MicroRNA‐488 inhibits endometrial glandular epithelial cell proliferation, migration, and invasion in endometriosis mice via Wnt by inhibiting FZD7

Abstract Endometriosis is a chronic inflammatory syndrome and nearly 6%‐10% of women are affected by it during the reproductive period. Previous studies have proved that microRNAs (miRNAs) are implicated in the pathogenesis of ovarian endometriosis. In this study, we aimed to investigate that restored miR‐488 would effectively inhibit the development of endometriosis. The microarray‐based data analysis was performed to screen endometriosis‐related differentially expressed genes (DEGs). The mouse model in endometriosis syndrome was established by being subcutaneously injected with Estradiol benzoate, and the ectopic endometrial tissues and normal endometrial tissues were collected. Additionally, the endometrial glandular epithelial cells were extracted from the endometrial glandular epithelial tissues from normal and endometriosis mice. In order to examine the role of miR‐488 in mice with endometriosis, we measured miR‐488 expression and expression levels of Frizzled‐7 (FZD7), cyclinD1, β‐catenin, and c‐Myc in vivo and in vitro. Finally, we detected the effect of miR‐488 on cell proliferation, apoptosis, migration and invasion in vitro. FZD7 was upregulated in human endometriosis. The data showed higher expression levels of FZD7, β‐catenin, c‐Myc and cyclinD1, and lower miR‐488 expression in mouse endometrial tissues. FZD7 was the target gene of miR‐488. Furthermore, elevated miR‐488 in isolated mouse endometrial glandular endometrial cells inhibited FZD7, the translocation of β‐catenin to nucleus, the activation of Wnt pathway, and the cell proliferation, migration and invasion. Collectively, these findings indicated that up‐regulated miR‐488 may reduce the proliferation, migration and invasion of endometrial glandular epithelial cells through inhibiting the activation of Wnt pathway by down‐regulating FZD7.


| INTRODUC TI ON
Endometriosis is a chronic inflammation disease characterized by the allotopic endometrial glands and stroma, which appear outside the uterine cavity. 1 The pathogenesis of endometriosis is still largely unknown, and a study has provided evidence for the possibility that the progression of endometriosis may be attributed to inflammation, oxidative stress and immunological dysfunctions. 2 The well-known symptoms of endometriosis include dyschezia, deep dyspareunia, dysuria and dysmenorrhoea. 3 The amount of women with endometriosis during reproductive years reaches 10%, and those who have pelvic or infertility are more likely to suffer from endometriosis. 4 Although, a previous report indicated that the prevalence of endometriosis had decreased in a certain degree, the incidence of endometriosis was higher than several decades ago. 5 A great deal of health-care resources and persistent therapy are needed for the patients with endometriosis, since once the current medical therapies are discontinued, the alleviative symptoms are likely to recur and extra interventions are required. 6 It has been indicated that gene therapy holds significant promise in the endometriosis treatment for patients who have very limited treatment options at present. 7 MicroRNAs (miRNAs) are a group of endogenous small noncoding RNAs that regulate translation of various target mRNAs, involving in most cellular and developmental processes as a novel and potent gene expression regulator. 8 Recent study has revealed that an abnormal miRNA expression profile is responsible for the endometriosis by promoting the implantation of endometrial cells at aberrant sites through high angiogenic and proteolytic activities. 9 miR-488 is a kind of miRNA, which has been demonstrated to play positive role in ovarian cancer by regulating the mitochondrial function. 10 Also, the potential role of miR-488 as a tumour suppressor by inhibiting cell proliferation, cell migration, colony information, and the cell cycle in non-small-cell lung cancer and gastric cancer. 11 Besides, it has been suggested by some researchers that the activation of Wnt/β-catenin signalling pathway can be a potential mechanism under the fibrosis in endometriosis. 5 Frizzled-7 (FZD7), a member of the Frizzled family of transmembrane proteins, is a Wnt receptor that can activate the canonical and/or the non-canonical Wnt signalling pathways. 12 It has been demonstrated that the overexpressed FZD7 can contribute to the proliferation and growth of gliomas cells, contrarily, the low expression of FZD7 enormously inhibits the proliferation ability of gliomas cells. 13 In this study, we hypothesised that the miR-488 can regulate the proliferation, migration, and invasion of endometrial glandular epithelial cells through the Wnt signalling pathway by targeting FZD7, and we aim to explore new novel therapeutic targets for endometriosis.

| Ethics statement
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Affiliated Obstetrics and Gynecology Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital.

| Microarray-based data analysis
NCBI is a public platform for storing gene expression datasets, original sequences and records. By retrieving endometriosis microarrays, we found the GSE5108 and GSE23339 chips and downloaded the data from Gene Expression Omnibus (GEO). It was found that GSE5108 included 11 endometriosis samples and 11 normal control samples, and GSE23339 included 10 endometriosis samples and nine normal control samples. Next, Bioconductor-based "limma" package in r language was employed to select important differentially expressed genes (DEGs) with the empirical Bayes method. Finally, the DEGs were annotated using the "annotate" package. P < 0.05 was considered statistically significant. The miRNA target prediction web-

| Model establishment
A total of 70 healthy sexually-mature specific-pathogen-free female mice of Institute of Cancer Research (ICR) weighting between 20 and 25 g with 6-8 weeks of age were purchased from Shanghai Sippr BK laboratory animal Co., Ltd. (Shanghai, China). The mice were checked in fixed time every day for a week before the injection, and were performed with the ovariectomy to control the differences of oestrogen induced by different mice. The normal mice were chosen as study subjects, and they were subcutaneously injected with Estradiol benzoate (E2, 0.1 mg) in the first 3 days and anaesthetised by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg). One to two mice were chosen as donors, and their uterus were taken, washed with sterile D-Hank's solution for several times to remove the blood clot and mucus, and placed in the Dulbecco's modified Eagle's medium (DMEM) and Ham's nutrient mixture F-12 at room temperature. The endometrium was separated and the myometrium were removed in the culture dish containing normal saline. The endometrial tissues were cut into 2-3 mm 3 pieces for the operation. The normal mice were anaesthetised and fixed on the operation panel, the abdomen was disinfected and a 5 mm-incision on the abdomen skin was made by using an ophthalmic scissors.
The subcutaneous tissues were separated and implanted with two prepared 2-3 mm 3 endometrial tissues in the two sides of ventrimeson, respectively (no suture), and lastly the incision was sutured.
After disinfection, the mice were placed in the cages. The emergence of subcutaneous node of endometriosis indicated the success of model establishment. 14 The review was conducted 3 weeks after model establishment, the skin of model mice was opened as the previous operation to observe the growth and survival of implants. The criteria for the success of model establishment observed by naked eyes 15 were as follows: the volume of implants increased, the implants were transparent nodositas and cystiform, the supernatant liquid accumulated, the implants were soft, covered by connective tissue, and blood vessels were formed.

| Hematoxylin eosin staining
A total of 30 mice were sacrificed by air embolism method 6 weeks after model establishment. The uterus was removed in sterile environment, and the endometrial tissues were taken under dissecting microscope (2354353, Olympus Optical Co., Ltd., Tokyo, Japan). Part of tissue was taken and fixed in 4% formaldehyde, embedded in paraffin and cut into 5-8 μm-thick sections. The sections were dewaxed by xylene two times with 5 minutes each time, dehydrated with gradient ethanol (100%, 95%, 80%, and 75%) for 1 minute, respectively, and then washed under running water for 2 minutes. Next, the sections were stained with hematoxylin for 2 minutes, washed under running water for 10 seconds, and differentiated with 1% hydrochloric ethanol for 10 seconds. The sections were washed with distilled water for 1 minute, stained with eosin for 1 minute, washed with distilled water for 10 seconds, and then dehydrated with 95% ethanol two times and 100% ethanol two times with 1 minute each time. Finally, the sections were cleared with xylene, mounted with neutral balsam and observed at the microscope.

| Immunohistochemistry
Samples were fixed with 4% formaldehyde, embedded with paraffin, cut into 4 μm serial sections and then placed in an incubator at 60°C

| Dual-luciferase reporter gene assay
The biological prediction website microRNA.org was performed to analyse the target gene of miR-488, and the dual-luciferase reporter gene assay was performed to prove whether FZD7 was the target gene of miR-488. The 3'UTR gene fragment of amplified FZD7 was cloned, and the product of polymerase chain reaction (PCR) was cloned to the multiple clone sites of luciferase gene of pmirGLO (E1330, Promega, Madison, WI, USA) and named as pFZD7-wild type (Wt). Next, according to the predicted binding site of miR-488 and its target gene, PCR site-directed mutagenesis was conducted and pFZD7-mutant type (Mut) vector was constructed. The pRL-TK vector (E2241, Promega) expressing ranilla luciferase was used as internal reference to adjust the differences of cell number and efficiency of transfection. Lastly, miR-488 mimic and corresponding NC were co-transfected with luciferase reporter vector into epithelial cells of ectopic endometrium respectively. The activity of dual luciferase was measured according to the method provided by Promega. inhibitor and si-FZD7). All the sequences were listed in Table 1. The plasmids used for transfection were all purchased from Sangon biotech Co., Ltd. (Shanghai, China). The cells in logarithmic growth phase that grew well were chosen with the concentration being adjusted to 1.2 × 10 5 cells/mL and seeded in a 24-well plate with 500 μL in each well until the cell confluency reached 50%.
Another 250 μL serum-free medium Opti-MEM was used to dilute 5 μL lipofectamine 2000, and the mixture was incubated for 5 minutes at room temperature. The two mixtures were mixed, incubated for 20 minutes at room temperature, and added into culture plate, which was shaken gently. The cells were incubated in CO 2 incubator for 6-8 hours at 37°C and then cultured in complete medium for 48 hours for following experiments.

| Immunofluorescence assay
Cells were regularly detached, transfected, counted and spread in immunofluorescence chamber with 2 × 10 5 cells in each well. When the cell confluency reached about 90%, the cells were washed with PBS three times on the ice. The cells were fixed by 4% paraformaldehyde (1 mL for each well), and then allowed to stand at room temperature for 15 minutes. After cells were washed with PBS three times, 0.3% Triton was used to perforate cells. Ten minutes later, the cells were washed with PBS three times, blocked with goat serum, and allowed to stand for 30 minutes. The cells were incubated with β-catenin antibody (1:500, ab32572, Abcam) at 4°C overnight. The cells were washed with PBS three times, and incubated with Alexa Fluor 488 conjugated goat anti rabbit IgG (1:500, ab150077, Abcam) for 1 hour at room temperature avoiding exposure to light. After being washed three times, cells were added with DAPI to stain for 15 minutes avoiding exposure to light, and washed with PBS three times. Lastly, cells were mounted with fluorescence quenching agent and observed and photographed under a fluorescence microscope.

| Western blot analysis
The total protein of cells was extracted respectively, and the con-  This experiment was repeated three times.

| Scratch test
After 48-hour transfection, cells were seeded into a 6-well plate.
When cells adhered to wall, they were cultured by serum-free DMEM. When cell confluence reached 90%-100%, a 10 μL-pipette was used to perpendicularly and slowly scratch on the bottom of the culture plate, and 4-5 wounds with the same width were created in each well. The cells were washed with PBS three times to remove scratched cells and then incubated in incubator. After 0 and 24 hours of scratch, inverted microscope was performed to observe the migration distance of cells, and cells were photographed under several randomly-selected fields. This experiment was repeated three times.

| Transwell assay
Transwell chambers (Corning, New York, USA) with a 24-well plate and under randomly-selected fields to calculate the mean value. The method above was applied to assess the invasion of tumour cells.

| Statistical analysis
Statistical analyses were conducted by using spss 19.0 (IBM, Armonk, NY, USA). Measurement data were expressed as mean ± SD.
Comparison between two groups was performed by t test.
Comparison among multiple groups was conducted by one-way anova. Results were expressed as percentage and analysed using chisquare test. P < 0.05 was considered significantly different.

| FZD7 is identified as an upregulated gene in endometriosis
We downloaded the gene expression datasets GSE5108 and role in many human cancers. However, the exact function of FZD7 in human endometriosis was unclear; thus FZD7 gene became a candidate gene that we were interested in. We aimed to predict the function and clinical significance of FZD7 in endometriosis. Moreover, FZD7 ranked highest among the most DEGs. Figure 1A is a heat map of DEGs in the GSE23339 chip and Figure 1B is a heat map of DEGs in the GSE5108 chip. From Figure 1A,B, it was found that the FZD7 gene was upregulated in endometriosis. In order to study the upstream of the DEG, FZD7, we predicted the miRNAs capable of regulating FZD7 through the bioinformatics website, combining with the use of a Venn map. The result showed that only one gene was co-expressed, namely, miR-488 ( Figure 1C,D).

| The endometrial tissues appear lesions in mice with endometriosis
Initially, hematoxylin eosin (HE) staining was performed to observe the detailed differences in tissues of ectopic endometrium. The results indicated that the nidus of most of mice with endometriosis appeared typical endometrioid tissues, which had similar morphological structure as normal endometrium ( Figure 2). Meanwhile, the nidus appeared atypical endometrial tissues, and then mainly appeared fibrous connective tissue, accompanied by endometrial stroma and glandular epithelium with increasing number of weeks. Therefore, mice with endometriosis had pathological changes in endometrial tissues.

| FZD7 is a target gene of miR-488
According to the online bioinformation analysis website microRNA.
org, the target binding site of FZD7 and miR-488 existed ( Figure 4A) and the target sequences of FZD7-wild type (Wt) and FZD7-Mut are shown in Figure 4B. Besides, the results of dual-luciferase reporter gene assay indicated that compared with the NC group, the co-transfection of miR-488 mimic and Wt-miR-488/FZD7 group had lower luciferase activity (P < 0.05) ( Figure 4B), while that of the Mut-miR-488/FZD7 had no significant difference (P > 0.05). These results suggested that miR-488 could bind to FZD7 mRNA specifically.

| MiR-448 inhibits the activation of Wnt signalling pathway via suppression of the FZD7
Luciferase reporter gene of firefly was found in the TOP-Flash There was no significant difference of the expression of cyclinD1, β-catenin and c-myc between the blank and NC groups (P > 0.05).
Compared with the blank and NC groups, the miR-488 mimic and si-FZD7 groups showed lower mRNA and protein expressions of cyclinD1, β-catenin and c-myc, while the miR-488 inhibitor group showed obviously higher mRNA and protein expressions of these genes (P < 0.05). There was no significant difference of the expressions in the miR-488 inhibitor + si-FZD7 group (P > 0.05). Therefore, it can be concluded that elevated miR-488 inhibits the FZD7 expression and the activation of the Wnt signalling pathway.

| Overexpression of miR-488 inhibits the proliferation, migration and invasion of ectopic endometrium cells
MTT assay, scratch test and Transwell assay were performed to detect the proliferation, migration and invasion of ectopic endometrium cells. According to the results of MTT assay shown in Figure 6A, the proliferation rate of cells in the other groups was higher than that in the normal group. There was no significant difference in cell proliferation between the blank and NC groups (P > 0.05). Compared with the blank and NC groups, the miR-488 inhibitor group had obviously higher activity of cell proliferation, 5′ migration and invasion (P < 0.05). There was no significant difference in the miR-488 inhibitor + si-FZD7 group when compared with the blank and NC groups (P > 0.05). These results suggested that overexpressed miR-488 and FZD7 gene silencing suppressed the proliferation, migration and invasion of endometrial glandular epithelial cells.

| D ISCUSS I ON
It is well known that endometriosis is a chronic disease that has affected about 5-10% of reproductive-age women, and is defined as the abnormal presence of endometrial glands and stroma outside the uterus, primarily in the pelvic cavity. 2 Recent studies have identified miRNAs as key regulators of gene expression, and dysregulated miRNA expression may be involved in the endometriosis pathogenesis. 9,21,22 In this study, we aimed to find out the mechanism that    The result of a recent study demonstrated that miR-488 expression was inhibited in gastric cancer, compared with tissues without tumour, and the proliferation and migration of gastric cancer cell were suppressed by the up-regulated miR-488. 27 In the study of Dengdi Hu et al, the unusually low expression of miR-488 was also observed in hepatocellular carcinoma tissues, and the important role of miR-488 in suppressing proliferation, colony formation, cell invasion and EMT process has been reported. 11 FZD7, which is widely known as the most commonly reporter of Wnt, has been identified as a target for cancer therapy, since it can play an important role in controlling endothelial cell proliferation through the inhibition of the Wnt/β-catenin signalling regulators. 28 According to the previous studies, the overexpressed FZD7 can be found in multiple solid cancers and implicated in the carcinogenesis and tumour progression, thus leads to the shorter survival of colorectal and gastric cancer patients. 12 In accord with some previous studies, our study demonstrated that the inhibition of FZD7 was associated with blocked Wnt signalling pathway. It is well documented that Wnt/β-catenin signalling plays an important role in promoting oncogenic activities of cell proliferation, as well as cell invasion in glioma, and inhibiting apoptosis of cells. 13 The study conducted by Ana M. Sanchez et al have revealed that the Wnt/β-catenin signalling may play a crucial role in suppressing cell apoptosis. 29 Hence, we suggest that FZD7 may regulate Wnt signalling pathway, which is controlled by miR-488.
In summary, according to what has been concluded in the present study, we suggests that overexpressed miR-488 inhibits the Wnt signalling pathway by targeting FZD7, thus suppressing proliferation, migration, invasion of endometrial cells and act as a protective role in mice with artificial endometriosis (Figure 7). Given that low level of miR-488 is confirmed in relation to mouse endometriosis progression, our discovery outlines a potential molecular target for endometriosis treatment.

ACK N OWLED G EM ENTS
We would like show sincere appreciation to the reviewers for critical comments on this article.

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest in this work.

R E FE R E N C E S
F I G U R E 7 The mechanism of miR-488 involved in endometriosis; microRNA-488 exerted inhibitory effects on endometrial glandular epithelial cell proliferation, migration, and invasion of mice in endometriosis via the Wnt signaling pathways by inhibiting FZD7