RESUMO
In this study, to search for novel preeclampsia (PE) biomarkers, we focused on microRNA expression and function in the human placenta complicated with PE. By comprehensive analyses of microRNA expression, we identified 22 microRNAs significantly upregulated in preeclamptic placentas, 5 of which were predicted in silico to commonly target the mRNA encoding hydroxysteroid (17-ß) dehydrogenase 1 (HSD17B1), a steroidogenetic enzyme expressed predominantly in the placenta. In vivo HSD17B1 expression, at both the mRNA and protein levels, was significantly decreased in preeclamptic placentas. Of these microRNAs, miR-210 and miR-518c were experimentally validated to target HSD17B1 by luciferase assay, real-time PCR, and ELISA. Furthermore, we found that plasma HSD17B1 protein levels in preeclamptic pregnant women reflected the decrease of its placental expression. Moreover, a prospective cohort study of plasma HSD17B1 revealed a significant reduction of plasma HSD17B1 levels in pregnant women at 20 to 23 and 27 to 30 weeks of gestation before PE onset compared with those with normal pregnancies. The sensitivities/specificities for predicting PE at 20 to 23 and 27 to 30 weeks of gestation were 0.75/0.67 (cutoff value=21.9 ng/mL) and 0.88/0.51 (cutoff value=30.5 ng/mL), and the odds ratios were 6.09 (95% CI: 2.35-15.77) and 7.83 (95% CI: 1.70-36.14), respectively. We conclude that HSD17B1 is dysregulated by miR-210 and miR-518c that are aberrantly expressed in preeclamptic placenta and that reducing plasma level of HSD17B1 precedes the onset of PE and is a potential prognostic factor for PE.
Assuntos
Estradiol Desidrogenases/metabolismo , MicroRNAs/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/metabolismo , Complicações Cardiovasculares na Gravidez/diagnóstico , Complicações Cardiovasculares na Gravidez/metabolismo , Adulto , Biomarcadores/metabolismo , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Hipóxia/metabolismo , Placenta/citologia , Valor Preditivo dos Testes , Gravidez , Segundo Trimestre da Gravidez/metabolismo , Terceiro Trimestre da Gravidez/metabolismo , Prognóstico , Estudos Prospectivos , Curva ROC , Trofoblastos/citologia , Trofoblastos/metabolismoRESUMO
In this study, we performed small RNA library sequencing using human placental tissues to identify placenta-specific miRNAs. We also tested the hypothesis that human chorionic villi could secrete miRNAs extracellularly via exosomes, which in turn enter into maternal circulation. By small RNA library sequencing, most placenta-specific miRNAs (e.g., MIR517A) were linked to a miRNA cluster on chromosome 19. The miRNA cluster genes were differentially expressed in placental development. Subsequent validation by real-time PCR and in situ hybridization revealed that villous trophoblasts express placenta-specific miRNAs. The analysis of small RNA libraries from the blood plasma showed that the placenta-specific miRNAs are abundant in the plasma of pregnant women. By real-time PCR, we confirmed the rapid clearance of the placenta-specific miRNAs from the plasma after delivery, indicating that such miRNAs enter into maternal circulation. By using the trophoblast cell line BeWo in culture, we demonstrated that miRNAs are indeed extracellularly released via exosomes. Taken together, our findings suggest that miRNAs are exported from the human placental syncytiotrophoblast into maternal circulation, where they could target maternal tissues. Finally, to address the biological functions of placenta-specific miRNAs, we performed a proteome analysis of BeWo cells transfected with MIR517A. Bioinformatic analysis suggests that this miRNA is possibly involved in tumor necrosis factor-mediated signaling. Our data provide important insights into miRNA biology of the human placenta.
Assuntos
Vilosidades Coriônicas/metabolismo , Exossomos/metabolismo , MicroRNAs/metabolismo , Gravidez/sangue , Trofoblastos/metabolismo , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Hibridização In Situ , Reação em Cadeia da Polimerase , Proteômica , Análise de Sequência de RNARESUMO
More than 700 microRNAs (miRNAs) have been cloned, and the functions of these molecules in developmental timing, cell proliferation, and cancer have been investigated widely. MiRNAs are analyzed with Northern blot and sequential colony evaluation; however, reverse transcription-polymerase chain reaction (RT-PCR)-based miRNA assay remains to be developed. In this report, we describe improved real-time RT-PCR methods using specific or non-specific RT primer for the semi-quantitative analysis of miRNA expression. The use of the new methods in a model study revealed differential expression of miRNA-1 (miR-1) and miR-124 in mouse organs. Specifically, our methods revealed that miR-124 concentrations in the mouse central nervous system (CNS; cerebral cortex, cerebellum, and spinal cord) were more than 100 times those in other organs. By contrast, miR-1 expression in the CNS was 100-1000 times lower than that in skeletal muscle and heart. Furthermore, we revealed anatomically regional differences in miR-124 expression within the CNS: expression ratios versus the cerebral cortex were 60.7% for the cerebellum and 35.4% for the spinal cord. These results suggest that our RT-PCR-based methods would be a powerful tool for studies of miRNA expression that is associated with various neural events.
Assuntos
Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica/genética , MicroRNAs/análise , MicroRNAs/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Sistema Nervoso Central/anatomia & histologia , Cerebelo/anatomia & histologia , Cerebelo/metabolismo , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/metabolismo , Clonagem Molecular/métodos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Medula Espinal/anatomia & histologia , Medula Espinal/metabolismoRESUMO
It is generally believed that cell-to-cell cross-talk and signal transduction are mediated by cell surface molecules that play diverse and important regulatory roles in spermatogenesis and fertilization. Recently, we identified a novel plasma membrane-associated protein, TES101-reactive protein (TES101RP, or TEX101), on mouse testicular germ cells. In this study, we investigate Tex101 mRNA expression in the adult mouse testis using in situ hybridization, and we examine the fate of TEX101 during sperm transport by immunohistochemical and Western blot analyses. Tex101 mRNA was expressed in a stage-specific manner in spermatocytes and in step 1-9 spermatids of the testis, but not in spermatogonia. Although the TEX101 protein remained on the cell surfaces of step 10-16 spermatids and testicular sperm, it was shed from epididymal sperm located in the caput epididymidis. The results of this study provide additional information on germ cell-specific TEX101 expression during spermatogenesis and post-testicular sperm maturation.