RESUMEN
BACKGROUND: The protein plasminogen activator inhibitor-1 (PAI-1), an inhibitor specific for urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA), has been shown to have a key role in cancer metastases. Currently, it is unknown as to whether the exocellular inhibition of PAI-1 can inhibit the migration of cancer cells. METHODS: By fusing the mutated serine protease domain (SPD) of uPA and human serum albumin (HSA), PAItrap3, a protein that traps PAI-1, was synthesized and experiments were conducted to determine if exocellular PAItrap3 attenuates PAI-1-induced cancer cell migration in vitro. RESULTS: PAItrap3 (0.8 µM) significantly inhibited the motility of MCF-7, MDA-MB-231, HeLa and 4T1 cancer cells, by 90%, 50%, 30% and 20%, respectively, without significantly altering their proliferation. The PAI-1-induced rearrangement of F-actin was significantly inhibited by PAItrap3, which produced a decrease in the number of cell protrusions by at least 20%. CONCLUSIONS: In vitro, PAItrap3 inhibited PAI-1-induced cancer cell migration, mainly through inhibiting the rearrangement of F-actin. Overall, these results, provided they can be extrapolated to humans, suggest that the PAItrap3 protein could be used as an exocellular inhibitor to attenuate cancer metastases.
Asunto(s)
Actinas/genética , Movimiento Celular/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/farmacología , Inhibidor de Proteína C/farmacología , Actinas/antagonistas & inhibidores , Actinas/metabolismo , Sitios de Unión , Línea Celular , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HeLa , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Células MCF-7 , Oligopéptidos/genética , Oligopéptidos/metabolismo , Pichia/genética , Pichia/metabolismo , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Unión Proteica , Inhibidor de Proteína C/química , Inhibidor de Proteína C/genética , Inhibidor de Proteína C/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Circulating miR-195-5p has been proposed as a promising peripheral biomarker for the diagnosis, prognosis and severity assessment of various diseases. However, the demand for its sensitive and convenient quantification has not been met yet. Herein, we proposed a one-pot isothermal approach, in which the target signal acquisition, amplification and conversion (fluorescence read-out) system was integrated by a triple strand displacement amplification (SDA) cascade. Using this triple SDA strategy, miR-195-5p can be at least detected at 1 aM, and the linear dynamic range (from 100 aM to 1 pM) is wide enough to meet the detection needs of clinical miRNA level. A proof-of-principle study, using this novel methodology to directly analyze the spiking serum samples with different levels of miR-195-5p, demonstrated the potential of circulating miR-195-5p detection for clinical point-of-care assay. This one-pot isothermal triple SDA approach, we believe, will be a simple and feasible tool for ultrasensitive quantification of circulating miR-195-5p, and may promote the wide application of this potential biomarker in non-invasive clinical diagnosis.
Asunto(s)
MicroARNs , Bioensayo , Biomarcadores , Humanos , MicroARNs/sangre , MicroARNs/genética , Pruebas en el Punto de AtenciónRESUMEN
AIMS: The study aims to investigate the effect of plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of fibrinolytic process, on blood glucose in type 2 diabetes mellitus (T2DM) and its mechanism. MATERIALS AND METHODS: We developed a highly potent and highly specific PAI-1 inhibitor, named PAItrap3, based on the inactivated urokinase. Meanwhile, a single point mutation of PAItrap3 (i.e., PAItrapNC) was parallelly prepared as negative control. PAItrap3 was intravenously injected into type 2 diabetic (T2D) mice and its effect on metabolic system was evaluated by measuring the levels of blood glucose, PAI-1, and tumor necrosis factor alpha (TNF-α) in T2D mice. KEY FINDINGS: PAItrap3 significantly reduced the high blood glucose level and PAI-1 level in streptozotocin-induced T2D mice. PAItrapNC did not have any hypoglycemic effect at all on T2D mice. Mechanistically, both PAI-1 and TNF-α levels were attenuated by the administration of PAItrap3. In addition, we observed that PAItrap3 reduced the amount of fat droplets in adipocytes. SIGNIFICANCE: These findings provide clear evidence for PAI-1 to participate in inflammation and obesity mediated hyperglycemia, and open up a new prospect for the treatment of T2DM by PAI-1 inhibition.