Selective estrogen receptor modulator ormeloxifene suppresses embryo implantation via inducing miR-140 and targeting insulin-like growth factor 1 receptor in rat uterus

Accepted Manuscript Title: Selective estrogen receptor modulator Ormeloxifene suppresses embryo implantation via inducing miR-140 and targeting insulin-like growth factor 1 receptor in rat uterus Authors: Vijay K. Sirohi, Kanchan Gupta, Rohit Kumar, Vinay Shukla, Anila Dwivedi PII: DOI: Reference:

S0960-0760(18)30007-4 https://doi.org/10.1016/j.jsbmb.2018.01.006 SBMB 5102

To appear in:

Journal of Steroid Biochemistry & Molecular Biology

Received date: Revised date: Accepted date:

1-9-2017 1-12-2017 4-1-2018

Please cite this article as: Sirohi VK, Gupta K, Kumar R, Shukla V, Dwivedi A, Selective estrogen receptor modulator Ormeloxifene suppresses embryo implantation via inducing miR-140 and targeting insulin-like growth factor 1 receptor in rat uterus, Journal of Steroid Biochemistry and Molecular Biology (2010), https://doi.org/10.1016/j.jsbmb.2018.01.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Selective estrogen receptor modulator Ormeloxifene suppresses embryo implantation via inducing miR-140 and targeting insulin-like growth factor 1 receptor in rat uterus Vijay K. Sirohi, Kanchan Gupta, Rohit Kumar, Vinay Shukla and Anila Dwivedi#

Running title: Ormeloxifene induces miR-140 in rat uterus #

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Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India

Correspondence: A. Dwivedi, Division of Endocrinology, CSIR-Central Drug Research

Institute, Lucknow-226031, Uttar Pradesh, India. Tel: +91 0522 277 2486; Fax: +91 0522 2771941

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E-mail: [email protected]

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Graphical abstract

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Highlights

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Ormeloxifene induces miR-140 expression in rat uterus. Expression of miR-140 is regulated by estradiol. Over-expression of miR-140 inhibited embryo implantation in rats. Both ormeloxifene and miR-140 suppressed the migration and invasion of endometrial epithelial cells. Ormeloxifene potentially downregulates the expression of IGF1R, integrin-β3, FAK and p-FAK via miR-140 in uterus.

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Abstract

Ormeloxifene, the non-steroidal SERM contraceptive, inhibits endometrial receptivity and

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embryo implantation via countering nidatory estrogen. However, the molecular mechanism of

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ormeloxifene action responsible for its contraceptive efficacy still remains unclear. Herein, we

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aimed to identify the miRNAs modulated under the influence of ormeloxifene and to explore

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their role in endometrial receptivity and embryo implantation. By doing microRNA sequencing analysis, a total of 168 miRNAs were found to be differentially expressed in uterine tissue of

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ormeloxifene-treated rats, on day 5 (10:00 h) of pregnancy i.e. peri-implantation period. Out of

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differentially expressed miRNAs, miR-140 expression was found to be elevated in ormeloxifene administered groups and was selected for detailed investigation. In-vivo gain-of-function of miR-

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140 resulted in a significant reduction of implantation sites indicating its role in embryo

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implantation. The experiment on delayed implantation showed that estradiol caused downregulation of miR-140. It also suppressed the attachment and outgrowth of BeWo spheroids to

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RL95-2 endometrial cells. In transwell migration assay, miR-140 was found to be involved in suppression of migration and invasion of endometrial epithelial cells. The ormeloxifene treatment caused up-regulation of miR-140 along with down-regulated expression of its target IGF1R in endometrial epithelial and stromal cells which also led to the suppression of downstream effectors integrin β3 and FAK. In mimic miR-140 receiving horn, the reduced

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expression of IGF1R along with suppressed downstream integrin β3 and FAK was observed similar to that observed in uteri of ormeloxifene- treated rats. Taken together, these findings suggest that ormeloxifene-induced inhibition of embryo implantation occurs via inducing miR-

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140 and altering its target IGF1R in rat uterus. Keywords: Ormeloxifene; Embryo implantation; MicroRNA-140; Insulin-like growth factor receptor; Migration. 1. Introduction

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Embryo implantation to the receptive uterus is a prerequisite process for the establishment of

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successful pregnancy. Uterus becomes receptive for the competent blastocyst during specific

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time period designated as "window of implantation”, thereafter the uterus becomes refractory to

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the embryo [1]. Various growth factors and signaling molecules are known to be involved in

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regulation of processes required for successful implantation at feto-maternal interface [2-4].

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MicroRNAs (miRNAs) can regulate diverse biological functions including cell proliferation, apoptosis, differentiation, metabolism, organ development, embryo formation and implantation

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[5, 6]. Recently, microRNAs gained much importance in implantation physiology as they target various genes important for embryo implantation [7-9]. MiR-21, one of the implantation-specific

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miRNA in rodents, was down-regulated by progesterone in ovariectomized mice, which may explain the detrimental effect of prematurely elevated progesterone on endometrial receptivity in

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IVF programmes [10]. MiR-133b has been found to reverse the hydrosalpinx-induced downregulation of HOXA10 through directly targeting SGK1 and improves embryo attachment invitro [11]. MiR-212 regulates the expression of olfactomedin 1 and C-terminal binding protein1 in human endometrial epithelial Ishikawa cells and enhances spheroid attachment [12]. The

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impairment of endometrial receptivity in-vitro via regulating miR-30d expression and epithelial mesenchymal transition via endocrine disrupter polychlorinated biphenyls has also been observed [13]. It is clear from the literature that till date most of the work has been focused on

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miRNA profiling in the reproductive tissues. However, the expression, regulation and function of miRNAs in specific cell types and during various reproductive phases, still needs to be established.

Ormeloxifene (trans-7-methoxy-2, 2-dimethyl-3-phenyl-4-[4-(2- pyrrolidinoethoxy) phenyl]

chroman hydrochloride) is a non-steroidal, selective estrogen receptor modulator (SERM) class of

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drug [14]. Ormeloxifene is once-a-week oral contraceptive which inhibits implantation in rats via

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suppressing endometrial receptivity at the dose of 1.25 mg/kg, administered on day 1 of pregnancy

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[15]. Ormeloxifene manifests its contraceptive efficacy by producing asynchrony between ovum

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transport and uterine receptivity [16, 17]. Although, ormeloxifene binds to estrogen receptors [18,

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19] and exerts its effects in tissue selective manner, the precise molecular mechanism of regulation

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of gene expression induced by ormeloxifene, responsible for its contraceptive action still remains unclear. Therefore, the current investigation was aimed to identify the miRNAs modulated by

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ormeloxifene and to explore their role in rat uterus, during the period of endometrial receptivity. 2. Materials and Methods

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2.1. Animal preparation, Ormeloxifene administration and tissue collection

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All experiments were performed according to the standards specified by the Institutional Animal Ethics Committee of CSIR-Central Drug Research Institute, Lucknow, India. Adult rats (Sprague–Dawley strain; females 150 g, males 200 g) were housed in a controlled environment at 24 ±10 C on an illumination schedule of 12L: 12D. Female and male rats were co-caged and the mated females were identified by the presence of sperms in vaginal smear on following

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morning and that day was considered as day 1(D1) of pregnancy. In one group, rats were fed with contraceptive dose i.e., 1.25 mg/kg of ormeloxifene (procured from HLL Lifecare Ltd., India) prepared in gum acacia, on D1 [16] and vehicle alone was given to rats of second group.

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Animals from both groups were sacrificed on day 5 10:00 h (D5 M). The implantation sites were visualized by intravenous injection of 1% Evans blue dye (0.2 ml/rat), and the regions between the blue bands defined as inter-implantation sites. Uteri were removed, stripped of fat/

connective tissue and washed with PBS and were immediately transferred into vials containing 1 ml RNAlater (Ambion, Huntingdon, U.K.) and incubated overnight at 4 0C. Next day, the uterine

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samples were stored at -800C until processing for miRNA expression analysis.

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Animals were further divided into three groups for assessment of miRNA in uterine tissue during

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window of implantation. Animals were sacrificed on D4 10:00 h or D5 M (10:00 h) or D5 E

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(18:00 h). Uterine horns were immediately dissected out and flushed with saline, and

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immediately stored in -80 0C till further analysis.

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2.2. Delayed implantation model

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To induce delayed implantation, the pregnant rats were ovariectomized on D3 and progesterone (5 mg/rat; Sigma-Aldrich) was injected subcutaneously, to maintain delayed implantation on D5,

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D6 and D7 [20]. In another group progesterone-primed rats were injected with 17β-estradiol (0.5μg/ rat; Sigma-Aldrich) on D7 to terminate delay in implantation. Uterine flushings were

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collected on D7 to confirm that the rats receiving only progesterone were in a state of delayed implantation, and examined for the presence of hatched blastocysts. Rats were sacrificed by cervical dislocation and the implantation segments in uterine specimens were separated by dissection and the endometrial tissue was stripped of fat. Samples were immediately stored in -80o C. Three separate rats were prepared for each group.

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2.3. MicroRNA sequencing MicroRNA next generation sequence profiling was done for the identification of differentially expressed miRNAs from ormeloxifene treated and vehicle treated control rat uteri collected on day

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5 10:00 h of pregnancy. Total RNA was isolated and quality was checked using an Agilent Technologies 2100 Bioanalyzer with an RNA integrity number value greater than or equal to 8. Small RNA library was prepared using TruSeq small RNA library prep kit (Illumina Inc, USA). Indexed Small RNA libraries are multiplexed in equimolar amounts, denatured and loaded on GAIIx flow cell lanes for cluster generation using cBot station and Illumina cluster generation kits.

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Raw data generated in FastQ format was filtered for high quality (>Q20 bases) reads and low

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quality reads were removed following which adaptor sequences were trimmed. Reads less than 18

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bases were discarded as they were too short. We selected only diffrentially expressed miRNAs

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showing raw reads ≥5 in either of sample and p value below 0.05. MicroRNA target prediction

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algorithms TargetScan and miRWalk 2.0 were used to predict the target genes of identified

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miRNAs.

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2.4. Quantitative RT-PCR of miRNA and mRNA Total RNA was isolated with TRIzol reagent (Invitrogen, USA) from uterine horns collected

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from female rats on day 5 of pregnancy treated with ormeloxifene and vehicle (control). The concentration of total RNA was determined using a NanoDrop-2000 spectrophotometer (Thermo

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Scientific, USA). Quality of isolated RNA was checked by taking the absorbance ratio at A260/280. One microgram of total RNA was used to synthesize cDNA using miScript-II RT kit according to manufacturer’s protocol (Qaigen, CA, USA). MiRNA expression was measured by quantitative RT-PCR using the miScript SYBR green PCR kit (Qiagen, CA, USA) as per manufacturer’s protocol. Briefly, miScript SYBR green PCR kit which contains miScript

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universal primer (reverse primer) and quantiTect SYBR green PCR master mix, was used to prepare real-time PCR reactions. Primer sequences are given in supplementary table 1. Amplification was programmed in a CFX96TM instrument (Bio-Rad, CA, USA). Cycling

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conditions for real-time PCR included an initial activation step at 95°C for 15 min to activate HotStar Taq DNA polymerase and 40 cycles of denaturation (at 94 °C for 15 s), annealing (at 55 °C for 30 s) and extension (at 70 °C for 30 s). Fluorescence data was collected at the holding stage of the extension step. Specificity and identity were verified by melting curve analyses. Threshold cycles values (CT) were exported as an excel file for analyses.

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For mRNA expression, SYBR Green qPCR master mix (GeneSureTM, Genetix Biotech Asia Pvt. Ltd.)

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was used. Cycling conditions for real-time PCR comprised the initial activation step at 95°C for 10 min

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and next 40 cycles of denaturation (94 °C for 15 s), annealing (58 °C for 30 s) and extension (72 °C for

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30 s). Expression of the investigated gene was normalized to the steady expression of a housekeeping

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gene GAPDH. Experiments were performed in triplicates.

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2.5. Endometrial cell culture, ormeloxifene-treatment and over-expression of miR-140

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Rats were sacrificed on D5 of pregnancy, uterus was collected in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich, USA) after stripping of fat. Uterine tissue was minced with

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scissors in DMEM containing 1mg/ml of collagenase and incubated for 1 h at 370 C in a shaker at 110 rpm. Cell suspension was allowed to settle for debris and supernatant was collected.

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Supernatant was centrifuged to collect pellet of endometrial cells. Endometrial epithelial and stromal cells were separated by using 40 μm cell strainer [21]. Cultured epithelial and stromal cells were treated with Orm for 48 h at 100 nM concentration. Epithelial cells were also transfected with miR-140 mimic (50 nM) and miR-140 inhibitor (100 nM) using RNAiMAX reagent (Invitrogen, CA, USA). Total RNA and protein were isolated after 48 h of transfection.

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2.6. Intra-uterine administration of miR-140 mimic in rats An in vivo jetPEI/miRNA complex was prepared according to manufacturer’s protocol to obtain the N/P ratio 8. To achieve this, 3 μg of miR-140 mimic and 0.48 μl in vivo jetPEI (Polyplus-

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transfection®, New York, USA) was added with 5% glucose solution to obtain a final volume of 40 μl. Rats underwent mini laprotomy under anesthesia on D3 of pregnancy and received miR140 mimic in one of the uterine horn while other horn received mimic negative control. In a

separate group of rats, miR-140 inhibitor was injected in one of the uterine horns and the other horn received inhibitor control. On D5 and D8 of pregnancy, animals were sacrificed and uteri

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2.7. Spheroid attachment and expansion assay

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were photographed to record the number of implanted embryos.

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Human trophoblast BeWo cell line was obtained from cell repository of National Centre for Cell Science, India. Human endometrial epithelial RL95-2 cells were purchased from American Type

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Culture Collection (Manassas, VA, USA). BeWo trophoblast cell spheroids were prepared by

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procedure previously described [22] with slight modifications. Briefly, for the preparation of 100

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spheroids, 5 × 104 BeWo cells were stained with cell tracker dye CMFDA in PBS for 30 min in dark. Cells were collected by centrifugation and washed with PBS. The pellet was re-suspended

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in 9.65 ml of F-12 Ham medium containing 10% FBS and 350 μl of 1% methylcellulose (SigmaAldrich) solution was added. The cell suspension was seeded 100 μl per well in a non-adherent

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clear round bottom 96-well plate (Corning, USA) and allowed to incubate for 16 h at 370C in 5 % CO2. Human endometrial epithelial RL95-2 cells were seeded in 96-well plates at a density of 1× 104 cells per well and incubated in 370 C in a humidified 5% CO2 incubator. After 24 h of incubation, cells were grouped in four different groups (control, mimic miR-140, Orm with and without inhibitor of miR-140). After 48 h, spheroids (n=15) were transferred to the monolayer of

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RL95-2 cells. After 1h of co-culture non-adherent spheroids were removed by centrifugation (with the cell spheroid surface facing down) at 10 ×g for 10 min. Attached spheroids were counted again using a fluorescence or phase-contrast microscope. Percent attachment rate of

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spheroids was calculated by using the formula: 100 × number of attached spheroids/number of total spheroids seeded.

In another set of experiment, BeWo spheroids were allowed to outgrow on the monolayer of RL95-2 cells for 24 h. All images were captured by inverted microscope (Nikon ECLIPSE

TE2000-S, Nikon, Singapore). Spheroid spread area was measured by using Image J software

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(National Institute of Health, USA).

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2.8. Migration and invasion assay

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Scratch wound healing and transwell chamber migration experiments were performed to study the effect of miR-140 on migration of endometrial epithelial cells. Matrigel coated transwell

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chambers were used for endometrial epithelial cell invasion study. For scratch wound assay,

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endometrial epithelial cells were seeded in a 6-well plate. Next day, cells were washed with PBS

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and miR-140 mimic (50 nM) with RNAiMAX reagent was given to cells for 7 h in a serum free media. In a separate group Orm was given with/or without miR-140 inhibitor. After 24 h of

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transfection, a vertical scratch was made using 200 μl tip. Images were taken immediately after wound generation at 0 h and 24 h under bright field inverted microscope. The percent wound

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closure was calculated by using the following formula % wound closure = wound at 0 h – wound at T h/wound at 0 h×100

Where T denotes time in hour

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To analyze the effect of miR-140 on transwell migration and invasion, endometrial epithelial cells were knocked-down as described above. After 48 h, cells were collected by trypsinization and seeded in with/without matrigel coated chambers. Briefly, miR-140 mimic transfected and

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Orm-treated with/without miR-140 inhibitor epithelial cells were seeded in chambers. After 48 h, migrated and invaded cells were fixed with chilled methanol and stained with crystal violet. Cells were counted from three different fields and images were captured under bright field inverted microscope.

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2.9. Enzyme-linked immunosorbent assay of IGF1

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To measure the IGF1 in blood serum, enzyme-linked immunosorbent assay was performed.

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Female and male rats were bred, the mated females were identified by the presence of sperms in

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vaginal smear on subsequent morning and that day was considered as day 1(D1) of pregnancy. In one group, rats were fed with 1.25 mg/kg of ormeloxifene prepared in gum acacia on D1 and

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vehicle alone was given to rats of second group. Serum was collected from control and

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ormeloxifene treated rats on D5 of pregnancy, diluted 500 fold in assay diluent buffer. ELISA

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was performed according to manufacturer’s protocol (Thermo Scientific, USA). 2.10. Immunoblotting

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Anti-IGF1R (sc-463), FAK (sc-558), p-FAK (sc-11765) and β-actin (sc-1616) antibodies were purchased from Santa Cruz Biotechnology, USA. Integrin-β3 (#13166) antibody was procured

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from Cell Signaling Technology, USA. Control and treated tissue and cells from different groups were homogenized and lysed in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (Sigma-

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Aldrich) and 1 mM PMSF. Supernatant was collected by centrifugation at 12,000 ×g at 40 C for 10 min. Equal amounts of protein were separated by SDS-Polyacrylamide gel electrophoresis and then transferred to Immuno-BlotTM PVDF membrane (Millipore, USA). The membrane was

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blocked with 5% skimmed milk and then incubated with appropriate primary antibody overnight at 4°C. The membrane was then washed and incubated with a secondary peroxidase conjugated antibody for 2 h. Antibody binding was detected using enhanced chemiluminescence detection system (GE Healthcare). After developing, the membrane was stripped and re-probed with

another primary antibody of interest or β-actin to confirm equal loading. Each experiment was

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performed three times to assess for consistency of results.

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Quantitation of band intensity was performed by densitometric analysis using Quantity One

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software (v.4.5.1) and a Gel Doc imaging system (Bio-Rad). 2.11. Statistical analysis

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All values are presented as the mean ± SEM, as determined from at least three independent

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experiments. Statistical significance was assessed by one-way ANOVA and Newmann Keul’s

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3. Results

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test or Student’s t-test. P< 0.05 was considered statistically significant.

3.1. MicroRNA profiling

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To examine differential expression of miRNAs in rat uterus between ormeloxifene treated and control group, miRNA sequencing analysis was performed. A total of 283 miRNAs were obtained from sequencing out of which 168 miRNAs were significantly differentially expressed showing raw reads ≥5 in either of sample (p<0.05). Expression of 92 miRNAs was found to be significantly increased (P < 0.01) and that of 76 miRNAs was found to be significantly decreased

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in rat uterus in Orm-treated over vehicle control rat uterus. Compared with control group, 17 miRNAs were up-regulated by at least two- fold, and 27 miRNAs were down-regulated up to minimum of two-fold in the Orm treated rat uterus (Fig.1). Taken together, these results suggests

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that ormeloxifene is potentially involved in regulating miRNAs changes on D5 of pregnancy i.e., window of implantation in rat uterus.

3.2. Validation of differentially expressed microRNAs by quantitative RT-PCR

Among differentially expressed, 9 were selected for validation as obtained in miRNA sequencing

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analysis. These miRNAs were differentially expressed by at least 2-fold (up-regulated or down-

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regulated) in ormeloxifene-treated rat uterus over control. Expression of miR-31a-3p, miR-145-

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5p, miR-140-5p, let-7i-3p, miR-196a-5p, miR-665, miR-540-3p, miR-132-5p and miR-505-5p

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was analyzed by real time PCR. Results showed an up-regulation of miR-31a-3p, miR-145-5p, miR-140-5p, let-7i-3p and miR-196a-5p in Orm- treated uterine samples. Expression of miR-

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665, miR-540-3p, miR-132-5p and miR-505-5p was found to be down-regulated in Orm-treated

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group of rats on D5 of pregnancy (Supplementary Fig.1 & Supplementary Table 2).

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3.3. Expression of miR-140 during peri-implantation period, its hormonal regulation and effect of ormeloxifene in uterine tissue and endometrial cells

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The expression of miR-140 was studied during window of implantation in rat uterus i.e., on D4 (pre-implantation), D5 M (peri-implantation) and D5 E (post-implantation) of early pregnancy.

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The miR-140 was found to be significantly down-regulated on D5 M (p<0.001) as compared to that on D4 of pregnancy (Fig. 2 A). These results suggested that the down-regulation of miR-140 might be essential for the establishment of embryo implantation. We further analyzed whether miR-140 expression was restricted to implantation sites. We observed no change in expression

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pattern of miR-140 in implantation and inter-implantation sites (Fig. 2 B).To identify the hormonal regulation of miR-140 which showed down-regulation in D5 M (peri-implantation period), uterine samples from delayed implantation and estradiol terminated delayed

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implantation (active) rats were collected and analyzed for miR-140 expression. Higher expression of miR-140 was observed in the uterus under delayed implantation condition (delayed by P4 treatment), which was significantly suppressed after implantation was restored with

estrogen treatment (Fig. 2 C). The results from delayed implantation experiment suggested that

down-regulated expression of miR-140 is dependent on blastocyst activation by estrogen during

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peri-implantation period.

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The effect of ormeloxifene on miR-140 expression in different uterine cell population was also

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studied in-vitro. Rat endometrial epithelial and stromal cells were cultured and treated with

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ormeloxifene at a concentration of 100 nM for 48 h. It was observed that ormeloxifene

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significantly up-regulated the expression of miR-140 in epithelial as well as in stromal cells (Fig.

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2 D).

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3.4. Functional validation of miR-140 in-vivo Further, we elucidated whether miR-140 up-regulation affects the embryo implantation in rats in-

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vivo. Intra-uterine administration of miR-140 mimic significantly reduced the number of implantation sites in rat uterus as observed by Evans blue staining on D5 and by direct

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visualization of embryos on D8 of pregnancy (Fig. 3 A & B). We also measured the expression of miR-140 in mimic-transfected and control uterine horn using quantitative RT-PCR to confirm the miR-140 over-expression. Here miR-140 was found to be over-expressed on D5 of pregnancy over control uterine horn (Fig. 3 B). These results demonstrated that miR-140 was involved in implantation in rats.

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3.5. Ormeloxifene and miR-140 significantly reduced the attachment and outgrowth of trophoblast spheroids on RL95-2 cells To evaluate specifically whether ormeloxifene affects the trophoblast attachment, we performed

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in-vitro attachment assay. For this, we used BeWo trophoblast spheroids co-cultured on RL95-2 endometrial epithelial cell monolayer. Over-expression of miR-140 by mimic and by

ormeloxifene treatment to RL95-2 cells significantly reduced the percent attachment of spheroids (p<0.01, p<0.001) (Fig. 4 A). Moreover, spheroids outgrowth was significantly suppressed in

miR-140 mimic-transfected and also in ormeloxifene-treated RL95-2 cells (p<0.01). The relative

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area spreaded in miR-140 mimic- and also in Orm- treated groups was reduced by approximately

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3.5 fold as compared to the area spreaded in control. However, no significant changes were

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found in Orm+inhibitor miR-140 treated group (Fig. 4 B & C). 3.6. Ormeloxifene and miR-140 suppresses the migration and invasion of endometrial

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epithelial cells

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Cell migration and invasion are important processes during embryo implantation. To know

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whether miR-140 and Orm affects endometrial epithelial cell migration, we performed wound healing and transwell migration assay. Both miR-140 and ormeloxifene significantly suppressed

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the percent wound closure and percent migrated cells from 8 μm pore size membrane migration chamber (p<0.001) (Fig. 5 A). Further, both conditions led to inhibition of the percent matrigel

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invaded endometrial epithelial cells. Interestingly, co-treatment of miR-140 inhibitor along with ormeloxifene reversed the effect of ormeloxifene on migration and invasion of endometrial epithelial cells (Fig. 5 B).

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In addition, to ascertain whether inhibitory effects on migration and invasion of EECs were due to cytotoxic effect of Orm on these cells, we performed the cytotoxicity assay in endometrial epithelial cells. The results suggested that at 100 nM of concentration more than 93% cells were

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live after 48 h of treatment. The IC50 of ormeloxifene was found to be 9.25 μM in these cells (Supplementary Fig. 3).

3.7. Insulin-like growth factor-1 receptor (Igf1r) is a target of miR-140

Targets of miR-140 were predicted using microRNA target prediction tools which were designed

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algorithms of miRNA binding complementary sites and free energies to 3’ UTR regions of target

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mRNA. Although these tools only predict the target genes but these targets may/may not be the

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functionally validated targets. Out of several predicted targets, we have selected the target gene

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Igf1r whose binding site is conserved in mammalian species (Fig. 6 A). The expression of predicted target of miR-140, Igf1r was measured in endometrial epithelial cells over-expressing

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miR-140. Upon miR-140 mimic administration, Igf1r expression was found to be down-

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regulated in these cells (Fig. 6 B). Immunoblotting results suggested that IGF1R protein expression was suppressed in case of miR-140 over-expression and was found to be elevated

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upon miR-140 inhibition in endometrial epithelial cells (Fig. 6 C).

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The expression of Igf1r mRNA was found to be down-regulated in uterine tissue on D5 in ormeloxifene treated group, over control (Fig. 7 A) (p<0.001). Its expression was up-regulated at

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D5 compared with that of D4 of early pregnancy (p<0.05) (Fig. 7 B). In in-vitro culture, ormeloxifene treatment significantly suppressed the expression of Igf1r mRNA in endometrial epithelial and stromal cells (p<0.01, p<0.05) (Fig. 7 C). In order to know the effect of ormeloxifene on IGF1 level in serum, we performed ELISA to measure the concentration of

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IGF1. The ormeloxifene was found to significantly attenuate the serum IGF1 concentration on D5 of pregnancy as compared to that of control rats (Fig. 7 D). 3.8. MiR-140 down-regulated IGF1R expression and suppresses the expression of integrin

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β3, FAK and p-FAK in rat uterus To know the effect of miR-140 over-expression on downstream signaling molecules to IGF1R in rat uterus, we performed immunoblotting. IGF1R expression was found to be significantly

suppressed in miR-140 mimic receiving uterine horn (p<0.001). In addition, the downstream

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signaling molecules integrin β3, FAK and p-FAK were also found to be down-regulated in

mimic receiving horn over control horn (p<0.01) (Fig. 8). Similarly, down-regulated expression

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of integrin β3, FAK and p-FAK were observed in ormeloxifene treated rat uteri as compared to

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vehicle treated control rats (p<0.01).

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4. Discussion

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To our knowledge this is the first report of microRNA profiling in rat uterus under the influence of contraceptive agent ormeloxifene, which also helped to identify the miRNAs involved in

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embryo implantation. Previously, miRNA microarray analysis has been reported in rat uterus where the up-regulated expression of miR-29a during receptive phase was suggested to be

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involved in embryo implantation via targeting pro- and anti- apoptotic factors [23]. In the present study, we have identified 44 differentially expressed miRNAs which were modulated by at least

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2- fold under the influence of ormeloxifene, during peri-implantation period in rat uterus. Here, we measured the uterine expression pattern of top 5 miRNAs which were up-regulated by ormeloxifene i.e., miR-31a, miR-96, miR-140, miR-7i and miR-145 during window of implantation in normal animals. Among these, miR-31a, miR-96, miR-140 and miR-145 showed

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a down-regulated pattern in peri- and post-implantation phase as compared to pre-implantation phase. Among these, miR-140 showed time- specific temporal changes showing minimum expression at D5 M (Fig. 2 A), while other 3 miRNAs showed continuous down-regulation as

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analyzed till D5 E (data not given). The miR-140 was selected for detailed functional analysis as its role has not been identified and explored in embryo implantation so far. Hence, the study was undertaken to explore the role of miR-140 in embryo implantation and its modulation by

contraceptive agent ormeloxifene to dissect its miRNA- mediated mechanism involved in

suppression of endometrial receptivity and embryo implantation. The miR-140 was found to be

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significantly up-regulated (D5 M) in rats receiving ormeloxifene which might be responsible for

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its contraceptive activity.

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Embryo implantation process is tightly regulated by ovarian steroids i.e., estrogen and

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progesterone in endometrium [24, 25] and microRNAs expression are also reported to be influenced by these hormones via effecting their biogenesis and maturation to cytoplasmic form

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[8, 20, 26, 27]. In our study, miR-140 was found to be regulated by estradiol-activated blastocyst

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in endometrium in rats where implantation was experimentally delayed. We have analyzed the

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effect of ormeloxifene on uterine epithelial and stromal cells separately and results suggested that ormeloxifene significantly induced miR-140 expression in both the cell types. Similarly, the

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over-expression of miR-140 led to reduction in implantations and caused embryo implantation failure in rats. However, no significant change was observed in number of implantation sites

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after miR-140 inhibitor administration (Supplementary Fig. 2). As miR-140 was found to be regulated by estradiol, we speculate that ormeloxifene being an anti-estrogenic agent [18, 19], is able to induce miR-140 via altering the estrogen-regulated miRNA biogenesis enzymes [27] in rat uterus during early pregnancy.

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Since embryo implantation comprises of various phases such as apposition, adhesion and invasion of blastocyst to the endometrium, we analyzed the effect of miR-140 and/or ormeloxifene on BeWo spheroid attachment and its outgrowth in in-vitro assay system. Reduced

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number of attached spheroids on RL95-2 monolayer after 1 h of co-culture and also fewer outgrowths of spheroids observed on miR-140 transfected RL95-2 monolayer indicates that

ormeloxifene suppressed the adhesion and invasion of spheroids via miR-140 up-regulation. Similar effects were observed in RL95-2 cells with over-expressed miR-140.

Ormeloxifene is reported to be involved in inhibition of epithelial to mesenchymal transition

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(EMT) which suppresses prostate tumor growth and metastatic phenotypes [28]. In another

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study, ormeloxifene was found to suppress breast cancer metastasis by reversing EMT via down-

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regulation of HER2/ERK1/2/MMP-9 signaling [29]. Epithelial to mesenchymal transition in

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endometrium is the crucial process for successful embryo implantation [1, 13]. It is characterized

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by the loss of cell to cell contact, migration and invasion of endometrial cells [30]. MiR-140 is

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also reported to repress cell migration and invasion in human non-small cell lung cancer and gliomas [31, 32]. We here hypothesized that miR-140 may be involved in suppression of

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migration and invasion of endometrial epithelial cells which might be the reason to inhibit embryo attachment and invasion to the endometrium. In our study, cells with over-expression of

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miR-140 or treated with ormeloxifene, significantly suppressed the migration and invasion of

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endometrial epithelial cells. Whereas, co-treatment with miR-140 inhibitor reversed the ormeloxifene-induced suppression of these processes. Thus, it is probable that ormeloxifene suppressed endometrial epithelial cell migration and invasion through the elevated expression of miR-140.

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In the cytoplasm, mature miRNAs regulate their target by binding to 2-8 nucleotide sequence to the 3’UTR of target mRNA [33]. In search of 3’ UTR target of miR-140, we identified Igf1r using target prediction software target scan. The seed binding site of miR-140 for 3’UTR of Igf1r

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mRNA is conserved in different mammalian species as well. IGF1R expression has also been previously reported during peri-implantation period in mouse uterus [34] and its mRNA

expression is also high in human endometrium of secretory phase [35]. Recently, IGF1R protein expression was demonstrated in all phases of menstrual cycle but its expression was highly abundant in luminal epithelium of mid and late-secretory endometrium [36]. In in-vivo

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experiments, the higher expression of Igf1r mRNA was evident during peri-implantation phase

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(D5 M) than in pre-implantation phase (D4) which was suppressed in ormeloxifene treated rats.

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Interestingly, the serum level of IGF1 was also found to be down-regulated in ormeloxifene

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treated rats on D5 of pregnancy. In in-vitro experiments, ormeloxifene also down-regulated the expression of Igf1r in both endometrial epithelial and stromal cells. Over-expression of miR-140

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in endometrial epithelial cells, was able to suppress the expression of IGF1R whereas, miR-140

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inhibitor transfection resulted in elevated expression of IGF1R in these cells. These findings

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revealed the endogenous regulation of IGF1R expression by miR-140 in endometrial epithelial cells and also suggested the ormeloxifene-induced miR-140 -mediated regulation of IGF1R.

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To investigate the molecular mechanism of miR-140-mediated regulation of IGF1R involved during implantation, we evaluated the down -stream signaling molecules viz., integrin β3 and

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FAK [37]. Integrin β3 has been reported in the endometrium during menstrual cycle and in embryo implantation [38, 39]. Its increased expression has been reported in rat blastocyst and in human endometrial epithelial Ishikawa cells during attachment [40, 41]. In human, integrin β3 expression is up-regulated in endometrial epithelium at the time of implantation whereas loss of

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integrin β3 is the major cause of infertility in women with unexplained infertility [42]. The FAK activation is mediated by integrin β3 and is reported to be involved in adhesion of rat blastocysts to human uterine epithelial Ishikawa cells [37, 41]. Further, FAK facilitates the invasion of

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competent blastocyst to the receptive endometrial epithelium in rat [43]. In our study, downregulated IGF1R expression resulted in loss of integrin β3 protein expression in uteri with overexpressed miR-140 similar to that observed in ormeloxifene-treated rat uterus during peri-

implantation period. Over all, these results suggested the possible mechanism of ormeloxifene

involving miR-140- mediated down-regulation of IGF1R which in turn suppresses the expression

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of integrin β3 and FAK leading to inhibition of adhesion and subsequent invasion of blastocyst

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into the uterus.

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5. Conclusion

In conclusion, miR-140 exerted its effect via targeting IGF1R in endometrium which further

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suppressed the expression of adhesion molecules integrin β3 and FAK leading to embryo

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implantation failure (Fig. 9). This study revealed the mechanism of contraceptive action of ormeloxifene occurring via inducing miR-140 and down-regulating its target IGF1R. In addition,

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the role of miR-140 has been established in embryo implantation which further expanded our

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knowledge of microRNA -regulated phenomenon of uterine receptivity in rodent model. Conflict of Interest

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Authors have declared no conflict of interest. Acknowledgments Authors wish to thank Director, CSIR-CDRI for taking keen interest in this work. Financial support was provided by Council of Scientific and Industrial Research. One of the authors

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(V.K.S.) is recipient of senior research fellowship from Council of Scientific and Industrial Research, New Delhi. This is CDRI communication number 02/2017-AD. References

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Figure legends

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Fig. 1. MicroRNA profiling. Profiling of miRNAs in rat uterus on D5 of pregnancy showing ≥ 2 fold change in ormeloxifene treated group as compared to control group. Red color indicates

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relative high expression and green color indicates relative low expression over control (left

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panel). Expression of miR-140 in rat uterus using real-time RT-PCR (right panel). Data are presented as relative fold change in expression after normalization to the mean value of internal

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control U6. For comparative analysis, the expression values for miRNA were set as 1 in control (vehicle treated) group. Significant fold changes are marked by ***p < 0.001 vs. control. Fig. 2. Expression of miR-140 during window of implantation, its hormonal regulation and effect of ormeloxifene on miR-140 expression. (A) Relative Expression of miR-140 during the period of early pregnancy in rat uterus on D4 (day 4), D5 M (day 5 10:00 h) and D5 E (day 5

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18:00 h). For comparative analysis, the expression values miRNA were set as 1 in D4 group. Significant fold changes are marked by ***p < 0.001, **p < 0.01 vs. D4. (B) Relative expression of miR-140 in implantation and inter-implantation sites of rat uterus on D5. (C) Relative

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expression of miR-140 in delayed implantation condition (delayed, P4-treated), as compared to estrogen terminated delayed implantation (active, E2+P4 treated) group in rats ovariectomized on D3; details given in ‘materials & methods’ section. Significant fold changes are marked by

***p < 0.001 vs. delayed, P4-treated. (D) Expression of miR-140 in endometrial epithelial (left panel) and stromal (right panel) cells treated with vehicle and Orm (48h), determined by real-

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time PCR. Data are presented as relative fold change in expression after normalization by

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internal control U6. Significant fold changes are marked by ***p < 0.001 vs. vehicle control.

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Fig. 3. Effect of miR-140 over-expression on embryo implantation in rats.

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(A) Representative images of rat uteri showing implantation sites on D5 and D8 from the miR-

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140 transfected group; the right horn was injected with miR-140 mimic and the left horn was

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injected with mimic control. (B) Graph showing the number of implantation sites in control mimic- and miR-140 mimic- transfected uterine horn on D5 of pregnancy (left panel). Relative

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expression of miR-140 in control mimic and miR-140 mimic transfected uterine horn on D5 of

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pregnancy (right panel). Significant fold changes are marked by ***p < 0.001 vs. mimic control. Fig. 4. Effect of miR-140 mimic and ormeloxifene on adhesion and expansion of BeWo

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spheroids. (A) Graph showing the percent attachment of trophoblast spheroids on RL95-2 cells. Briefly, RL95-2 cells were seeded in 96-well plate, transfected with miR-140 mimic and inhibitor with or without ormeloxifene, for 48 h. Spheroids were co-cultured for 1 h and the percent attachment was calculated using the formula described in ‘materials & methods’ section. (B) Graph showing spheroid area spread after 24 h of co-culture, relative to 1 h, on RL95-2 cells.

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(C) Representative images of BeWo spheroids outgrowth on RL95-2 monolayer. The spheroid images were photographed at 1 h and 24 h after co-culture. Significant fold changes are marked by ***p < 0.001, **p < 0.01 and *p < 0.05 vs. mimic control.

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Fig. 5. Effect of miR-140 mimic and ormeloxifene on migration and invasion of primary rat endometrial epithelial cells. Enometrial epithelial cells were treated with miR-140 mimic, Orm and miR-140 inhibitor+Orm to perform scratch wound assay (A) and transwell migration and invasion assay (B). Cells migrated and invaded to the lower chamber were fixed, stained and counted by light microscopy. Details have been given in ‘materials & methods’ section. The

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graph in the lower panel shows the percent wound closure, percent migrated and invaded cells.

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Significant fold changes are marked by ***p < 0.001 and **p < 0.01 vs. mimic control.

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Fig. 6. The prediction and confirmation of the miR-140 target gene Igf1r. (A) MiR-140 binding sites in the 3'-UTR region of Igf1r was compared in different mammalian species. (B)

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Relative expression of miR-140 after transfection of miR-140 mimic to endometrial epithelial

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cells after 48 h and its target Igf1r mRNA in rat endometrial epithelial cells. (C) The effect of miR-140 on endogenous IGF1R expression in rat endometrial epithelial cells. Relative IGF1R

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protein expression in miR-140 mimic (left panel) and miR-140 inhibitor (right panel)-treated

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endometrial epithelial cells were detected by immunoblotting. The densitometric quantitation of band intensities was showing in graphs. Significant fold changes are marked by ***p < 0.001, *p

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< 0.05 vs. mimic control and ***p < 0.001 vs. inhibitor control. Fig. 7. Expression of Igf1r mRNA and serum IGF1 level. (A) Relative expression of Igf1r mRNA on D5 in ormeloxifene-treated (1.25 mg/kg, on D1) and vehicle-treated rat uterus. (B) Relative expression of Igf1r mRNA during window of implantation in rat uterus. (C) Expression of Igf1r mRNA in Orm-treated rat endometrial epithelial (EECs) and stromal cells (ESCs) over

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control. (D) Serum IGF1 level on D5 in ormeloxifene-treated and vehicle-treated rats. Data are presented as relative fold change in expression after normalization by internal control GAPDH. Significant fold changes are marked by *p < 0.05, **p < 0.01, ***p < 0.001 vs. control and *p <

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0.05 vs. D4. Fig. 8. Effect of miR-140 and ormeloxifene on IGF1R, Integrin β3, FAK and p-FAK

expression. Western blot analysis to check the effect of miR-140 mimic and ormeloxifene on

Igf1r and its downstream signaling molecules. Rats uterine horns were transfected with miR-140 mimic or control mimic on D3. In another group, rats received the oral dose of ormeloxifene

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(1.25 mg/kg body weight) on D1. Animals were sacrificed on D5. Representative images of

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immunoblotting showing expression of IGF1R, integrin β3, FAK and p-FAK in uterine tissues

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(upper panel). The densitometric quantitation of protein expression levels are shown as fold

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changes (lower panel). Results are expressed as mean ± SEM, p values are ***p < 0.001 and **p

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< 0.01 vs. control.

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Fig. 9. Schematic representation of mechanism of action of ormeloxifene involved in suppression of endometrial receptivity and embryo implantation. Ormeloxifene induces miR-140

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expression which in turn down-regulates the expression of IGF1R and its downstream signaling

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molecules integrin β3 and FAK and leads to inhibition of embryo implantation in rat uterus.

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Supplementary Fig. 1. Expression of the 5 up-regulated and 4 down-regulated miRNAs in the

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Orm-treated rat uterus as compared to control using real-time RT-PCR. Data are presented as relative fold change in expression after normalization to the mean value of internal control U6.

For comparative analysis, the expression values for each miRNA were set as 1 in control group. Significant fold changes are marked by *p < 0.05, **p < 0.01 and ***p < 0.001 vs. vehicle control.

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Supplementary Fig. 2. (A) Representative images of rat uteri showing implantation sites on D5

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and D8 from the miR-140 inhibitor transfected group; the right horn was injected with miR-140

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inhibitor and the left horn was injected with inhibitor control. (B) Graph showing the number of

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implantation sites in inhibitor control and miR-140 inhibitor transfected uterine horn on D5 of

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pregnancy.

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Supplementary Fig. 3. Effect of ormeloxifene on cell viability in primary rat endometrial epithelial cells. The endometrial epithelial cells were treated with varying doses of ormeloxifene,

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i.e., 10 nM, 100 nM, 1 μM, 10 μM and 20 μM for 48 h. Cell viability was determined by MTT

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assay. The percentage of viable cells was calculated as the ratio of treated cells to the control cells. Significant changes are marked by *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.

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Supplementary Table 1. List of miRScript primers used for the amplification of miRNAs in real-time PCR.

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Supplementary Table 2. Table represents comparative results (control and Orm treated groups) obtained in terms of fold change in miRNA sequencing and by RT-qPCR for the selected

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