RESUMEN
It is very difficult to gain a better understanding of the events in human pregnancy that occur during and just after implantation because such pregnancies are not yet clinically detectable. Animal models of human placentation are inadequate. In vitro models that utilize immortalized cell lines and cells derived from trophoblast cancers have multiple limitations. Primary cell and tissue cultures often have limited lifespans and cannot be obtained from the peri-implantation period. We present here two contemporary models of human peri-implantation placental development: extended blastocyst culture and stem-cell derived trophoblast culture. We discuss current research efforts that employ these models and how such models might be used in the future to study the "black box" stage of human pregnancy.
Asunto(s)
Blastocisto/metabolismo , Implantación del Embrión , Placentación , Trofoblastos/metabolismo , Femenino , Humanos , Embarazo , Células Madre/metabolismoRESUMEN
The chromatin associated transcription factor HMGA2 is a downstream target of let-7 miRNAs and binds to chromatin to regulate gene expression. Inhibition of let-7 miRNAs by RNA-binding proteins LIN28A and LIN28B is necessary during early embryogenesis to ensure stable expression of HMGA2. In addition to LIN28, HMGA2 is regulated by a BRCA1/ZNF350/CtIP repressor complex. In normal tissues, the BRCA1/ZNF350/CtIP complex binds to the HMGA2 promoter to prevent transcription. However, in many cancers the oncomiR miR-182 targets BRCA1, preventing BRCA1 translation and allowing for increased HMGA2. Little is known about the regulation of HMGA2 during early placental development; therefore, we hypothesized that both LIN28 and BRCA1 can regulate HMGA2 in placental cells. Using siRNA and CRISPR gene editing techniques, we found that knockdowns of both LIN28A and LIN28B increase HMGA2 levels in ACH-3P cells. These cells also demonstrated deficiencies in cell differentiation, seemingly differentiating solely towards the syncytiotrophoblast sublineage, secreting higher amounts of hCG, and displaying upregulated ERVW-1. Additionally, we found that a knockout of both LIN28A and LIN28B caused a significant increase of miR-182 and a decrease in BRCA1 allowing HMGA2 mRNA levels to increase and protein levels to remain the same. Using chromatin immunoprecipitation, we saw binding of the BRCA1 repressor complex to HMGA2. We also saw a decrease in binding to HMGA2's promoter in the LIN28A/B knockout cells. These findings suggest a novel role for BRCA1 during early human placental development.
Asunto(s)
Proteína BRCA1/fisiología , Proteína HMGA2/genética , Placenta/metabolismo , Proteínas de Unión al ARN/fisiología , Proteína BRCA1/genética , Células Cultivadas , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Proteína HMGA2/metabolismo , Humanos , Placenta/patología , Placentación/genética , Embarazo , Primer Trimestre del Embarazo/genética , Primer Trimestre del Embarazo/metabolismo , Proteínas de Unión al ARN/genética , Trofoblastos/metabolismo , Trofoblastos/patologíaRESUMEN
Chlorpromazine altered the mechanical and electrical activity of the isolated perfused guinea pig heart. While its effects on coronary flow were variable, chlorpromazine increased resting diastolic isometric tension and decreased the isometric systolic tension developed by spontaneously beating hearts. Heart rate was also decreased. The drug depressed conduction through the His-Purkinje system and ventricular muscle to a greater extent than it did atrial conduction time and AV nodal conduction time. From our data, we concluded that the greatest depressant action of chlorpromazine on the electrical activity of the isolated perfused guinea pig heart occurred within the specialized ventricular conduction system and ventricular muscle.