/reductase (SDR family) member 3

    H1FX
2.1
8971
H1 histone family, member X

Others

    SLC7A11
3.1
23657
Solute carrier family 7 (anionic amino acid transporter light chain, xc-system), member 11

    CTH
2.4
1491
Cystathionase (cystathionine gamma-lyase)

    HSPB8
2.2
26353
Heat shock 22 kda protein 8

Downregulated genes: 33 transcripts

Cytokines/membrane proteins/signalling factors

    CXCL3
−2.1
2921
Chemokine (C-X-C motif) ligand 3

    CAPRIN2
−2.0
65981
Caprin family member 2

Transporters

    COG5
−2.0
10466
Component of oligomeric golgi complex 5

Structural factors

    LMNB1
−5.5
4001
Lamin B1

    PNN
−2.9
5411
Pinin, desmosome associated protein

    MFAP5
−2.2
8076
Microfibrillar associated protein 5

    RPL37A
−2.2
6168
Ribosomal protein l37a

Transcription

    HIRA
−3.6
7290
HIR histone cell cycle regulation defective homolog A (S. Cerevisiae)

    YAP1
−2.4
10413
Yes-associated protein 1

    ZNF226
−2.4
7769
Zinc finger protein 226

    TAF15
−2.1
8148
TAF15 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 68 kda

    NFKBIZ
−2.1
64332
Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta

Ion binding

    ZDHHC11
−2.2
79844
Zinc finger, DHHC-type containing 11

    RNF145
−2.1
153830
Ring finger protein 145

Kinase

    AHSA2
−2.0
130872
AHA1, activator of heat shock 90 kda protein atpase homolog 2 (yeast)

Enzyme activity

    TNKS2
−7.7
80351
Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2

Nucleotide metabolism

    RAD23B
−2.0
5887
RAD23 homolog B (S. Cerevisiae)

    RCC1
−2.0
1104
Regulator of chromosome condensation 1

Others

    GOLGA8A
−5.4
23015
Golgin A8 family, member A

    LUC7L3
−3.8
51747
LUC7-like 3 (S. Cerevisiae)

    HNRNPA2B1
−2.9
3181
Heterogeneous nuclear ribonucleoprotein A2/B1

    XIST
−2.8
7503
X (inactive)-specific transcript (non-protein coding)

    SNORA33
−2.6
594839
Small nucleolar RNA, H/ACA box 33

    SNHG12
−2.5
85028
Small nucleolar RNA host gene 12 (non-protein coding)

    C20orf197
−2.4
284756
Chromosome 20 open reading frame 197

    EML2
−2.4
24139
Echinoderm microtubule associated protein like 2

    PNISR
−2.3
25957
PNN-interacting serine/arginine-rich protein

    KIAA0020
−2.3
9933
Kiaa0020

    HMMR
−2.2
3161
Hyaluronan-mediated motility receptor (RHAMM)

    NOP58
−2.2
51602
NOP58 ribonucleoprotein homolog (yeast)

    PILRB
−2
29990
Paired immunoglobin-like type 2 receptor beta

    NCAPG
−2
64151
Non-SMC condensin I complex, subunit G

    PICALM
−2
8301
Phosphatidylinositol binding clathrin assembly protein

3.2. Quantitative Real-Time RT-PCR (qRT-PCR)

To verify these changes, the transcript levels of the following genes were measured by qRT-PCR: CXCL-10, IL-15, ICAM-1, NLRC-5, and IRF-1 (Figure 1). The qRT-PCR results confirmed the substantial and significant changes in expression observed in the microarray analysis. Epithelial cells treated with the uNK cell-secretion medium showed increased expression levels of CXCL-10 (18.4-fold), IL-15 (11.7-fold), ICAM-1 (12.5-fold), NLRC-5 (31.5-fold), and IRF-1 (15.6-fold) (all ) compared with the control group.

(a)

(b)

(c)

(d)

(e)

Figure 1: Transcription levels of (a) CXCL-10, (b) IL-15, (c) ICAM-1, (d) NLRC-5, and (e) IRF-1 determined by qRT-PCR. The relative fold changes are shown in Section 3. The qRT-PCR results were consistent with the microarray analysis. The data are expressed as means SEM. for the comparison between the uNK and control groups; for the comparison between the ZDY and control groups.

We also confirmed that epithelial cells pretreated with ZDY and then treated with the uNK cell-secretion medium exhibited downregulated expression levels of CXCL-10 (0.9-fold; ) and NLRC-5 (1.0-fold;) and upregulated levels of IL-15 (1.1-fold; ), ICAM-1(1.1-fold; ), and IRF-1 (1.1-fold; ) compared with epithelial cells treated with the uNK cell-secretion medium. However, none of these changes were significant.

3.3. Western Blot

To confirm these effects at the protein level, the amount of ICAM-1 was analyzed by western blot (Figure 2). Epithelial cells treated with the uNK cell-secretion medium showed a 2.1-fold () increase in the expression of ICAM-1. However, the epithelial cells treated with ZDY and the uNK cell-secretion medium showed a 1.4-fold increase () in the expression of ICAM-1 compared with the epithelial cells treated with the uNK cell-secretion medium. Thus, there was no significant difference between the uNK and ZDY groups.

(a)

(b)

Figure 2: (a) Expression of endometrial ICAM-1 protein in endometrial epithelial cells. The GAPDH band was used as the internal loading control in each lane. (b) Comparison of the normalized expression intensity of endometrial ICAM-1 protein in the control, uNK, and ZDY groups. The data are expressed as means SEM. for the comparison between the uNK and control groups; for the comparison between the ZDY and control groups.

4. Discussion

Progesterone plays a central role in reproduction and is involved in implantation and pregnancy. When pregnancy occurs, high progesterone levels are critical not only for facilitating implantation but also for maintaining pregnancy by stimulating uterine growth [20, 21]. Several observations suggest that uNK cells also play an important role in reproduction: (a) these cells are hormonally regulated because they increase in number during the luteal phase when implantation occurs [7], (b) these cells are present in early gestation at the time placental cells invade the maternal arteries [22], and (c) these cells are particularly abundant around trophoblast cells [23]. The proliferation of uNK cells is the foundation of their reproductive function. One of the mechanisms underlying the increase in uNK cell numbers is the stimulation of the proliferation of existing uNK cells by progesterone. In mouse uterine tissues, the significant upregulation in the decidual NK population is regulated by progesterone [24]. Because there are no progesterone receptors on uNK cells, it is hypothesized that the paracrine communication between uNK and epithelial cells participates in this process. In this study, we used the uNK cell-secretion medium to stimulate epithelial cells in an attempt to find the interaction and/or paracrine effects between these cells. The data obtained in the present study demonstrate that uNK cells have a significant effect on the gene expression in epithelial cells in the nonpregnant state, and these paracrine effects also have a profound effect on uNK proliferation.

The largest group of upregulated genes induced in epithelial cells by the soluble factors derived from uNK cells induced cytokines and immunological factors, including IL-15 and IL-15RA. IL-15 is critical for NK cell differentiation in mice and human lymphoid tissue [25, 26]. IL-15 acts on precursor and immature NK cells. In humans, IL-15 transcription is more abundant during the secretory compared with the proliferative cycle phase, is maintained in early pregnancy, and localizes to the endothelium and decidual spiral arteries [27]. The correlative time strongly suggests that IL-15 participates in uNK cell proliferation and that IL-15 may also contribute to the localization of uNK cells. Our results show that uNK cells exhibit upregulated levels of both IL-15 and IL-15RA. This may activate a powerful proliferation loop because IL-15 is presented by IL-15RA on epithelial cells to promote uNK proliferation.

There are temporal and quantitative features associated with the activities of uNK cells and trophoblast cells in uterine spiral artery maturation. There is evidence that IL-15 upregulates the level of VEGF-C mRNA expression, which suggests that uNK cells may play an important role in endometrial angiogenesis and regeneration [28]. In addition, CXCL-10 is involved in uNK cell production, directs the migration and invasion of CXCR1+, CXCR3+, CXCR4+, and CCR3+ trophoblast [12], and promotes angiogenesis in the placental bed [29]. Furthermore, in the absence of uNK cells, spiral artery extension is delayed, and the tunica media remain intact [30], and these studies are consistent with an angiogenic/vascular remodeling role for uNK cells [4, 12, 31]. We found some evidence that uNK cells promote changes in endometrial epithelial cells in the nonpregnant state. The level of VEGF-C, which is upregulated in epithelial cells by the uNK cell-secretion medium, increases markedly during implantation and early pregnancy [32]. Our data suggest that uNK may prime some of the decidualization changes that occur in epithelial cells prior to the onset of full decidualization.

The exact function of uNK cells has not yet been unequivocally determined, but it is known that these cells express a different cytokine profile compared with peripheral NK cells. For example, these cells express colony-stimulating factor (CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-), interferon- (IFN-), and leukemia inhibitory (LIF) [33, 34]. During human pregnancy, these cells are found in close proximity to the implantation site and in close contact with the infiltrating extravillous trophoblasts. Furthermore, uNK cells increase their numbers in early pregnancy due to hormonal dependence and exhibit close proximity to trophoblasts. Taken together, these data support the conclusion that uNK cells play an important role in the regulation for the maternal immune response for the control of implantation and trophoblast invasion during human pregnancy. The summary of the paracrine effects of uNK cells on the epithelial cells is shown in Figure 3.

Figure 3: Model of the proliferation and function of uNK cells. During implantation and early pregnancy, the uNK paracrine signals stimulate endometrial epithelial cells to produce IL-15, VEGF, and other unidentified factors that may regulate uNK cell proliferation. The immune paracrine communication between uNK and endometrial epithelial cells may support implantation and trophoblast development.

In addition, this study provides the first demonstration of the paracrine communication between uNK and endometrial epithelial cells. Because our previous study showed that ZDY can improve the morphology of the endometrium in mice, we attempted to identify a relationship between the paracrine communication (uNK/epithelial cells) and the mechanism of action of ZDY. However, the majority of the up- and downregulated genes induced in the ZDY pretreated epithelial cells were of unknown function. To the best of our knowledge, there are no published studies on the effects of traditional Chinese medicine on the human endometrial paracrine communication. Our results suggest that ZDY may not regulate this process in vitro. ZDY may improve the morphology of the uterus through another mechanism.

5. Conclusions

Overall, this study provides the first detailed demonstration of the paracrine interaction between immune cells and epithelial cells in the endometrial environment. The findings indicate that these paracrine signals may contribute to uNK cell proliferation during implantation and early pregnancy. In addition, we failed to find evidence indicating that ZDY regulates the paracrine effects between uNK cells and epithelial cells in vitro.

Conflict of Interests

The authors declare that they have no conflict of interests.

Acknowledgments

This project was supported by the Natural Science Foundation of China. The project number is 81173292.

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