550 MOLECULAR MEDICINE REPORTS 10: 550-554, 2014 MicroRNA-155 inhibits migration of trophoblast cells and contributes to the pathogenesis of severe preeclampsia by regulating endothelial nitric oxide synthase XUELAN LI, CHUNFANG LI, XIN DONG and WENLI GOU Department of Obstetrics and Gynaecology, The First Affiliated Hospital, Xi'an Jiao Tong University, Xi'an, Shaanxi 710061, P.R. China Received September 29, 2013; Accepted March 21, 2014 DOI: 10.3892/mmr.2014.2214 Abstract. The aim of the present study was to characterize of the placenta (4) and the generally accepted opinion that the the role of microRNA (miR)-155 in the pathogenesis of severe abnormal development of the placenta at an early stage of gesta- preeclampsia (PE). A total of 19 severe preeclampsic and 22 tion initiates this disease, it is important to elucidate the role normal placentas were collected to measure miR-155 and endo- of miRNA in the development of PE which may facilitate the thelial nitric oxide synthase (eNOS) expression using quantitative understanding of the pathogenesis of the disease. (q)PCR and western blot analysis. The results demonstrated a miRNA, a class of ~22-nucleotide-long non-protein coding significant increase in the levels of miR-155 and decreased eNOS RNAs, are able to regulate gene expression by binding to the expression in the severe preeclampsic placentas, as compared 3' untranslated region (UTR) of target gene messenger RNA with the normal controls. In order to examine the function of (mRNA), resulting in translational repression and/or mRNA miR-155 in the human placenta, the HTR8/Svneo cell line was degradation (5). It is generally considered that ~1/3 of all human transiently transfected with an miR-155 mimic or its inhibitor, genes may be regulated by miRNA (6), and that microRNA has anti-miR-155. It was confirmed that miR-155 may suppress a key role in cellular activities, including cell proliferation, apop- the expression of eNOS in HTR-8/SVneo cells. Furthermore, tosis and immune responses (7-9). Therefore, understanding the a transwell insert invasion assay demonstrated that miR-155 regulatory network of miRNAs has attracted noteable attention inhibited cell invasion in trophoblast cells, and the effect was in recent years. rescued by over expression of eNOS. The present study revealed Investigating the expression of miRNA and endothelial that miR-155 has a negative regulatory role in the migratory nitric oxide synthase (eNOS) in PE is important for the following behavior of HTR-8/SVneo cells via modulating eNOS. reasons: i) Aberrant expression of eNOS may reduce the migra- tory capabilities of trophoblast cells (10) and the migratory Introduction capability is the key for deep placentation, the compromise of which may result in a wide spectrum of obstetric diseases, Preeclampsia (PE) is a medical condition characterized by including PE (11); ii) ENOS may directly exacerbate the PE-like hypertension and proteinuria in pregnant females following phenotype by interfering with the endothelin system (12); iii) 20 weeks of gestation. It is the most common cause of fetal functional polymorphisms in eNOS that alter enzymatic activity morbidity and mortality, which has no curative therapeutic in vivo were reported to be associated with an increased risk strategy, except for delivery of the placenta (1). The association for PE (13); iv) miR-155 was demonstrated to be an essential between PE and aberrant microRNA (miRNA) expression in regulator of eNOS expression and endothelium-dependent placentas was first reported in 2007 (2) and a number of other vasorelaxation, and inhibition of miR-155 may restore eNOS similar findings have since been demonstrated (3). Considering expression and improve endothelial dysfunction (14,15). the previously reported role of miRNAs in the development Based on the aforementioned evidence, it was hypothesized that miR-155 may function as a regulator of trophoblast cell behavior by modulating eNOS. To examine this hypothesis, the effect of miR-155 on the proliferation and invasion of tropho- blast cells as well as the rescue effect of eNOS was investigated. Correspondence to: Dr Wenli Gou, Department of Obstetrics The expression pattern of miR-155 and eNOS in preeclampsic and Gynaecology, The First Affiliated Hospital, Xi'an Jiao Tong placentas in comparison with the normal controls was also University, 277 Yanta West Road, Xi'an, Shaanxi 710061, P.R. China E-mail: [email protected] examined. Key words: microRNA-155, endothelial nitric oxide synthase, Materials and methods preeclampsia Sample collection. The present study was performed with placenta tissues collected from 22 normal pregnant females LI et al: miR-155 INHIBITS TROPHOBLAST CELL MIGRATION VIA MODULATING eNOS 551 and 19 severe PE patients at The First Affiliated Hospital, (Xi'an fied and inserted into a pcDNA4.0 vector (Invitrogen Life Jiao Tong University, Xi'An, Shaanxi, China). The study was Technologies) to construct the eNOS expression plasmid approved by the Research Ethics Committees of the hospital (pcDNA4-eNOS). and written informed consent were obtained from all of the subjects. Normal pregnancy was defined as a previously and Cell survival and proliferation assay. Untreated or pre-treated currently normotensive female during pregnancy who deliv- cells were cultured in serum-free RPMI-1640 medium for ered a healthy neonate following 37 weeks of gestation. Severe three days. To examine the viability, cells were incubated in PE was define as a female patient without a history of hyper- RPMI-1640 medium supplemented with 10% fetal bovine tension presenting with systolic blood pressure ≥160 mmHg or serum in 6-well plates for 48 h and the cell number was counted diastolic blood pressure ≥110 mmHg on at least two occasions, using the trypan blue exclusion method. Dead cells were defined combined with significant proteinuria following 20 weeks of as those stained with trypan blue dye. The percentage of living gestation. The pregnant females who had renal disease, spon- cells was calculated by the association between the number of taneous abortion, gestational diabetes, fetal chromosomal or viable cells and the total number of cells counted. congenital abnormalities were excluded from the present study. Transwell invasion and migration assay. A transwell insert RNA extraction and quantitative polymerase chain reac- invasion assay was performed in 24 well fitted inserts with tion (qPCR). Total RNA was isolated from the cultured membranes (pore size, 8 mm; Costar, Cambridge, MA, USA). cells or placenta tissues using TRIzol reagent (Invitrogen As described previously (16), cell invasion was examined using Life Technologies, Carlsbad, CA, USA) following the a polycarbonate membrane cell culture insert (Costar, Corning manufacturer's instructions with few modifications. Inc., Corning, NY, USA) coated with growth factor reduced Reverse transcription and qPCR were conducted using Matrigel (BD Biosciences, Bedford, MA, USA). The cells the primer set: 5'-CTGTTAATGCTAATCGTGATAG-3' transfected with the control, miR-155 mimic, anti-miR-155 and 5'-GCAGGGTCCGAGGT-3' for miR-155 detec- or eNOS, were treated with 10 mg/ml mitomycinC (Sigma- tion; primer set: 5'-CTCGCTTCGGCAGCACA-3' and Aldrich, St. Louis, MO, USA) for 2 h and placed on top of the 5'-AACGCTTCACGAATTTGCG T-3' for U6 detection; wells at a density of 2.5x105 cells/well. For the ‘rescue experi- primer set: 5'-CCCTTCAGTGGCTGGTACAT-3' and ment’, pDNA4-eNOS or its control plasmid was transfected 5'-CACGATGGTGACTTTGGCTA-3' for eNOS detection, into the cells, respectively. Invaded cells on the lower surface and the SYBR Green real time detection system (Bio-Rad, of the membrane were stained with crystal violet and counted. Hercules, CA, USA), respectively. U6, an internal control, was used to normalize miR-155 levels. Statistical analysis. The results of the transwell insert invasion assay, proliferation assay, qPCR, western blotting, densitometry Western blot analysis. Cell lysates were loaded onto an analysis and clinical characteristics of the severe PE group and SDS-polyacrylamide gel for electrophoresis and then the control group, were performed in triplicate and expressed as the proteins were transferred onto a polyvinylidene fluoride mean ± standard error of the mean. Student's t-test or one-way membrane (Beyotime Institute of Biotechnology, Haimen, analysis of variance was used to analyze the differences. The China), which was blocked with TBST (10 mM Tris-Cl pH 8.0, statistical analysis was conducted by SPSS 17.0 software (SPSS, 150 mM NaCl and 0.05% Tween-20) containing 5% non-fat dry Inc., Chicago, IL, USA). P<0.05 was considered to indicate a milk powder at room temperature for 1 h. The membrane was statistically significant difference. then incubated with the rabbit anti-eNOS polyclonal antibody (1:1500; Abcam, Cambridge, UK) at 4˚C overnight, followed by Results incubation with horseradish peroxidase-anti-rabbit secondary antibody (Abcam) at room temperature for 1 h. Chemical fluo- Expression levels of miR-155 and eNOS in preeclampsic rescence was detected using an enhanced chemiluminescence placentas. The expression levels of miR-155 and eNOS in kit (Amersham Biosciences, Piscataway, NJ, USA) according the placentas were measured and compared between the to the manufacturer's instructions. The target bands were two groups comprising of 19 patients with severe PE and 22 densitometrically analyzed and normalized by β-actin. healthy pregnant females. No significant differences