RESUMEN
Based on our experiences in bile acid profiling, this work developed and validated a liquid chromatography electrospray ionization tandem mass spectrometry method to separate endogenous bile acid isomers and quantitatively determine ursodeoxycholic acid (UDCA), glycoursodeoxycholic acid (GUDCA) and tauroursodeoxycholic acid (TUDCA) in human plasma. The separation was performed on a CORTECS C18 column with the mobile phase consisting of 1.0 mM ammonium acetate and acetonitrile-methanol (80:20, v/v). UDCA, GUDCA and TUDCA were detected in the negative mode on a triple-quadrupole mass spectrometer at the ion transitions of m/z 391 > 391, m/z 448 > 74, m/z 498 > 80, respectively. Phosphate buffer was employed as the surrogate matrix to establish the isotope internal standard corrected calibration curves of analytes. The background-method with a linearity range of 10-200 ng/mL was partially validated to determine the endogenous levels of analytes in blank human plasma, which was incorporated into the validation of bioequivalence-method with a linearity range of 50-10000 ng/mL. The bioequivalence (BE)-method was fully validated with special focus on matrix effects, which have been critically evaluated using the precision and accuracy of quality control samples prepared from the blank human plasma of 12 individuals. It is disclosed for the first time that the BE results of UDCA formulation may yield false results when the method is insufficient to separate UDCA from isoursodeoxycholic acid, a microbial metabolite of both endogenous and exogenous UDCA. The present method has established a milestone for the evaluation of UDCA formulations and is expected to provide a valuable reference for the bioanalytical development of endogenous medicinal products.
Asunto(s)
Ácidos y Sales Biliares , Ácido Ursodesoxicólico , Humanos , Equivalencia Terapéutica , Cromatografía Liquida/métodos , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodos , Cromatografía Líquida de Alta Presión/métodosRESUMEN
RNA-binding proteins (RBPs) are well-known to bind RNA via a set of RNA-binding domains (RBDs) and determine the fate and function of their RNA targets; inversely, some RBPs, in certain cases, may be modulated by the bound RNAs rather than regulate their RNA partners. Current proteome-wide studies reveal that almost half of RBPs have no canonical RBDs, and the discovery of tens of thousands of noncoding RNAs (ncRNAs), especially those with the size larger than 200 nt (namely long noncoding RNAs, lncRNAs), makes the crosstalk between RBPs and RNAs more complicated. It is clear that macromolecular complexes formed by RBP and RNA are not only a form of existence of their RBP and RNA components in cells, but also represent a functional entity through which those RBPs and regulatory ncRNAs participate in the construction of regulatory networks in organism. In this review, we summarize the multidimensional crosstalk between RBPs and ncRNAs in cancer and discuss how RBPs achieve their function via the bound ncRNAs in different aspects of gene expression as well as how RBPs direct modification and processing of ncRNAs, in order to better understand tumor biology and provide new insights into development of strategies for cancer therapy and early detection.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias/patología , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/metabolismo , Animales , Biomarcadores de Tumor/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Long noncoding RNAs (lncRNAs) participate in various biological processes such as apoptosis. The function of lncRNAs is closely correlated with their localization within the cell. While regulatory potential of many lncRNAs has been revealed at specific subcellular location, the biological significance of discrete distribution of an lncRNA in different cellular compartments remains largely unexplored. Here, we identified an lncRNA antisense to the pro-apoptotic gene PYCARD, named PYCARD-AS1, which exhibits a dual nuclear and cytoplasmic distribution and is required for the PYCARD silencing in breast cancer cells. The PYCARD-regulated apoptosis is controlled by PYCARD-AS1; moreover, PYCARD-AS1 regulates apoptosis in a PYCARD-dependent manner, indicating that PYCARD is a critical downstream target of PYCARD-AS1. Mechanistically, PYCARD-AS1 can localize to the PYCARD promoter, where it facilitates DNA methylation and H3K9me2 modification by recruiting the chromatin-suppressor proteins DNMT1 and G9a. Moreover, PYCARD-AS1 and PYCARD mRNA can interact with each other via their 5' overlapping region, leading to inhibition of ribosome assembly in the cytoplasm for PYCARD translation. This study reveals a mechanism whereby an lncRNA works at different cellular compartments to regulate the pro-apoptotic gene PYCARD at both the epigenetic and translational levels, contributing to the PYCARD-regulated apoptosis, and also sheds new light on the role of discretely distributed lncRNAs in diverse biological processes.