Análisis global de la expresión de genes de lenguado (Solea senegalensis) y de ratón (Mus musculus) mediante genotecas substractivas, microchips de DNA y qRT-PCR

Abstract

La obtención de genotecas substrativas en S. senegalensis permitió la identificación de genes diferencialmente expresados en respuesta a CuSO4. Para ello se combinaron a partes iguales RNAs procedentes de hígado de animales expuestos durante 6 y 24 horas. Se secuenciaron 229 clones, y los productos de las ESTs se identificaron mediante comparación con las bases de datos. De este modo, se detectaron 89 ESTs únicas, 78 de las cuales pudieron ser identificadas. Fundamentalmente, los genes diferencialmente expresados estuvieron relacionados con la respuesta de fase aguda. Adicionalmente, mediante esta técnica, se obtuvo la secuencia parcial de los 229 clones, lo que supone un avance considerable en el conocimiento genómico (de acceso público) del lenguado senegalés. El microchip desarrollado para la platija europea P. flesus (plataforma GENIPOL) resultó una herramienta muy útil para analizar la expresión transcripcional de S. senegalensis. Se estudió la respuesta hepática de lenguados expuestos durante 6 y 24 horas a CuSO4 o LPS. El análisis funcional de los resultados (software Blast2GO) permitió identificar genes de respuesta específica a CuSO4 (relacionados con las uniones celulares), a LPS (glutatión transferasas, respuesta inmune), o a ambos tratamientos (respuesta inmune, enzimas digestivas, unión a proteínas mal plegadas, transporte intracelular y secreción, y proteasoma). Asimismo, aunque este microchip se construyó con ESTs procedentes de hígado de platija, resultó válido para analizar la respuesta transcripcional del riñón cefálico en el lenguado, discriminando entre genes que codifican transcritos con patrones específicos de expresión hepática o renal (e.g., transcritos que codifican proteínas de fase aguda vs transcritos relacionados con la división celular, y el recambio de proteínas, entre otros). Dada la importancia económica de S. senegalensis y el limitado conocimiento a nivel genómico que existe de este organismo, este estudio ha proporcionado resultados muy relevantes de carácter genético, inmunológico y toxicológico. El análisis global de la expresión transcripcional en hígado de ratón en respuesta a paraquat (2 horas, 30 mg/kg, aprox. la mitad de la LD50) se efectuó empleando una plataforma comercial de microchips (Agilent) donde está representado el genoma completo del ratón. El análisis funcional (softwares GOstat e IPA) de los 310 genes diferencialmente expresados que mostraron cambios en su expresión >1,5 veces mostró que éstos se relacionan fundamentalmente con la respuesta a estrés, el metabolismo de xenobióticos, el crecimiento y muerte celular, y el metabolismo de lípidos. Asimismo, la detección de genes de función desconocida (e.g. Arrdc3) constituye un punto de partida en el estudio de las funciones de las proteínas que codifican. La presencia en este microchip de varias sondas para algunos de los genes diferencialmente expresados demostró que el análisis pormenorizado de la posición de las distintas sondas en la secuencia de un gen puede contribuir a la identificación de transcritos alternativos del mismo. El efecto del silenciamiento del gen Prdx1 sobre la expresión transcripcional global se analizó en células de hepatocarcinoma de ratón Hepa 1-6 expuestas a 30 μM de CdCl2 durante 2 horas. En las condiciones estudiadas, Prdx1 desempeña un papel más relevante como reguladora de la actividad celular que como proteína antioxidante, ya que los genes diferencialmente expresados se relacionaron principalmente con la presentación de antígenos, la señalización celular, y el crecimiento, proliferación y muerte celular. Prdx1 parece actuar como un oncogén, de manera que la inhibición de su expresión podría traducirse en la inducción de apoptosis. Se seleccionaron un total de 18 transcritos de lenguado y 17 de ratón en función de la relevancia fisiológica de sus productos y de la magnitud y especificidad de su respuesta para su cuantificación absoluta por RT-PCR en tiempo real, confirmándose, en general, los resultados obtenidos con las genotecas substractivas y con los microchips de DNA. Como cabía esperar, la cuantificación por qRT-PCR en tiempo real resultó ser una metodología más sensible ya que, en la mayoría de los casos, los cambios de expresión detectados con esta técnica fueron superiores a los obtenidos con los microchips. Asimismo se confirmó la importancia de la cuantificación absoluta de los transcritos frente a la relativa, que tiende a sobredimensionar las variaciones de los transcritos menos abundantes. La cuantificación absoluta de los transcritos seleccionados no se limitó a las muestras empleadas en al análisis global, sino que se amplió a muestras procedentes de diferentes individuos, órganos, tratamientos y tiempos de exposición, proporcionando nueva información acerca de los perfiles de expresión de los genes analizados y de su variabilidad interindividual. En general, cada transcrito presentó un perfil característico en lo concerniente tanto a sus niveles basales como a sus respuestas, distinguiendo entre patrones constitutivos, inducibles o reprimibles, de respuesta temprana o tardía, específicos o no del agente estresante etc., todo ello en función del órgano analizado.

Cells and tissues present characteristic patterns of gene expression, which become modified in stress situations with the aim of returning, as quickly as possible, to their normal conditions. The analysis of transcriptional expression patterns supplies valuable information on the coordinated functioning of the genes in response to different environmental, physiological and pathological variables. The current work has focused on two animal species of great relevance in the food, sanitary and biomedical areas. Thus, the Senegalese sole (Solea senegalensis) is a non model organism of enormous interest to aquaculture due to its great market expectations, but whose breeding is being made difficult by diverse pathologies and its susceptibility to different stress situations. For that reason, this species’ transcriptional expression in response to 2 mg/kg of CuSO4 (zoosanitary compound) or 25 mg/kg of LPS (a mimetic of bacterial infections) has been analyzed. On the other hand, the laboratory mouse (Mus musculus) is the model species most used in research due to its great physiological and genetic similarity to humans. Transcriptional expression studies have been carried out in M. musculus, both in vivo and in vitro, in response to oxidative stress given the relationship of this type of stress with many physiopathological processes, including cancer. The differential expression patterns and their functional analysis has been addressed from a multidisciplinary perspective, using different experimental and methodological approaches, such as substractive libraries, DNA microarrays, real time qRT-PCR and silencing of gene expression by RNA interference (RNAi). The construction of cDNA libraries by suppression substractive hybridization (SSH) constitutes a fundamental methodology in differential expression studies with non model species since it enables the identification of genes with no previous knowledge of their sequence. The DNA microarrays permit, in a single experiment, the analysis of the levels of thousands of transcripts, making them a high-throughput methodology in Functional Genomics studies. Moreover, the heterologous hybridization allows the use of microarrays manufactured from transcripts of one species to probe gene expression in another, related, species. Real time qRT-PCR has become a reference methodology for the detection and quantification of transcripts, and for validating the results obtained with other techniques like substractive libraries or microarrays. Since its discovery, the RNAi has revolutionized Functional Genomics studies. This methodology, nowadays a routine one and with multiple commercial options, is replacing more conventional methods such as gene disruption or the use of dominant-negative mutants, and it is being used, in combination with genome-wide techniques, for the identification of complex regulating pathways in numerous biological systems. The construction of substractive libraries in S. senegalensis permitted the identification of differentially expressed genes in response to CuSO4. For this, RNAs from the liver of animals exposed for 6 and 24 hours were combined in equal parts. 229 clones were sequenced and the products of the ESTs were identified by comparison with the databases. In this way, 89 unique ESTs were detected, 78 of which could be identified. Basically, the differentially expressed genes were related to the acute phase response. Additionally, by this technique, the partial sequence of the 229 clones was obtained, which represents a considerable advance in the genomic knowledge (for public access) of the Senegalese sole. The microarray developed for the European flounder P. flesus (GENIPOL platform) was a very useful tool for analyzing the transcriptional expression of S. senegalensis. The hepatic response of soles exposed for 6 and 24 hours to CuSO4 or LPS was studied. The functional analysis of the results (Blast2GO software) permitted the identification of response-specific genes to CuSO4 (related to cell junctions), to LPS (glutathione transferase, immune response), or to both treatments (immune response, digestive enzymes, unfolded protein binding, intracellular transport and secretion, and proteasome). Similarly, although this microarray was constructed with ESTs from flounder liver, it was valid for analyzing the transcriptional response of sole head kidney, discriminating between genes coding for transcripts with specific patterns of hepatic or renal expression (e.g. transcripts coding for acute phase proteins vs. transcripts related to cell division, and protein turnover, among others). Given the economic importance of S. senegalensis and the limited genomic knowledge of this organism, this study has provided results that might be highly relevant at a genetic, immunological and toxicological level. The global analysis of the transcriptional expression in mouse liver in response to paraquat (2 hours, 30 mg/kg., approx. half of the LD50) was performed by employing a commercial platform of microarrays (Agilent), in which the complete mouse genome is represented. The functional analysis (GOstat and IPA software) of the 310 differentially expressed genes which showed changes in their expression > 1-5 times revealed that they are mainly related to the response to stress, the metabolism of xenobiotics, cell growth and death, and lipid metabolism. Besides, the detection of genes of unknown function (e.g. Arrdc3) constitutes a starting point in the functional characterization of their products. The presence in this microarray of several probes for some of the differentially expressed genes demonstrated that the detailed analysis of the position of the different probes in the sequence of a gene can contribute to the identification of alternative spliced transcripts. The effect of silencing the Prdxl gene on the global transcriptional expression was analyzed in mouse hepatocarcinoma cells Hepa 1-6 exposed to 30 μM of CdCl2 for 2 hours. Under the studied conditions, Prdxl plays a more relevant role as a regulator of cell activity than as an antioxidant protein since the differentially expressed genes were mainly related to the presentation of antigens, cell signalling, and the growth, proliferation and cell death. Prdxl seems to act as an oncogene so that the inhibition of its expression could be translated into the induction of apoptosis. A total of 18 transcripts of sole and 17 of mouse were selected for absolute quantification by real time RT-PCR. This selection was based on the physiological relevance of their products and on the magnitude and specificity of their response. The qRT-PCR quantifications confirm, in general, the results obtained with the substractive libraries and the DNA microarrays. While microarray and qRT-PCR data corresponded qualitatively, the absolute quantification of transcripts was more sensitive as, in most cases, the magnitude of the changes detected by qRT-PCR was greater. Similarly, the relevance of the absolute quantification was highlighted when comparing with the conventional fold changes, which tends to overestimate the variations affecting to less abundant transcripts. The absolute quantification of the selected transcripts was not limited to the pooled samples used for global analysis but it was extended to samples coming from different individuals, organs, treatments and exposure times. In general, each transcript displayed a characteristic expression profile, distinguishing between constitutive or up-/down-regulated, early or late responsive, stressorspecific or not, etc., all this as a function of the organ analyzed.