CytoScan SRB cell cytotoxicity assay kit was purchased from G-biosciences (St. The miRNA and mRNA kits with appropriate real-time reagents and miRNEasy were purchased from Qiagen (Valencia, CA).
The villous 3A cytotrophoblast first trimester placental cell line (CRL-1584) was purchased from American Type Culture Collection (ATCC) (Manassas, VA). In the present study, we investigate the impact of oxidative stress on miRNA and mRNA expression profiles, with specific emphasis on mRNAs of genes known to be associated with PE, in villous first trimester 3A cytotrophoblast cell line. The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in PE is not fully elucidated. Given the reported changes in miRNAs observed in PE, early detection, protection, and regulation of miRNAs may help decrease the impact of this disease. Dysregulated expression of a number of other miRNAs, such as the oncogenic miR-17 family, occurs in preeclamptic placentas but the temporal change is unknown. In addition, miR-210, which regulates hypoxia-inducible factor 1, is commonly found to be upregulated in PE women. For example, dysregulation of C19MC expression is seen in preeclamptic placentas. Women with PE, eclampsia, and HELLP (Hemolysis, Elevated Liver enzymes, Low Platelets) syndrome have significant alterations in the placental miRNA profile. Expression of C14MC decreases while expression of C19MC increases as pregnancy progresses. This pattern includes two large imprinted miRNA clusters, one located at chromosome 19q13.41 (C19MC) and another at 14q32 (C14MC). There is a temporal and placental-specific pattern of miRNA expression. MicroRNAs (miRNAs) are short 20-22 single strand regulatory RNAs that function by inhibiting translation of their targets or promoting target RNA degradation. With disease risk on the rise but no effective way to predict its development, it is crucial to understand the early etiologic mechanisms of PE to develop early detection biomarkers and possible preventive measures for the disease. The age-adjusted incidence of PE in the United States increased almost 25% from 1987 to 2004. Therefore, the relationship between oxidative stress and PE is a vicious cycle where increased oxidative stress can induce PE and the occurrence of PE also exacerbates oxidative stress. Oxidative stress can induce endothelial dysfunction and vasoconstriction. Moreover, evidence supports an early increase in oxidative stress in the placenta by the end of the first trimester before the clinical development of PE. H 2O 2 levels are also significantly higher in preeclamptic placentas compared to normotensive placentas at term. Superoxide anions generated endogenously or exogenously can rapidly be converted to hydrogen peroxide (H 2O 2) and studies showed a significant elevation of H 2O 2 levels in the bloodstream of women with PE. PE is thought to result from a combination of many factors including shallow trophoblast invasion, failed maternal spiral artery remodeling, and an increase in endothelial activation leading to placental hypoxia, reactive oxygen species (ROS) generation, apoptosis and necrosis of trophoblasts, and systemic activation of inflammatory processes in the mother. It is a medical condition characterized by de novo hypertension in pregnancy (diastolic > 90 mm Hg) after 20-week gestation with high proteinuria (>300 mg). Preeclampsia (PE), which affects 3% to 8% of pregnant women, remains a major cause of short- and long-term maternal and neonatal morbidity and mortality.
In conclusion, short-term exposure of villous first trimester trophoblasts to low concentrations of H 2O 2 significantly alters miRNA profile and expression of genes implicated in placental development. Exposure to H 2O 2 altered mRNA expression of 22 out of 84 key genes involved in dysregulation of placental development. Tool for annotations of microRNAs (TAM) analysis indicated that these altered miRNAs fall into 43 clusters and 34 families, with 41 functions identified. Short-term exposure of 3A cells to low concentration of H 2O 2 (5% of IC 50) significantly altered miRNA profile as evidenced by significant changes in 195 out of 595 evaluable miRNAs. H 2O 2 exerted a concentration- and time-dependent cytotoxicity on 3A trophoblast cells. RNA was extracted after 4 h exposure to H 2O 2 for miRNA and gene expression profiling. Cytotoxicity, determined using the SRB assay, was used to calculate the IC 50 of H 2O 2. We investigated the impact of oxidative stress on miRNAs and mRNA expression profiles of genes associated with PE in villous 3A first trimester trophoblast cells exposed to H 2O 2 at 12 different concentrations (0-1 mM) for 0.5, 4, 24, and 48 h. The relationship between oxidative stress and miRNA changes in placenta as a potential mechanism involved in preeclampsia (PE) is not fully elucidated.