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.

ATM expression is attenuated by promoter hypermethylation in human ovarian endometriotic stromal cells.

A number of genes involved in the pathogenesis of endometriosis are silenced by the hypermethylation of their promoter regions. We assessed the effect and mechanism of the DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) (10 µM) on the cell cycle in human endometriotic cyst stromal cells (ECSCs) and normal endometrial stromal cells (NESCs) by flow cytometry. The DNA methylation status of G2/M checkpoint regulators were investigated by methylation-specific PCR. The expression of ataxia telangiectasia mutated (ATM) and the effect of 5-aza-dC on its expression were also evaluated by quantitative RT-PCR and western blotting analysis. 5-aza-dC treatment resulted in the cell cycle arrest of ECSCs at the G2/M phase. In contrast, 5-aza-dC did not affect the cell cycle of NESCs. The promoter region of the ATM gene was hypermethylated in ECSCs, but not in NESCs. ATM mRNA expression was attenuated in ECSCs compared to that in NESCs. Further, 5-aza-dC was found to restore ATM expression in ECSCs by its promoter demethylation. Our findings indicate that ATM promoter hypermethylation occurs in endometriosis, and that ATM silencing is involved in tumorigenesis during this disease; moreover, selective DNA demethylating agents and molecular target drugs against ATM silencing are promising for the treatment of endometriosis.

Arcyriaflavin a, a cyclin D1-cyclin-dependent kinase4 inhibitor, induces apoptosis and inhibits proliferation of human endometriotic stromal cells: a potential therapeutic agent in endometriosis.

BACKGROUND: We previously showed that microRNA-503 (miR-503) transfection into endometriotic cyst stromal cells (ECSCs) induced cell cycle arrest at the G0/G1 phase by suppressing cyclin D1. This finding prompted us to evaluate the potential therapeutic effects of cyclin D1 inhibitors in endometriotic cells. This study aimed to determine whether arcyriaflavin A, a representative inhibitor of cyclin D1-cyclin-dependent kinase 4 (CDK4), is beneficial in the treatment of endometriosis. METHODS: ECSCs were isolated from the ovarian endometriotic tissues of 32 women. The effects of arcyriaflavin A on cell viability and proliferation, vascular endothelial growth factor A expression, apoptosis, and cell cycle progression were evaluated using a modified methylthiazoletetrazolium assay, enzyme-linked immunosorbent assay (ELISA), Caspase-Glo® 3/7 assay, and flow cytometry. RESULTS: Arcyriaflavin A significantly inhibited cell viability, proliferation, and angiogenesis of ECSCs as assessed using the 5-bromo-2-deoxyuridine (BrdU) and methylthiazoletetrazolium bromide (MTT) assays, and vascular endothelial growth factor (VEGF) ELISA. Arcyriaflavin A induced apoptosis as shown in the Caspase-Glo® 3/7 assay and cell death detection ELISA whilethe cell cycle was arrested at the G0/G1 phase. CONCLUSION: The findings indicate that cyclin D1-CDK4 inhibitors may be promising candidates for the treatment of endometriosis. This is the first study to demonstrate the potential usefulness of arcyriaflavin A as a therapeutic agent for endometriosis. Further studies of the effects of cyclin D1-CDK4 inhibitors on endometriosis may provide useful information on pathogenesis and treatment.

miR-503, a microRNA epigenetically repressed in endometriosis, induces apoptosis and cell-cycle arrest and inhibits cell proliferation, angiogenesis, and contractility of human ovarian endometriotic stromal cells.

STUDY QUESTION: Is the micro-RNA (miRNA) miR-503, downregulated in endometriotic cyst stromal cells (ECSCs) and does this affect the cell cycle, cell proliferation, angiogenesis and contractility of these cells? SUMMARY ANSWER: miR-503 expression is downregulated in ECSCs by DNA hypermethylation and this contributes to their proliferation, resistance to apoptosis, extracellular matrix (ECM) contractility and angiogenesis through effects on cyclin D1, B-cell lymphoma/leukemia (Bcl)-2, Ras homology A  and vascular endothelial growth factor A (VEGF-A). WHAT IS KNOWN ALREADY: A variety of miRNAs are demonstrated to involve in the pathogenesis of endometriosis. miR-503 is a miRNA with tumor-suppressor functions, whose expression is suppressed in ECSCs. STUDY DESIGN, SIZE, DURATION: We isolated ECSCs and normal endometrial stromal cells (NESCs) from ovarian endometriotic tissues (n = 32) and eutopic endometrial tissues without endometriosis (n = 8), respectively. PARTICIPANTS/MATERIALS, SETTING, METHODS: We investigated the functions of miR-503 by using miR-503-transfected ECSCs and the DNA methylation status of miR-503 gene in ECSCs and NESCs by combined bisulfite restriction analysis. MAIN RESULTS AND THE ROLE OF CHANCE: In ECSCs, miR-503 is downregulated by the DNA hypermethylation of its gene. The transfection of miR-503 into ECSCs resulted in the inhibition of cell proliferation and induction of cell-cycle arrest at G0/G1 phase through the suppression of cyclin D1, the induction of apoptosis through Bcl-2 suppression, the inhibition of VEGF-A production and the attenuation of ECM contractility via the suppression of Rho/Rho-associated coiled-coil-forming protein kinase-pathways. LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: The present experiments were carried out only with the stromal component of endometriosis and eutopic endometrium. The experiments with the eutopic endometrial stromal cells from women with endometriosis are not performed. WIDER IMPLICATIONS OF THE FINDINGS: Our findings indicate that epigenetically repressed miR-503 in ECSCs is involved in the acquisition of endometriosis-specific cellular functions. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 13237327 to K.N., no. 26861335 to K.K. and no. 23592407 to H.N.) and the Kanzawa Medical Research Foundation (to K.K.). There are no conflicts of interest to declare.

Small-Cell Carcinoma of the Uterine Cervix Discovered With Quadriplegia Caused by Cervical Spine Metastasis.

Small-cell carcinoma of the uterine cervix is a rare and aggressive tumor, and the prognosis is poor compared with those of squamous cell carcinoma and adenocarcinoma of the uterine cervix, even when discovered at an earlier stage. We treated a patient with progressive small-cell carcinoma of the uterine cervix that metastasized to the cervical spine. The patient, a 73-year-old woman, presented with the symptom of numbness in her limbs. As she had difficulty moving her limbs (ie, quadriplegia), she was carried to an emergency room. A metastatic cervical spine tumor from the uterine cervical cancer was revealed by a computed tomography scan, and the patient was then transferred to our hospital's neurosurgery department for treatment. We performed a resection of the cervical spine tumor and fixation of the spinal bone. Because the patient's performance status was 4 and she remained bedridden 24 h/day, we could not perform systemic chemotherapy. We thus provided palliative care, including palliative radiotherapy, pain control, and rehabilitation to improve her limbs' functioning. The patient died of the uterine cancer within approx. 6 months after the initiation of treatment. There is no established treatment for small-cell carcinoma as a gynecological lesion. For patients with progressive uterine cancer, the optimal treatments, including palliative care, must be determined.

Chemokine expression profiles of ovarian endometriotic stromal cells in three-dimensional culture.

Endometriosis is a chronic inflammatory disease which is associated with aberrant chemokine expression. We have established a three-dimensional (3D) floating collagen gel culture of human endometriotic cyst stromal cells (ECSCs) as an in vitro model of early-stage fibrosis formation in endometriosis. We evaluated the gene expression profiles of 3D-cultured ECSCs using a gene expression microarray. We identified and confirmed with reverse transcription-polymerase chain reaction that mRNA levels of CXCL1, CXCL2, CXCL3, CXCL8, and CCL20 in 3D-cultured ECSCs were significantly higher than in 2D-cultured ECSCs. The protein levels of CXCL1, CXCL2, CXCL8, and CCL20 in the supernatant of 3D-cultured ECSCs were significantly higher than in 2D-cultured ECSCs. It has been suggested that the 3D-culture model of ECSCs is more suitable for in vitro endometriosis research than 2D-culture. This microarray data provides a new platform to identify the candidate genes involved in the pathogenesis of endometriosis which could be masked in conventional 2D-culture.

A case of successful laparoscopic surgery for tubal stump pregnancy after tubectomy.

The incidence of ectopic pregnancy is approximately 1.3-2% of all pregnancies, and more than 90% of ectopic pregnancies are detected in the ampulla of the fallopian tube. Ectopic pregnancy occurring in tubal stump after tubectomy is extremely rare, and the frequency of tubal stump pregnancy is approximately 0.4% of all pregnancies. We report one of these rare cases of ectopic pregnancy in a 26-year-old Japanese woman, gravida 4, parity 1. She had undergone laparoscopic tubectomy because of a tubal pregnancy two years ago. She was presented to our hospital with a positive pregnancy test, but no gestational sac was detected in the uterus by echography, even though the level of human chorionic gonadotropin (hCG) in the blood was elevated to 8,900 mIU/mL. Laparoscopic surgery for ectopic pregnancy was performed. During surgery, the position of the pregnancy was found to be in the tubal stump, where tubectomy had already been performed, and the gestational sac was successfully removed. After the surgery, the condition of the patient uneventfully improved and she was discharged from the hospital three days after the surgery. The diagnosis of tubal stump pregnancy is more difficult than that of the more common positions of an ectopic pregnancy in the fallopian tube, and so it is more important to carefully examine the patients with suspected ectopic pregnancy. Laparoscopic surgery is one of the options for tubal stump pregnancy if diagnosed early and if the condition of the patient is stable.

A Case of Lymphoepithelioma-like Carcinoma in the Uterine Cervix.

Lymphoepithelioma-like carcinoma occurring in the reproductive organs is a rare variant of squamous cell carcinoma, and this tumor of the uterine cervix accounts for 0.7% of all primary cervical uterine neoplasms. Associations with Epstein-Barr virus (EBV) and human papilloma virus (HPV) have been demonstrated in some studies. Some investigators suggested that EBV has an important role in the initiation of lymphoepitheliomalike carcinoma in Asian women. Here we report the case of a 45-year-old Japanese woman, gravida 2 and parity 2. She was admitted due to severe atypical genital bleeding caused by uterine cervical cancer. A >60-mm tumor was detected at the uterine cervix, and no distal metastasis or swallowing of lymph nodes was revealed by magnetic resonance imaging and a computed tomography scan. The cervical cancer stage FIGO Ib2 was diagnosed, and a radical hysterectomy was performed for this malignant tumor. The in situ hybridization for EBV was negative. HVP infection was strongly suspected because the squamous cell carcinoma was observed macroscopically in the uterine cervix. The prognosis of uterine lymphoepithelioma-like carcinoma is thought to be better than those of other cervical cancer types, but careful follow-up at fixed intervals is recommended. The patient has been followed up for 4 months since her surgery, and no evidence of recurrence has been detected.