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

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. In our previous study, using a miRNA microarray technique, we demonstrated that hsa-miR-100-5p was upregulated in ECSCs [ 4 ]. For the validation of the microarray data, we performed quantitative RT-PCR with NESCs ( n  = 6) and ECSCs ( n  = 6) as described previously [ 4 – 6 ]. hsa-miR-100-5p-specific (Assay ID: 000437, Applied Biosystems, Carlsbad, CA, USA) or endogenous control (RNU44)-specific (Assay ID: 001094, Applied Biosystems) reverse primers were used. The expression levels of hsa-miR-100-5p were normalized to those of RNU44, calculated by the ΔΔCT method, and were presented as the relative expression in ECSCs compared to that in NESCs. Precursor hsa-miR-100-5p (pre-miR miRNA precursor-hsa-miR-100-5p, Ambion, Austin, TX, USA), negative control precursor miRNA (pre-miR miRNA precursor-negative control #1, Ambion), SMARCD1 silener pre-designed siRNA (AM16708, Ambion) or Silencer® select negative control #1 siRNA (Ambion) were transfected into NESCs using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) and the reverse transfection method, as described before [ 4 – 6 ]. 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 Z-score ≥ 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 ). To elucidate the downstream target genes and signal pathways of hsa-miR-100-5p, datasets representing the genes with an altered expression profile derived from the microarray analyses were analyzed by the Ingenuity pathways analysis (IPA) software (Ingenuity Systems, Redwood City, CA, USA) with the IPA knowledgebase (IPA Summer Release 2015). Thereafter, predicted targets of hsa-miR-100-5p were confirmed by online public databases including miRDB ( http://mirdb.org/miRDB/ ), TargetScanHuman ( http://www.targetscan.org/ , Release 7.0), PicTar ( http://pictar.mdc-berlin.de/ ), and microRNA.org ( http://www.microrna.org/microrna/getGeneForm.do ). 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. 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. 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 – 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 ). Fig. 1 Downstream signaling pathway of hsa-miR-100-5p in NESCs. A gene expression microarray and pathway analyses of hsa-miR-100-5p-transfected NESCs revealed that hsa-miR-100-5p upregulated the motility of NESCs by direct inhibition of SMARCD1 expression followed by MMP1 activation. SMARCD1, SWItch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1; MMP1, matrix metallopeptidase 1; NESCs, normal endometrial stromal cells Downstream signaling pathway of hsa-miR-100-5p in NESCs. A gene expression microarray and pathway analyses of hsa-miR-100-5p-transfected NESCs revealed that hsa-miR-100-5p upregulated the motility of NESCs by direct inhibition of SMARCD1 expression followed by MMP1 activation. SMARCD1, SWItch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1; MMP1, matrix metallopeptidase 1; NESCs, normal endometrial stromal cells 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. 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. 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.

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). Fig. 2 hsa-miR-100-5p expression in NESCs and ECSCs. The relative hsa-miR-100-5p levels in ECSCs ( n  = 6) were significantly higher than those in the NESCs ( n  = 6). * p  < 0.0005 vs. NESCs (Student’s t -test). Data are shown as the mean ± SD. ECSCs, endometriotic cyst stromal cells; NESCs, normal endometrial stromal cells hsa-miR-100-5p expression in NESCs and ECSCs. The relative hsa-miR-100-5p levels in ECSCs ( n  = 6) were significantly higher than those in the NESCs ( n  = 6). * p  < 0.0005 vs. NESCs (Student’s t -test). Data are shown as the mean ± SD. ECSCs, endometriotic cyst stromal cells; NESCs, normal endometrial stromal cells As shown in Fig.  3 a, mature hsa-miR-100-5p expression in NESCs was significantly induced by hsa-miR-100-5p precursor transfection ( p  < 0.05). We thus considered this experimental model as appropriate for hsa-miR-100-5p functional analyses. Fig. 3 Effects of hsa-miR-100-5p transfection on the downstream target molecule expression in NESCs. ( a ) hsa-miR-100-5p expression after precursor miRNA transfection. Note that the vertical axis is expressed as a logarithmic scale. ( b ) SMARCD1 mRNA expression. ( c ) MMP1 mRNA expression. ( d ) Active MMP1 protein expression. * p  < 0.05, ** p  < 0.005, # p  < 0.0005 vs. controls (Student’s t -test). MMP1, matrix metallopeptidase 1; NESCs, normal endometrial stromal cells; SMARCD1, SWItch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 Effects of hsa-miR-100-5p transfection on the downstream target molecule expression in NESCs. ( a ) hsa-miR-100-5p expression after precursor miRNA transfection. Note that the vertical axis is expressed as a logarithmic scale. ( b ) SMARCD1 mRNA expression. ( c ) MMP1 mRNA expression. ( d ) Active MMP1 protein expression. * p  < 0.05, ** p  < 0.005, # p  < 0.0005 vs. controls (Student’s t -test). MMP1, matrix metallopeptidase 1; NESCs, normal endometrial stromal cells; SMARCD1, SWItch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 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. Table 1 List of mRNAs aberrantly expressed in miR-100-5p-transfected NESCs Gene family Gene symbol Control signal miR-100 precursor signal Z-score Ratio (A) Upregulated mRNAs  Cytokine CCL2 312.22 1041.65 6.87 4.32 IL11 1034.43 2782.35 5.98 3.11 LIF 128.77 261.89 3.80 2.46 RP2 838.00 1737.49 4.13 2.17  Growth factor BMP2 817.37 461.26 −4.41 0.45  Peptidase MMP1 49,499.07 74,041.18 7.47 2.40 CPXM1 70.96 120.43 2.27 2.40  Enzyme ASPH 1259.03 3722.52 6.99 3.81 CYBRD1 1067.64 1867.57.94 4.18 2.08 MTAP 440.73 714.19 3.88 2.18  Transcription regulator UBE2V1 2487.25 6026.87 5.85 2.87 SCML1 48.29 98.99 3.02 2.53 BATF3 147.71 291.50 3.05 2.02  Transmembrane receptor ITGA6 1383.58 2680.82 4.48 2.21 PVRL2 6699.21 13,211.87 4.72 2.04  Transporter ABCA1 134.70 270.47 3.29 2.12 BCAP29 1166.81 1994.48 4.09 2.03  Other TUBB2B 41.72 123.42 4.64 3.54 PKIA 192.01 593.66 5.84 3.20 PALM3 260.83 736.98 5.72 2.99 CEND1 196.72 412.79 4.30 2.80 EMC10 4713.79 9455.68 5.88 2.76 ERLIN2 225.65 451.23 3.82 2.41 TFPI2 752.91 1839.06 4.64 2.35 RDX 469.66 816.51 4.11 2.27 FAM131B 47.52 91.03 2.61 2.24 SWAP70 440.11 805.17 4.01 2.18 LOC728392 1643.31 3433.27 4.56 2.15 STBD1 104.93 162.95 3.20 2.15 SNRPC 10,269.42 17,744.24 4.89 2.12  Other CIDEC 488.37 852.48 3.68 2.05 ANGPTL4 1403.18 2829.40 4.28 2.01  Null LOC102723946 38.79 110.21 3.88 3.33 (B) Downregulated mRNAs  Enzyme HSD17B2 286.50 128.94 −5.76 0.26 PLCH1 190.28 88.66 −4.24 0.38 INMT 1404.19 713.49 −5.10 0.40  Ion channel KCNN2 118.10 32.10 −4.34 0.24 KCTD4 151.44 53.14 −5.41 0.28 KCTD10 1074.65 563.34 −4.14 0.47  Kinase FGFR3 239.75 121.71 −3.31 0.49  Peptidase ADAMTS5 3143.19 1023.39 −6.74 0.33 ADAM19 239.21 117.14 −3.26 0.47  Phosphatase LPPR4 184.80 73.50 −4.33 0.34  Transcription regulator SMARCD1 2210.80 932.50 −5.34 0.43 BAZ2A 288.00 135.67 −3.32 0.47  Transporter ATP6AP1 9337.60 3682.26 −6.29 0.40  Other EPDR1 3173.94 780.35 −8.19 0.27 MPZL3 125.86 59.79 −3.82 0.43 ZBED2 161.28 71.97 −3.74 0.43 SUDS3 568.38 289.31 −4.34 0.46 CGA 112.70 61.15 −3.01 0.46 TMEM30A 3229.37 1492.02 −4.80 0.47 DGCR2 4328.05 2074.04 −4.65 0.47 CTDSPL 748.62 362.73 −4.11 0.48 DEPTOR 398.87 187.27 −3.37 0.48 DNAJC11 1560.93 839.74 −3.46 0.49 CLDN11 1351.65 687.71 −3.46 0.49  Null SLC16A14 144.45 54.61 −3.92 0.35 PROSER2-AS1 35.66 16.12 −2.11 0.42 AREG 120.26 61.28 −3.53 0.43 ABCA1 ATP-binding cassette, sub-family A, member 1; ADAM19 ADAM metallopeptidase domain 19; ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif, 5; ANGPTL4 angiopoietin-like 4 transcript variant 1; AREG amphiregulin; ASPH aspartate beta-hydroxylase, transcript variant 3; ATP6AP1 ATPase, H+ transporting, lysosomal accessory protein 1; BATF3 basic leucine zipper transcription factor, ATF-like 3; BAZ2A bromodomain adjacent to zinc finger domain, 2A; BCAP29 B-cell receptor-associated protein 29, transcript variant 2; BMP2 bone morphogenetic protein 2; CCL2 chemokine ligand 2; CEND1 cell cycle exit and neuronal differentiation 1; CGA glycoprotein hormones, alpha polypeptide, transcript variant 2; CIDEC cell death-inducing DFFA-like effector c, transcript variant 3; CLDN11 claudin 11, transcript variant 1; CPXM1 carboxypeptidase X, member 1, transcript variant 1; CTDSPL CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like, transcript variant 1; CYBRD1 cytochrome b reductase 1, transcript variant 1; DEPTOR DEP domain containing MTOR-interacting protein, transcript variant 1; DGCR2 DiGeorge syndrome critical region gene 2, transcript variant 1; DNAJC11 DnaJ (Hsp40) homolog, subfamily C, member 11; EMC10 ER membrane protein complex subunit 10, transcript variant 1; EPDR1 ependymin related 1, transcript variant 1; ERLIN2 ER lipid raft associated 2, transcript variant 1; FAM131B family with sequence similarity 131, member B, transcript variant a; FGFR3 fibroblast growth factor receptor 3, transcript variant 1; HSD17B2 hydroxysteroid (17-beta) dehydrogenase 2; IL11 interleukin 11; INMT indolethylamine N-methyltransferase, transcript variant 2; ITGA6 integrin, alpha 6, transcript variant 2; KCNN2 potassium channel, calcium activated intermediate/small conductance subfamily N alpha, member 2, transcript variant 1; KCTD10 potassium channel tetramerization domain containing 10; KCTD4 potassium channel tetramerization domain containing 4; LIF leukemia inhibitory factor, transcript variant 1; LOC102723946, Zinc finger protein 695; LOC728392, uncharacterized LOC728392; LPPR4 lipid phosphate phosphatase-related protein type 4, transcript variant 1; MMP1 matrix metallopeptidase 1, transcript variant 1; MPZL3 myelin protein zero-like 3, transcript variant 1: MTAP methylthioadenosine phosphorylase; PALM3 paralemmin 3; PKIA protein kinase (cAMP-dependent, catalytic) inhibitor alpha, transcript variant 1; PLCH1 phospholipase C, eta 1, transcript variant 2; PROSER2-AS1 PROSER2 antisense RNA 1; PVRL2 poliovirus receptor-related 2, transcript variant delta; RDX radixin, transcript variant 3; RP2 retinitis pigmentosa 2; SCML1 sex comb on midleg-like 1, transcript variant 1; SLC16A14 solute carrier family 16, member 14; SMARCD1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1, transcript variant 2; SNRPC small nuclear ribonucleoprotein polypeptide C, transcript variant 1; STBD1 starch binding domain 1; SUDS3 suppressor of defective silencing 3 homolog; SWAP70 SWAP switching B-cell complex 70 kDa subunit, transcript variant 1; TFPI2 tissue factor pathway inhibitor 2, transcript variant 1; TMEM30A transmembrane protein 30A, transcript variant 1; TUBB2B tubulin, beta 2B class IIb; UBE2V1 ubiquitin-conjugating enzyme E2 variant 1, transcript variant 4; ZBED2 zinc finger, BED-type containing 2 List of mRNAs aberrantly expressed in miR-100-5p-transfected NESCs ABCA1 ATP-binding cassette, sub-family A, member 1; ADAM19 ADAM metallopeptidase domain 19; ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif, 5; ANGPTL4 angiopoietin-like 4 transcript variant 1; AREG amphiregulin; ASPH aspartate beta-hydroxylase, transcript variant 3; ATP6AP1 ATPase, H+ transporting, lysosomal accessory protein 1; BATF3 basic leucine zipper transcription factor, ATF-like 3; BAZ2A bromodomain adjacent to zinc finger domain, 2A; BCAP29 B-cell receptor-associated protein 29, transcript variant 2; BMP2 bone morphogenetic protein 2; CCL2 chemokine ligand 2; CEND1 cell cycle exit and neuronal differentiation 1; CGA glycoprotein hormones, alpha polypeptide, transcript variant 2; CIDEC cell death-inducing DFFA-like effector c, transcript variant 3; CLDN11 claudin 11, transcript variant 1; CPXM1 carboxypeptidase X, member 1, transcript variant 1; CTDSPL CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase-like, transcript variant 1; CYBRD1 cytochrome b reductase 1, transcript variant 1; DEPTOR DEP domain containing MTOR-interacting protein, transcript variant 1; DGCR2 DiGeorge syndrome critical region gene 2, transcript variant 1; DNAJC11 DnaJ (Hsp40) homolog, subfamily C, member 11; EMC10 ER membrane protein complex subunit 10, transcript variant 1; EPDR1 ependymin related 1, transcript variant 1; ERLIN2 ER lipid raft associated 2, transcript variant 1; FAM131B family with sequence similarity 131, member B, transcript variant a; FGFR3 fibroblast growth factor receptor 3, transcript variant 1; HSD17B2 hydroxysteroid (17-beta) dehydrogenase 2; IL11 interleukin 11; INMT indolethylamine N-methyltransferase, transcript variant 2; ITGA6 integrin, alpha 6, transcript variant 2; KCNN2 potassium channel, calcium activated intermediate/small conductance subfamily N alpha, member 2, transcript variant 1; KCTD10 potassium channel tetramerization domain containing 10; KCTD4 potassium channel tetramerization domain containing 4; LIF leukemia inhibitory factor, transcript variant 1; LOC102723946, Zinc finger protein 695; LOC728392, uncharacterized LOC728392; LPPR4 lipid phosphate phosphatase-related protein type 4, transcript variant 1; MMP1 matrix metallopeptidase 1, transcript variant 1; MPZL3 myelin protein zero-like 3, transcript variant 1: MTAP methylthioadenosine phosphorylase; PALM3 paralemmin 3; PKIA protein kinase (cAMP-dependent, catalytic) inhibitor alpha, transcript variant 1; PLCH1 phospholipase C, eta 1, transcript variant 2; PROSER2-AS1 PROSER2 antisense RNA 1; PVRL2 poliovirus receptor-related 2, transcript variant delta; RDX radixin, transcript variant 3; RP2 retinitis pigmentosa 2; SCML1 sex comb on midleg-like 1, transcript variant 1; SLC16A14 solute carrier family 16, member 14; SMARCD1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily d, member 1, transcript variant 2; SNRPC small nuclear ribonucleoprotein polypeptide C, transcript variant 1; STBD1 starch binding domain 1; SUDS3 suppressor of defective silencing 3 homolog; SWAP70 SWAP switching B-cell complex 70 kDa subunit, transcript variant 1; TFPI2 tissue factor pathway inhibitor 2, transcript variant 1; TMEM30A transmembrane protein 30A, transcript variant 1; TUBB2B tubulin, beta 2B class IIb; UBE2V1 ubiquitin-conjugating enzyme E2 variant 1, transcript variant 4; ZBED2 zinc finger, BED-type containing 2 To investigate the underlying mechanisms of hsa-miR-100-5p functions, we investigated the expression levels of SMARCD1 and MMP1. As shown in Fig. 3 b, SMARCD1 mRNA expression was significantly attenuated by hsa-miR-100-5p transfection ( p  < 0.05). In contrast, as shown in Fig. 3 c and d, the expression levels of MMP1 mRNA, and active MMP1 protein were upregulated by hsa-miR-100-5p transfection ( p  < 0.005 and p  < 0.0005, respectively). 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.  4 a, SMARCD1 mRNA expression was significantly suppressed by SMARCD1 siRNA transfection ( p  < 0.005). As shown in Fig. 4 b, the expression levels of MMP1 mRNA was significantly upregulated by SMARCD1 siRNA transfection ( p  < 0.05). Fig. 4 Effects of SMARCD1 siRNA transfection on the MMP1 mRNA expression in NESCs. ( a ) SMARCD1 mRNA expression after SMARCD1 siRNA transfection. ( b ) MMP1 mRNA expression. * p  < 0.05, ** p  < 0.005 vs. controls (Student’s t -test). MMP1, matrix metallopeptidase 1; NESCs, normal endometrial stromal cells; SMARCD1, SWItch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 Effects of SMARCD1 siRNA transfection on the MMP1 mRNA expression in NESCs. ( a ) SMARCD1 mRNA expression after SMARCD1 siRNA transfection. ( b ) MMP1 mRNA expression. * p  < 0.05, ** p  < 0.005 vs. controls (Student’s t -test). MMP1, matrix metallopeptidase 1; NESCs, normal endometrial stromal cells; SMARCD1, SWItch/sucrose non-fermentable-related matrix-associated actin-dependent regulator of chromatin subfamily D member 1 As shown in Fig.  5 a 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). Fig. 5 Effects of hsa-miR-100-5p transfection on the motility of NESCs. ( a ) Results of transwell invasion assay. ( b ) Representative photographs of transwell invasion assay. ( c ) Results of in-vitro wound repair assay. ( d ) Representative photographs of in vitro wound repair assay. * p  < 0.05, ** p  < 0.0005 vs. controls (Student’s t -test). NESCs, normal endometrial stromal cells Effects of hsa-miR-100-5p transfection on the motility of NESCs. ( a ) Results of transwell invasion assay. ( b ) Representative photographs of transwell invasion assay. ( c ) Results of in-vitro wound repair assay. ( d ) Representative photographs of in vitro wound repair assay. * p  < 0.05, ** p  < 0.0005 vs. controls (Student’s t -test). NESCs, normal endometrial stromal cells 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. 5 c and d, the repaired area was significantly increased by hsa-miR-100-5p transfection ( p  < 0.0005).

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 – 5 ]. To understand the mechanism(s) responsible for the pathogenesis of endometriosis, we have previously investigated microRNA (miRNA) expression levels in endometriosis [ 4 – 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.

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. hsa-miR-100-5p can act as either a tumor suppressor gene or an oncogene, depending on the tumor type in different cancers [ 19 , 20 ]. For example, hsa-miR-100-5p overexpression has been demonstrated in nasopharyngeal cancer [ 21 ], esophageal squamous cell carcinoma [ 22 ], colon cancer [ 19 , 23 ], and gastric cancer [ 24 ]. In these tumors, this miRNA contributes to tumor progression. In contrast, hsa-miR-100-5p expression is suppressed in epithelial ovarian cancer [ 25 ], endometrial cancer [ 26 ], bladder carcinoma [ 27 ], renal cell carcinoma [ 28 ], prostate cancer [ 29 ], breast carcinoma [ 30 ], hepatocellular carcinoma [ 31 ], and non-small cell lung cancer [ 32 ]. In these tumors, this miRNA behaves as a tumor suppressor. The reported target genes of hsa-miR-100-5p include polo-like kinase 1 [ 21 , 32 ], insulin-like growth factor (IGF) [ 33 ], IGF-1 receptor [ 34 ], mammallian target of rapamycin (mTOR) [ 34 ], fibroblast growth factor receptor 3 [ 35 ], ataxia telangiectasia mutated (ATM) [ 36 ], Argonaute 2 [ 37 ], isoprenylcysteine carboxyl methyltransferase (ICMT) [ 38 ], nuclear factor-κB3 [ 39 ], ras-related C3 botulinum toxin substrate 1 (Rac1) [ 38 ], and β-tubulin [ 40 ]. 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.

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.