hsa-miR-100-5p, an overexpressed miRNA in human ovarian endometriotic stromal cells, promotes invasion through attenuation of SMARCD1 expression

Background A number of microRNAs are aberrantly expressed in endometriosis and are involved in its pathogenesis. Our previous study demonstrated that has-miR-100-5p expression is enhanced in human endometriotic cyst stromal cells (ECSCs). The present study aimed to elucidate the roles of has-miR-100-5p in the pathogenesis of endometriosis. Methods Normal endometrial stromal cells (NESCs) were isolated from normal eutopic endometrium without endometriosis. Using hsa-miR-100-5p-transfected NESCs, we evaluated the effect of hsa-miR-100-5p on the invasiveness of these cells by Transwell invasion assay and in-vitro wound repair assay. We also investigated the downstream signal pathways of hsa-miR-100-5p by microarray analysis and Ingenuity pathways analysis. Results hsa-miR-100-5p transfection enhanced the invasion and motility of NESCs. After hsa-miR-100-5p transfection, mRNA expression of SWItch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 (SMARCD1) was significantly attenuated. Whereas, the expression of matrix metallopeptidase 1 (MMP1) mRNA and active MMP1 protein levels was upregulated. Conclusion We found that SMARCD1/MMP-1 is a downstream pathway of hsa-miR-100-5p. hsa-miR-100-5p transfection enhanced the motility of NESCs by inhibiting SMARCD1 expression and MMP1 activation. These findings suggest that enhanced hsa-miR-100-5p expression in endometriosis is involved in promoting the acquisition of endometriosis-specific characteristics during endometriosis development. Our present findings on the roles of hsa-miR-100-5p may thus contribute to understand the epigenetic mechanisms involved in the pathogenesis of endometriosis.


Background
Endometriosis belongs to estrogen-dependent benign tumors and occurs in 6-10% of the women of reproductive age [1]. The microscopic features of endometriotic tissues resemble those of proliferative-phase endometrial tissues [1]; however, molecular studies have revealed a number of differences at the epigenetic, genetic, transcriptional, and posttranscriptional levels [2][3][4][5].
To understand the mechanism(s) responsible for the pathogenesis of endometriosis, we have previously investigated microRNA (miRNA) expression levels in endometriosis [4][5][6][7]. Our previous microarray study detected a repertoire of aberrantly expressed miRNAs in endometriosis [4]. Of these aberrantly expressed miRNAs, we demonstrated that upregulation of hsa-miR-210 [5] and downregulation of hsa-miR-196b [4] and hsa-miR-503 [6] contribute to the pathogenesis of endometriosis. Hsa-miR-210 induced the cell proliferation and vascular endothelial cell growth factor (VEGF) production of human normal endometrial stromal cells (NESCs) and inhibited apoptosis of these cells [5]. Whereas, hsa-miR-196b induced the apoptosis of human endometriotic cyst stromal cells (ECSCs) and inhibited the proliferation of these cells [4]. hsa-miR-503 also induced the cell-cycle arrest at G0/G1 phase and apoptosis and inhibited the cell proliferation, VEGF production, and contractility of ECSCs [6].
SWItch/sucrose non-fermentable (SWI/SNF)-related matrix-associatedactin-dependent regulator of chromatin subfamily D member 1 (SMARCD1) belongs to the SWI/SNF chromatin remodeling complex family of proteins which regulate the target gene transcription by altering the local chromatin structure around those genes [8,9]. SMARCD1 is often involved in somatic rearrangement in tumorigenesis [10]. The chromatin remodeling activity of SMARCD1 is essential for tumor suppression [11,12]. We speculated that SMARCD1 supression may induce tumorigenesis in endometriosis.
Matrix metallopeptidase 1 (MMP1) is a key enzyme that promotes the breakdown of extracellular matrix during physiological and pathological processes such as embryonic development, reproduction, and tissue remodeling, as well as tumor invasion and metastasis. MMP-1 is the most ubiquitously expressed interstitial collagenase that cleaves the interstitial collagen, types I, II, and III [13]. MMP1 is overexpressed in endometriotic tissues, suggesting its involvement in the pathogenesis of endometriosis [14].
In the present study, we evaluated the role of hsa-miR-100-5p, a miRNA that is upregulated in ECSCs, regarding the pathogenesis of endometriosis [4]. Using hsa-miR-100-5p-transfected NESCs, we assessed the effect of hsa-miR-100-5p on the invasiveness of these cells and the expression of SMARCD1 and MMP1, which are downstream targets of hsa-miR-100-5p, in these cells.

Human NESC and ECSC isolation procedures and cell culture conditions
Normal endometrial tissues were collected at the time of hysterectomies from patients with subserous or intramural leiomyoma who had regular menstrual cycle and had no evidence of endometriosis (n = 13, age 31-53 yrs.), as described previously [15]. Whereas, ovarian endometrioma tissues were obtained at the time of surgical treatment from patients with regular menstrual cycles (n = 6, age 22-42 yrs.), as described before [6,7,15]. None of the patients had received the hormonal treatments for at least 2 years prior to the surgery. Pathological examination and/or menstrual records confirmed that all the specimens were in the mid-to-late proliferative phases. This study was approved by the Institutional Review Board (IRB) of the Faculty of Medicine, Oita University (registration number: P-16-01), and written informed consent was obtained from all the patients.
ECSCs and NESCs were isolated from ovarian endometrioma and normal endometrial tissues, respectively, by enzymatic digestion, and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 IU/ml of penicillin (Gibco-BRL, Gaithersburg, MD, USA), 50 mg/ml of streptomycin (Gibco-BRL), and 10% charcoal-strippedheat-inactivated fetal bovine serum (FBS) (Gibco-BRL) at 37°C in 5% CO 2 in air, as described previously [6,7,15]. This culture condition is free of ovarian steroid hormones. Each experiment was performed in triplicate and was repeated at least three times with cells isolated from separate patients.

Gene expression microarray
Forty-eight hours after transfection, total RNA was extracted from cultured NESCs transfected with precursor hsa-miR-100-5p (n = 4) and NESCs transfected with negative control precursor miRNA (n = 4) using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) and subjected to gene expression microarray analyses with a commercially available human mRNA microarray (G4851A, SurePrint G3 Human Gene Expression Microarray 8x60K v2, Agilent Technologies, Santa Clara, CA, USA), as described previously [5]. To identify the upregulated and downregulated genes, the Z-scores and ratios (non-log scaled fold-change) from the normalized signal intensities of each probe were calculated to compare between NESCs transfected with precursor hsa-miR-100-5p and NESCs transfected with negative control precursor miRNA [5]. We established the following criteria for the regulated genes: at least 3 out of 4 samples has Zscore ≥ 2.0 and ratio ≥ 2.0-fold for upregulated genes, and Z-score ≤ − 2.0 and ratio ≤ 0.5 for downregulated genes. All the gene expression microarray data are available at the Gene Expression Omnibus through the NCBI under Accession No. GSE139954 (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE139954).

Transwell invasion assay
The invasive properties of hsa-miR-100-5p-transfected NESCs were evaluated by Transwell invasion assay, as described previously [16,17]. NESCs after miRNA transfection (2 × 10 5 cells) were cultured in DMEM supplemented with 10% charcoal-strippedheat-inactivated FBS on the growth factor-reducedMatrigel-coated Transwell inserts with 8-μm pores (Corning Inc., New York, NY, USA). After 48 h, the membranes were fixed with 100% methanol, and the number of cells appearing on the undersurface of the polycarbonate membranes after Giemsa staining was scored visually at × 200 magnification using a light microscope.
The data from triplicate samples were calculated and presented as the percent values obtained for the NESCs transfected with precursor hsa-miR-100-5p relative to those transfected with the negative control precursor miRNA.

In vitro wound repair assay
Cell motility was also determined by an in vitro wound repair assay, as described previously [16,17]. NESCs grown to confluence in 6-well plates (Corning Inc.) were challenged overnight with serum-free medium and then transfected with the miRNA precursor. The monolayer was wounded using a cell scraper and the plates were incubated in DMEM plus 0.1% BSA for 48 h. The cells were then fixed with 3% paraformaldehyde and stained with Giemsa solution. Areas with lesions were photographed, and wound repair was assessed by calculating the repaired area in square micrometers between the lesion edges at 0 h and 48 h using the public domain software Image J 1.44 developed at the U.S. National Institutes of Health (Bethesda, MD, USA).
The data from triplicate samples were calculated and presented as the percent values obtained for the NESCs transfected with precursor hsa-miR-100-5p relative to those transfected with the negative control precursor miRNA.

RT-PCR for mRNA expression
The effects of hsa-miR-100-5p on the expression levels of possible downstream target genes were evaluated in ECSCs by quantitative RT-PCR, as described [4][5][6]. SMARCD1 and MMP1 were selected as candidate genes because SMARCD1 was confirmed to be the predicted target of hsa-miR-100-5p in the online public database, TargetScanHuman (http://www.targetscan.org/, Release 7.2). MMP1 is known to be the downstream target of SMARCD1 [18] and promotes cell motility (Fig. 1).
In brief, 48 h after miRNA transfection, total RNA from miRNA-transfected NESCs was extracted as described above and subjected to quantitative RT-PCR with the following specific primers (all from Applied Biosystems): SMARCD1 (Assay ID: Hs00161980_m1), MMP1 (Assay ID: Hs00899658_m1), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID: Hs02758991_g1). The expression levels of candidate mRNAs relative to those of GAPDH mRNA were calculated using a calibration curve. The data were calculated from triplicate samples and are presented as percent values obtained for NESCs after hsa-miR-100-5p transfection relative to those transfected with the negative control precursor miRNA.

ELISA for active MMP1
Culture media of miRNA-transfected NESCs were collected 48 h after miRNA transfection and subjected to Human Active MMP-1 Fluorescent Assay (F1M00, R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions. The data from triplicate samples were calculated and presented as the percent values obtained for NESCs transfected with precursor hsa-miR-100-5p relative to those transfected with the negative control precursor miRNA.

Statistical analysis
All data were obtained from triplicate samples and are presented as percent values relative to the corresponding controls in the form of mean ± SD. Data were appropriately analyzed by the Student's t-test using the Statistical Package for Social Science software (IBM SPSS statistics 24; IBM, Armonk, NY, USA). P-values < 0.05 were considered statistically significant.

Expression of hsa-miR-100-5p
To validate the miRNA microarray data [4], we evaluated the hsa-miR-100-5p expression levels in NESCs and ECSCs using quantitative RT-PCR. As shown in Fig. 2, the relative hsa-miR-100-5p levels in the ECSCs were significantly higher than those in the NESCs (p < 0.0005). Thus, the results of quantitative RT-PCR for hsa-miR-100-5p expression were consistent with our previous miRNA microarray data [4]. Age of the patients did not affect the expression of hsa-miR-100-5p (data not shown).
Identification of hsa-miR-100-5p-regulated genes and predicted pathways in NESCs As shown in Table 1, gene expression microarray analyses detected 33 upregulated and 27 downregulated mRNAs using the criteria described above. Using the online public databases, we focused on SMARCD1 involved in the pathogenesis of endometriosis. The IPA software then identified MMP1 as a downstream target of SMARCD1 (Fig. 1). Regarding the known function of MMP1, we evaluated the cell motility of NESCs using the following experiments.

Modulation of MMP1 expression by SMARCD1 siRNA transfection
To confirm that the MMP1 expression is regulated by SMARCD1, we investigated the expression levels of MMP1 mRNA after SMARCD1 siRNA transfection. As shown in Fig. 4a, SMARCD1 mRNA expression was significantly suppressed by SMARCD1 siRNA transfection (p < 0.005). As shown in Fig. 4b, the expression levels of MMP1 mRNA was significantly upregulated by SMARCD1 siRNA transfection (p < 0.05).

Cell motility
As shown in Fig. 5a and b, the transwell invasion assay revealed that the number of invaded cells was significantly increased by hsa-miR-100-5p transfection (p < 0.05).
We also investigated the effects of hsa-miR-100-5p on the motility of NESCs by an in vitro wound healing assay. As shown in Fig. 5c and d, the repaired area was significantly increased by hsa-miR-100-5p transfection (p < 0.0005).

Discussion
To understand the role of hsa-miR-100-5p, which is upregulated in ECSCs, in the pathogenesis of endometriosis, we evaluated its expression in both ECSCs and NESCs. We also evaluated the hsa-miR-100-5p-mediated effects on the cellular functions of NESCs and sought to determine the underlying mechanisms of hsa-miR-100-5p action in those cells. With the present study, we found the following: (1) Expression of hsa-miR-100-5p in ECSCs was upregulated compared to that in NESCs. (2) hsa-miR-100-5p transfection enhanced the motility of NESCs. (3) hsa-miR-100-5p promoted these cellular functions through downregulation of SMARCD1 mRNA and induction of MMP1 expression. This suggests that hsa-miR-100-5p overexpression induces NESCs to acquire the highly motile characteristics of endometriosis and is involved in promoting the development and progression of this disease.
To our knowledge, there is no report which evaluated the expression and function of SMARCD1 in endometriosis. Whereas, overexpression of MMP1 is reported in endometriotic tissues [14], however, the roles of MMP1 regarding the pathogenesis of endometriosis has not been elucidated yet. MMP1 gene polymorphisms may also affect the motility of ECSCs [13]. In the present study, we demonstrated that transfection with hsa-miR-100-5p induced MMP1 expression in NESCs through downregulation of SMARCD1 and that MMP1 accelerated the migration of NESCs.
A limitation of the present study is that the experiments were performed only with the stromal cells of  endometriosis and the eutopic endometrium of women without endometriosis. Due to difficulties in obtaining samples, the expression of hsa-miR-100-5p in the eutopic endometrium of women with endometriosis was not evaluated. Future study is necessary on this point.

Conclusions
In summary, we confirmed that hsa-miR-100-5p expression is upregulated in ECSCs. By transfecting hsa-miR-100-5p into NESCs, we observed that SMARCD1/MMP-1 is the downstream pathway of hsa-miR-100-5p. Inhibition of SMARCD1 mRNA expression, followed by MMP1 activation, enhanced the motility of NESCs. These findings suggest that enhanced expression of hsa-miR-100-5p in endometriosis is involved a role in promoting the acquisition of endometriosis-specific characteristics during the development of endometriosis. Our present findings on the roles of hsa-miR-100-5p may thus contribute to understand the epigenetic mechanisms involved in the pathogenesis of endometriosis.