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Research Article Cell Biology International 10.1002/cbin.10429

Expression and Function of Fatty Acid-binding Protein 4 in Epithelial

Cell of Uterine Endometrium

Running Title: FABP4 is necessary for endometrial cell

Qiuyuan Zhu, Yan Jin, Peng Wang, Hanzhi Wang, Bingjian Lu

Zhengping Wang, Minyue Dong

Womens Hospital, School of Medicine, Zhejiang University

Key Laboratory of Reproductive Genetics, Ministry of Education

Key Laboratory of Womens Reproductive Health of Zhejiang Province

Key words: biology, endometrial epithelial cell, fatty acid-binding protein 4, function, maintenance, proliferation Corresponding Author Minyue Dong, MD/PhD Womens Hospital, School of Medicine, Zhejiang University Add: 1 Xueshi Road, Hangzhou, Zhejiang Province, 310006, China Tel: (+86) 571 8999 1011(work) Fax: (+86) 571 8706 1878 Email:mydong.cn@hotmail.com This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/cbin.10429] This article is protected by copyright. All rights reserved Received: 21 June 2014; Revised: 27 November 2014; Accepted: 19 December 2014

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Abstract

The aims of this study were to delineate the expression of fatty-acid binding protein

(FABP) 4 in human uterine endometrium and its function in the regulation of

proliferation, migration and invasion of epithelial cells. Immunohistochenistry,

immunofluorence and Western blotting were used to determine the expression and

cellular localization of FABP4 in endometrium and endometrial epithelial cell lines.

Interference of small ribonuclear acid (siRNA) and specific FABP4 inhibitor were used

to inhibit FABP4. The proliferation, migration and invasion of epithelial cells were

evaluated with CCK-8 assay, wound-healing test and transwell analysis respectivley. We

found that FABP4 was expressed by epithelial cells of proliferative endometrium and

epithelial and stromal cells of secrectory endometrium. Epithelial cell lines Ishikawa and

RL-952 expressed FABP4 and this expression was decreased by FABP4 siRNA. FABP4

siRNA and specific FABP4 inhibition significantly decreased the proliferation, migration

and invasion of epithelial cell lines. We concluded that FABP4 is functionally expressed

in endometrial epithelium and is necessary for maintaining the cell function of epithelial

cells of endometrium.

Key words: biology, endometrial epithelial cell, fatty acid-binding protein 4, function,

maintenance, proliferation

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1 Introduction

Fatty acid binding protein(FABP) 4 (FABP4, also named adipocyte FABP or aP2) is a

member of FABP superfamily consisting of 9 highly conserved cytosolic proteins that are

abundantly expressed in a tissue-specific manner with some overlap(Zimmerman and

Veerkamp, 2002). FABPs are capable of binding a variety of hydrophobic ligands, such

as long-chain fatty acid, encosanoids, leukotrienes, and postglandins, and are critical in

several cellular processes including uptake and trafficking of fatty acid, regulation of

gene expression, as well as cell proliferation and differentiation (Zimmerman and

Veerkamp, 2002). FABP4 has been originally identified as an adipocyte-specific protein

and have been considered a marker of adipogenesis and is important in the maintenance

of glucose and lipid metabolism (Kralisch and Fasshauer, 2013). It had been

demonstrated that FABP4 is constitutively expressed in human macrophage (Layne et al.

, 2001, Makowski et al. , 2001, Makowski et al. , 2005), bronchial epithelial cell (Shum et

al. , 2006), endothelial cell (Elmasri et al. , 2009), skeletal muscle (Fischer et al. , 2006),

vascular smooth muscle cell (Girona et al. , 2013), cardiomyocyte (Lamounier-Zepter et

al. , 2009), and trophoblast (Biron-Shental et al. , 2007). These observations indicate that

FABP4 is more widely expressed than originally thought and may have additional

biological role in other cell types.

Tian et al (Tian et al. , 2011) described that FABP4 messenger ribonuclear acid (mRNA)

level in human uterine endometrium was significantly higher on day LH+7 (day of

embryonic implantation) than that on day LH+2 (day of fertilization) and FABP4 was

highly expressed in mouse deciduas and significantly induced by in vitro decidualization.

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To further characterize the expression and function of FABP4 in human endometium, we

performed immunohistochemical analysis of FABP4 in human endometrium and found

that FABP4 is constitutively expressed in epithelial cells. Thus, we hypothesized that

FABP4 is important in maintaining the function of epithelial cells. To verify our

hypothesis, we observed the effects of FABP4 on the proliferation, migration and

invasion of epithelial cells of human uterine endometrium.

2 Materials and Methods

2.1 Tissue collection and Immunohistochemistry

Uterine samples were routinely fixed in formalin solution after hysterectomy, and

endometrial tissues were grossly examined and cut by a pathologist for

immunohistochemistry from patients with benign ovarian cyst or uterine myoma. The

tissue splices were processed with a standard protocol by the Leica Tissue Processor

ASP300S (Leica Biosystems, Heidelberger, Germany), and embedded in paraffin by the

Leica Embedding Workstation EG1160 (Leica Biosystems, Heidelberger, Germany).

The paraffin blocks were cut at 4 m with the Microm rotary microtome X-ActCut 4050

(Microm International GmbH, Walldorf, Germany). Heamotoxylin and Eosin staining

was performed for pathological examination with the Sakura Tissue-Tek DRS 2000 A-

D1 Autostainer (Sakura Finetek Co., Ltd, Tokyo, Japan). Additional 4 m sections were

stained with anti-human FABP4 antibody (Cell Signaling Technology, USA; catalog

number: 3544) using the 2-step En Vision method according to the manufacturers

instructions and visualized with 3-diaminobenzidine tetrachloride (Sigma Co., St Louis,

MO, USA). The second monoclonal antibodies and the detection kit were from DAKO

(DAKO Inc., Glostrup, Denmark). The omission of primary bodies was used as the

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negative control. Brown cytoplasmic staining was considered positive for FABP4. The

protocol of the current investigation was approved by the Ethics Committee of Womens

Hospital, School of Medicine, Zhejiang University and informed consents were obtained

from sample providers.

2.2 Cell culture and Immunofluorescence

Ishikawa cell was cultured in RPMI-1640 medium (phenol red free) and RL952 cell in

Dulbecco's Modified Eagle Media: Nutrient Mixture F-12 (F12/DMEM 1:1; phenol red

free) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin

at 37C in a humidified atmosphere of 5% CO2. For immuno- fluorescence, the cells

were fixed with 4% formaldehyde for 15 min, washed with PBS, incubated with 5% BSA

for 30 min at room temperature and with anti-human FABP4 antibody (Cell Signaling

Technology Co.) overnight at 4oC. The cells were incubated with goat FITC-conjugated

anti-mouse secondary antibody for 2 h at 4oC and then incubated with 4', 6-diamidino-2-

phenylindole (DAPI) for 2 min at room temperature, washed twice with phosphate-

buffered solution (PBS), and observed under an inverted fluorescence microscope.

2.3 Transfection of siRNA and functional inhibition of FABP4

Cells were transfected with FABP4 siRNA (Santa Cruz Inc., CA, USA; catalog number:

sc-43592; concentration: 10 mol/mL) or negative control siRNA (Santa Cruz; sc37007)

using Lipofectamine 2000 (Life Technologies Co., Taipei, Taiwan) according to the

manufacturers protocol. The transfection medium was replaced with complete medium 6

h after transfection, and the cells were incubated for further experiment. To inhibit the

function of FABP4, a selective inhibitor of FABP4 (EMD Millipore Inc., Darmstadt,

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Germany; catalog number: 341310), ((2-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1-

biphenyl)-3-yl)oxy)-acetic acid, which targets fatty acid binding pocket, was added to the

medium at the indicated concentrations for 12 hours and then the medium was replaced.

2.4 Western blotting

Cells were collected and lyzed in 50 l cell lysis buffer containing protease inhibitors.

The protein concentration was quantified using the BCA Protein Kit (Applygen Inc. Ltd,

Beijing, China). The cell lysates were separated on 10% SDS-PAGE and the proteins

were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore,

Massachusetts, USA), blocked with Tris-buffered saline (TBS) and 0.1% Tween 20

(TBS/T) containing 5% bovine serum albumin, and then incubated with anti-human

FABP4 antibody (Cell Signaling Technology, USA; diluted at 1:1000 in TBS/T) or anti-

human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Cell Signaling

Technology Co.; diluted at 1:2000 in TBS/T) ) at 4C overnight. The membranes were

washed three times with TBS/T and then incubated with the appropriate HRP-conjugated

secondary antibodies for 1 hour at room temperature. The protein bands were detected by

enhanced-chemiluminescence and visualized by autoradiography. The relative expression

of FABP4 was normalized against internal control GAPDH.

2.5 Cell proliferation assay

Cells were seeded in 96-well plates at a density of 5000 cells/well. The medium was

replaced with the corresponding serum-free medium for 24 h to synchronize the cells, and

then the culture media was replaced with complete medium containing the drugsat at the

indicated concentrations for 48 h. Then, 10 L CCK8 solution (Dojindo Inc., Kumamoto,

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Japan) was added, the plates were incubated for 3 h, and absorbance was measured at 450

nm using a MRX II microplate reader (BioTek Inc., Vermont, USA). Cell proliferation

was calculated as the percentage of control cells.

2.6 Wound-healing assay

Cell migration was analyzed with the in vitro scratch assay. Cells were seeded in six-well

plates at a density of 3 105 cells/well after FABP4 was quiescence or inhibited and

cultured with medium containing 10% FBS overnight, after which, the medium was

changed to corresponding FBS-free medium and the cells were cultured for a further 24 h

until >90% confluence. A single scratch wound was created in the middle of cell

monolayer by the gentle removal of attached cells with a sterile plastic pipette tip.

Micrographs were taken with an inverted phase contrast microscope (Olympus, Tokyo,

Japan; magnification, 40) at indicated time intervals. The ratio of the healed area

relative to the initial wound area was calculated and the wound area was quantified using

software Image-Pro Plus v 6.0.

2.7 Transwell analysis

After transfection with FABP-4 siRNA (10 mol/mL) or treatment with FABP-4

inhibitor (25 mole/L), the cells were seeded at a density of 2105 cells/well in the upper

chamber of a Transwell 24-insert plate with medium. The upper chambers were coated

with Matrigel and the lower chamber contained 10% FBS medium. After 24 h, the

bottom of the inserts were fixed in methanol for 10 min and stained with hematoxylin and

eosin (H&E). The cells that had invaded to the lower surface were photographed and

counted using an inverted phase contrast microscope (40).

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2.8 Statistical analysis

The Kolmogorov-Smirnov test was used to evaluate the distribution of data. The data

were presented in means SD of at least 3 separate experiments. Student t-test and one-

way analysis of variance (ANOVA) followed by a Dunnetts posthoc test were used for

the comparison of normally distributed data. SPSS statistical package (Statistical

Analysis System, Chicago, IL, USA) was used for the statistic analysis. Values of P

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(Figure 4B; P

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Tian et al (Tian, Zhao, 2011) described that FABP4 is expressed in human and mouse

uterine endometrium and is critical for the decidualization. They thought FABP4 is

present in stromal cells of deciduas but did not determine its cellular localization. In this

study, we reveled with immunohistochemistry that FABP4 is constitutively expressed in

the epithelial cells of endometrium at proliferative and secretory phases while FABP4 is

positive in stromal cells of secretory phase. Basically, our findings were in accordance

with those of Tian. We also confirmed Tians finding that FABP4 mRNA expression is

higher on day LH+7 than that on day LH+2 (data not shown). We also observed that

FABP4 expression was up-regulated by estradiol and further up-regulated by the

combination of estradiol and progesterone (data not shown). Taken together, our findings

indicate FABP4 may play a role in the biological function. To clarify whether FABP4 is

involved in the regulation of cell function of endometrial epithelium, we further observed

the effects of silence of FABP4 and selective inhibition of FABP4 on the proliferation,

migration and invasiveness and found FABP4 interfering or inhibition suppressed the

proliferation, migration and invasiveness, suggesting FABP4 is necessary for the

maintenance of cell function of endometrial epithelium.

Proliferation and migration are important cell function of endometrial epithelium and are

important for the periodic maturation of endometrium and menstruation. Apposition and

adhesion are initial steps of embryonic implantation occurring on the endometrium

(Wang and Dey, 2006), and then FABP4 may have a role in embryonic implantation and

thus involved in pregnancy loss. When the roles of FABP4 on cell function of

endometrial epithelium is considered, our findings imply FABP4 may also participate the

development of endometrial pathogenesis, such as dysfunctional uterine bleeding,

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hyperplasia, carcinoma, endometriosis and adenomyosis.

In summary, FABP4 is functionally expressed in human endometrial epithelium and

necessary for the maintenance of cell function of epithelial cells. It would be interesting

to clarify the potential roles of FABP4 in the regulation of cell function and biological

function of endometrial epithelium, and subsequently in the development of pathogenesis

of endometrium.

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trophoblasts. Am J Obstet Gynecol. 2007;197:516 e1-6.

Elmasri H, Karaaslan C, Teper Y, Ghelfi E, Weng M, Ince TA, et al. Fatty acid binding

protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells.

FASEB J. 2009;23:3865-73.

Fischer H, Gustafsson T, Sundberg CJ, Norrbom J, Ekman M, Johansson O, et al. Fatty

acid binding protein 4 in human skeletal muscle. Biochem Biophys Res Commun.

2006;346:125-30.

Girona J, Rosales R, Plana N, Saavedra P, Masana L, Vallve JC. FABP4 induces vascular

smooth muscle cell proliferation and migration through a MAPK-dependent pathway.

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Kralisch S, Fasshauer M. Adipocyte fatty acid binding protein: a novel adipokine

involved in the pathogenesis of metabolic and vascular disease? Diabetologia.

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Kralisch S, Stepan H, Kratzsch J, Verlohren M, Verlohren HJ, Drynda K, et al. Serum

levels of adipocyte fatty acid binding protein are increased in gestational diabetes

mellitus. Eur J Endocrinol. 2009;160:33-8.

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macrophage-expressed adipocyte fatty acid binding protein in the development of

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Makowski L, Boord JB, Maeda K, Babaev VR, Uysal KT, Morgan MA, et al. Lack of

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5 Funding

This work was supported by Natural Scientific Foundation of China (81170572) and

National Basic Research Program of China (2012CB944903).

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Figure Legends

Figure 1: The expression and cellular localization of FABP4 in human uterine

endometrium. The positive staining of FABP4 (brown) was localized to epithelial cells of

proliferative (A) and secretary (B) endometrium. FABP4 was negative in stromal cells of

proliferative endometrium (A) but positive in stromal cells of secretary endometrium (B).

Figure 2: FABP4 expression by cell lines of human uterine endometrial epithelium.

Immunefluorence showed positive cytosol signal of FABP4 in Ishikawa and RL-952 cells

(A). Western blotting revealed specific bands of FABP4 in Ishikawa (B) and RL-952

cells (C). FABP4 siRNA significantly reduced FABP4 expression of Ishikawa (B) and

RL-952 cells (C).

Figure 3: The effect of FABP4 siRNA and inhibition on cell proliferation. There were

significant differences in cell proliferation among control, negative siRNA and FABP4

siRNA (P

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experiments of Ishikawa.

Figure 5: The effect of FABP4 siRNA and inhibition on cell invasion. There were

significant differences in the cell numbers invaded to low chamber among control,

negative siRNA and FABP4 RNA (P

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5