Systematic Analysis of Hsa-Mir-363 Gene Overexpression Pattern in Endometrial Stromal Cells
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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 42: 2793-2800, 2018 Systematic analysis of hsa-miR-363 gene overexpression pattern in endometrial stromal cells WENQU LI1*, XUEMEI FAN1*, MI ZhANG1*, LEI hUANG2, ShANShAN LV3, LUyU WANG4, yING WU1, ChENChENG DAI1, JUAN XU1, PENGFEI XU1, ZIYI FU1, 1 1 XUEMEI JIA and XIAOYAN SHI 1Department of Gynecology, Affiliated Obstetrics and Gynecology hospital of Nanjing Medical University, Nanjing Maternal and Child health Care hospital, Nanjing, Jiangsu 210004; 2Department of General Surgery, The Affiliated Drum Tower hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008; 3Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210029; 4Soochow University, Soochow, Jiangsu 215004, P.R. China Received March 9, 2016; Accepted January 18, 2018 DOI: 10.3892/ijmm.2018.3840 Abstract. Endometriosis is a benign disease, but has invasion which could expand the insights into the miR-363 function and and metastasis characteristics similar to malignant tumors. molecular mechanisms in endometriosis. Clinically, it is a difficult problem of gynecological clinical treatment for its high recurrence rate. It has been confirmed Introduction that miR-363 was downregulated in endometriosis tissues and miR-363 overexpression inhibited the invasion ability of endo- Endometriosis is a benign gynecological disease, which is metrial stromal cells (ESCs). In order to explore the potential characterized by the presence of functional endometrial mechanism of miR-363-reduced ESC migration and invasion glands and stroma outside the uterine cavity. The character- progression, we sought to demonstrate the targeted mRNA istics of endometriosis, for example invasion and metastasis expression levels of miR-363 through microarray, and performed ability, are similar to the malignant tumors, and endometriosis cluster analysis to identify potential functions of these targeted affects up to 20% of women of reproductive age who suffer genes in ESCs. The wound migration assay showed that there from dysmenorrheal, chronic pelvic pain, subfertility and was an observable trend of cell migration potential decrease dyspareunia (1). It decreases the life quality of the affe cted after transfection with hsa-miR-363. The qRT-PCR result women, and the clinical prognosis of endometriosis is still showed that compared to miR-363 negative control cell group, poor due to the high recurrence rate. Present medications only miR-363 was upregulated 3,264.58-fold after miR-363 lentiviral ameliorate symptoms and cause significant side effects such transfection in miR-363 mimics group. The microarray data as osteoporosis at the same time. Therefore, novel therapeutic showed that compared to ESCs miR-363 negative control cell agents that suppress endometriotic lesion establishment and group, 249 genes were upregulated in ESCs miR-363 mimics growth to reduce the high burden conferred by this disease are cells group, and 139 genes were downregulated. Gene Ontology required (2). The pathogenesis of endometriosis is not fully analysis and the pathway analysis data demonstrated that these characterized. Sampson's theory of retrograde menstruation, target genes are mainly involved in cell migration, cell adhesion first described in 1927, remains the most accepted theory for and invasion, proliferation, apoptosis, alteration of endometrial the origin of this disease (3). According to Sampson's theory, cells and some related signaling pathways. Our study explored endometrial stromal cells (ESCs) could escape from the the gene expression pattern after miR-363 overexpression, uterine cavity and adhere to the peritoneal surface to mediate the invasion and metastasis to the target organs (4). In the last decade, we found that various cellular physi- ological and pathological processes (including cell cycle, Correspondence to: Dr Xuemei Jia or Dr Xiaoyan Shi, Department metabolism, cell-cell communication, cell survival and of Gynecology, Affiliated Obstetrics and Gynecology hospital of apoptosis, immune responses and oncogenesis) were induced Nanjing Medical University, Nanjing Maternal and Child health or promoted due to dozens of genes targeted by a single Care hospital, 123 Tianfeixiang street, Mochou Road, Nanjing, miRNA through the perfect or partial base pairing with the Jiangsu 210004, P.R. China 3'-untranslated region (3'-UTR) of the target mRNAs (5,6). E-mail: [email protected] Additionally, studies suggest that miRNAs play an important E-mail: [email protected] role in endometriosis (7-9). For example, miR-210 can promote *Contributed eqully the proliferation, resistance to apoptosis and VEGF production of endometriosis through STAT3 activation (6). It can be seen Key words: gene microarray, endometriosis, endometrial stromal that miRNAs have important roles in pathological processes cell, miR-363 of endometriosis, though the mechanisms responsible for endometriosis remain largely unknown. 2794 LI et al: gene expression pattern in endometrial stromal cells with hsa-miR-363 overexpression miRNAs belong to a large class of small, single-stranded, after miRNA-363 overexpression (11). After ESC cells were non-coding RNAs, molecules approximately 18 to 25 nucleo- tran sfected with hsa-miR-363 mimics or counterpart negative tides in length. They can bind to mRNA, preventing translation control lentivirus vector for 72 h, respectively, total RNA was and accelerating mRNA deadenylation and subsequent degra- isolated using TRIzol reagent (Invitrogen Life Technologies, dation, thus having a gene silencing effect (10). In a previous Carlsbad, CA, USA) according to the manufacturer's instruc- study (ref. ?), miR-363 was found significantly downregulated tions. Total RNA was extracted using Qiagen RNeasy Mini kit in endometriosis tissues than in normal tissues by microRNA and RNase-free DNase I (both from Qiagen Gmbh, hilden, chip technology. We supposed that miR-363 plays a signifi- Germany) according to the manufacturer's instructions. The cant role in endometriosis mediating target genes. However, purity and concentration of total RNA were measured by the the mechanism of miR-363 in endometriosis process remains UV absorbance at 260 and 280 nm (260/280 nm). Global gene largely unexplored. expression was detected using Affymetrix GeneChip human In this study, we sought to determine the expression pattern Gene 1.0 ST array (Affymetrix, Inc., Santa Clara, CA, USA). of miR-363 targeted mRNAs by sequence-specific base About 500 ng of total RNA were employed in each experiment. pairing on the 3'-UTR, and explored their potential functions Briefly, total RNA of each sample was amplified using the and pathways, which are involved in the biological behavior Ambion WT Expression kit (Applied Biosystems, Foster City, of human endometriosis. This study may afford new clues for CA, USA) according to the manufacturer's instructions. understanding the pathogenesis of endometriosis, and miR-363 Approximately 5.5 µg of generated cDNA was fragmented may serve as a therapeutic option in endometriosis treatment. and labeled using GeneChip3 WT Terminal Labeling kit and Control kit (both from Affymetrix, Inc.). Labeled cDNA Materials and methods target was hybridized to the Affymetrix GeneChip human Gene 1.0 ST array using GeneChip3 hybridization, Wash, and Cell lines and cell culture. The human ESC line was obtained Stain kit (Affymetrix, Inc.) at 45˚C for 16 h, then hybridized from the American Type Culture Collection (ATCC; Manassas, arrays were washed and stained on the Affymetrix GeneChip VA, USA). Cells were cultured in Dulbecco's modified Eagle's Command Console (AGCC; Affymetrix, Inc.), and scanned medium (DMEM) supplemented with 10% (v/v) fetal bovine on a GeneChip2 Scanner 3000 7G (Affymetrix, Inc.). The serum (FBS), 100 U/ml penicillin and 100 U/ml streptomycin acqui red a r ray i mages were a na lyzed by A f f ymet r i x GeneCh ip (all from Gibco Life Technologies, Carlsbad, CA, USA) at Operating Software (GCOS). Affymetrix expression console 37˚C in a humidified atmosphere containing 5% CO2. software was used to perform quantile normalization and subsequent data processing. Data transformation was applied Lentiviral transfection of ESC cells. ESC cells were seeded to set all negative raw values at 1.0, followed by quantile into 6-well plates and incubated overnight, reached 60-80% normalization. A filter on low gene expression was used confluence before transfection. Transient transfection of to keep only the probes expressed in at least one sample. ESC with mimics for hsa-miR-363 or counterpart negative Differentially regulated genes were identified via fold change control used lentiviral vector (LV-3-GFP-PURO; Shanghai filtering. GenePharma Co., Ltd., Shanghai, China) according to the manufacturer's instructions. The experiments were classified Real-time PCR quantification of miR-363 and mRNAs. Total into two groups, hsa-miR-363 (or miR-363 mimics) group, RNA was isolated from cultured cells using TRIzol reagent and negative control (NC) group. After 24 h post-transfection, (Invitrogen Life Technologies) according to the manufacturer's the medium was removed and the fresh culture medium with instructions. miR-363 was reverse transcribed to cDNA by using 10% FBS was injected. The transfection efficiency of lentiviral TaqMan MicroRNA Reverse Transcription kit with specific vector was observed by fluorescence microscope at 48 and probes according to the TaqMan MicroRNA assay protocol 72 h after transfection. (both from Applied Biosystems), and human