Alteraciones de la expresión génica en la infección por SARS-CoV-2 en distintos modelos de estudio / Alterations of gene expression in SARS-CoV-2 infection in different study models

Objetivo: La pandemia por SARS-CoV-2 ha supuesto un auténtico reto para el mundo científico debido a la rápida transmisión y elevada mortalidad que produce este nuevo coronavirus. La enfermedad asociada se ha denominado COVID-19 y abarca desde casos asintomáticos hasta graves que evolucionan rápidamente a síndrome de distrés respiratorio agudo, alteraciones multisistémicas y la muerte. La comunidad científica ha aunado esfuerzos para tratar de conocer mejor el proceso fisiopatológico de la infección con la intención de combatir de forma más eficaz la enfermedad. En este trabajo presentamos un estudio para conocer las alteraciones de la expresión génica provocadas por la infección. Metodología: Se han usado tres modelos de estudio distintos: cultivos de células epiteliales bronquiales, organoides de las vías respiratorias y muestras obtenidas de autopsias en pacientes, con y sin infección por SARS-CoV-2. Se han analizado los perfiles de expresión alterados por la infección en cada modelo, así como las categorías funcionales enriquecidas. Resultados: Solo 4 genes son comunes en los tres tipos de modelos de estudio, siendo el modelo de autopsias el más dispar. Dentro de los genes comunes en los modelos de cultivo celular y organoide de pulmón encontramos funciones relacionadas con procesos inflamatorios. Conclusiones: Los estudios in vitro son un buen modelo para tener una foto fija de las alteraciones en los patrones de infección, mientras que las autopsias no son un buen modelo debido al sesgo provocado por la necrosis. (AU)

Short summary: Three different study models have been used to study the gene expression profiles produced by SARS-CoV-2: bronchial epithelial cell cultures, airway organoids, and autopsy samples from patients with and without SARS-infection. CoV-2. Basis: The SARS-CoV-2 pandemic has been a real challenge for the scientific world due to the rapid transmission and high mortality caused by this new coronavirus. The associated disease has been named COVID-19 and ranges from asymptomatic to severe cases that rapidly progress to acute respiratory distress syndrome, multisystem disorders, and death. The scientific community has joined efforts to try to better understand the pathophysiological process of the infection with the intention of combating the disease more effectively. In this work we present a study to determine the alterations in gene expression caused by the infection. Methods: Three different study models have been used: bronchial epithelial cell cultures, airway organoids, and samples obtained from autopsies in patients with and without SARS-CoV-2 infection. The expression profiles altered by the infection in each model have been analyzed, as well as the functional categories enriched. Results: Only 4 genes are common in the three types of study models, the autopsy model being the most disparate. Within the common genes in cell and organoid culture models of the lung, we find functions related to inflammatory processes. Conclusions: In vitro studies are a good model to have a snapshot of alterations in infection patterns, while autopsies are not a good model due to bias caused by necrosis. (AU)

Nuevos biomarcadores de cáncer de pulmón basados en miRNA / [New biomarkers of lung cancer based on miRNA]

Objetivo: Determinar si existen diferencias de expresión entre los miRNA de tejido sano y tumoral de adenocarcinoma de pulmón y carcinoma epidermoide de pulmón, con lo que podrían ser usados como biomarcadores. Material y métodos: Se ha extraído y secuenciado el miRNA de tejido tumoral y sano adyacente de muestras de adenocarcinoma y carcinoma epidermoide de dieciséis pacientes intervenidos en el Hospital Regional de Málaga. Esas secuencias se han analizado con un flujo de trabajo bioinformático específico que conlleva varios pasos: 1o) preprocesar las lecturas, 2o) mapearlas sobre el genoma humano de referencia, 3o) determinar la expresión de los miRNA en cada una de las muestras, 4o) calcular su expresión diferencial entre el tejido sano y el tumoral de cada paciente, 5o) realizar un análisis funcional de los miRNA encontrados. Resultados: Hemos analizado los miRNA con expresión diferencial en cada uno de los tipos histológicos estudiados. El análisis del adenocarcinoma y carcinoma epidermoide de pulmón ha dado como resultado un total de 82 y 360 miRNA diferencialmente expresados (miDE), respectivamente. Hemos encontrado 50 miRNA comunes a los dos tipos histológicos, y su análisis funcional indica que están implicados en el crecimiento, desarrollo y movimiento celular, que se produce tanto en la célula normal como en el cáncer. Conclusiones: Los miDE encontrados son una fuente de biomarcadores, al tener una reprogramación específica en cáncer, y ser obtenibles de forma no invasiva. (AU)

Objective: Determine if there are differences in expression between the miRNAs of healthy and tumor tissue of lung adenocarcinoma and squamous cell carcinoma of the lung, with which they could be used as biomarkers. Material and methods: miRNA has been extracted and sequenced from tumor and adjacent healthy tissue from samples of adenocarcinoma and squamous cell carcinoma from sixteen patients operated at the Regional Hospital of Malaga. These sequences have been analyzed with a specific bioinformatic workflow that involves several steps: 1st) preprocessing the reads, 2nd) mapping them onto the reference human genome, 3rd) determining the expression of the miRNAs in each of the samples, 4th) calculate its differential expression between the healthy and tumor tissue of each patient, 5th) perform a functional analysis of the miRNAs found. Results: We have analyzed the miRNAs with differential expression in each of the histological types studied. Analysis of adenocarcinoma and squamous cell carcinoma of the lung has resulted in a total of 82 and 360 differentially expressed miRNAs (miDE), respectively. We have found 50 miRNAs common to the two histological types, and their functional analysis indicates that they are involved in cell growth, development and movement, which occurs both in normal cells and in cancer. Conclusions: The miDEs found are a source of biomarkers, as they have a specific reprogramming in cancer, and are obtainable non-invasively. (AU)

Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

Estudio de la desregulación de los transposones en el carcinoma epidermoide, el adenocarcinoma y el carcinoma microcítico de pulmón: en busca de nuevos biomarcadores / Study on the deregulation of transposons in squamous cell carcinoma, adenocarcinoma and small cell carcinoma of the lung: in search of new biomarkers

OBJETIVO: Determinar si existen biomarcadores entre los transposones, tanto para diagnóstico como pronóstico de adenocarcinoma, carcinoma epidermoide y carcinoma microcítico de pulmón, así como valorar diferencias y similitudes entre estos tres tipos histológicos. MATERIAL Y MÉTODOS: Se ha secuenciado el RNA total del tejido tumoral y sano adyacente de muestras de adenocarcinoma y carcinoma epidermoide de dieciséis pacientes intervenidos en el Hospital Regional de Málaga. En el caso del carcinoma microcítico, se han utilizado pacientes externos cuyos datos proceden de los repositorios genómicos disponibles para la comunidad científica. Esas secuencias se han analizado con un flujo de trabajo bioinformático específico que conlleva varios pasos: 1º) Preprocesar las lecturas, 2º) Mapearlas sobre el genoma humano de referencia, 3º) Determinar la expresión de los transposones en cada una de las muestras, y 4º) Calcular su expresión diferencial entre el tejido sano y el tumoral de cada paciente. RESULTADOS: En un primer paso, hemos analizado los transposones con expresión diferencial en cada uno de los tipos histológicos estudiados por separado. El análisis del adenocarcinoma, carcinoma microcítico y carcinoma epidermoide de pulmón ha dado como resultado un total de 7, 72 y 12 transposones diferencialmente expresados (TDE), respectivamente. Hemos encontrado transposones comunes a los tres tipos histológicos y otros cuyo comportamiento es específico en cada uno de ellos. CONCLUSIONES: Los transposones se reprograman específicamente cuando una célula normal del pulmón se vuelve cancerosa. Esta reprogramación es una fuente de biomarcadores que podría ayudar al diagnóstico precoz del cáncer

OBJECTIVE: To determine whether biomarkers exist among transposons for both the diagnosis and prognosis of adenocarcinoma, squamous cell carcinoma and small cell carcinoma of the lung, as well as to evaluate differences and similarities between these three histological types. MATERIAL AND METHODS: The total RNA was sequenced for the tumor tissue and adjacent healthy tissue in adenocarcinoma and squamous cell carcinoma samples from sixteen patients being treated at the Hospital Regional de Málaga. In the case of small cell carcinoma, external patients were used whose data came from genomic repositories available to the scientific community. These sequences were analyzed with a specific bioinformatic workflow which includes several steps: 1) Pre-process readings, 2) Map them on the reference human genome, 3) Determine transposon expression in each of the samples, and 4) Calculate the differential expression between the healthy and tumor tissue in each patient. RESULTS: In the first step, we analyzed the transposons with differential expression in each of the histological types studied separately. The analysis of adenocarcinoma, small cell carcinoma and squamous cell carcinoma of the lung has resulted in a total of 7, 72 and 12 differentially expressed transposons, respectively. We found transposons common to all three histological types and others whose behavior is specific to each type. CONCLUSIONS: Transposons are specifically reprogrammed when a normal lung cell becomes cancerous. This reprogramming is a source of biomarkers that could help with the early diagnosis of cancer

Potencial uso biomarcador de los retrotransposones en el adenocarcinoma de pulmón / Potential use of retrotransposons as a biomarker in lung adenocarcinoma

HIPÓTESIS Y OBJETIVO: partiendo de la hipótesis de que la reprogramación de los transposones en el cáncer nos puede orientar sobre su desarrollo, en este trabajo se pretende determinar qué transposones podrían servir de biomarcadores con futuros propósitos diagnósticos y pronósticos. Material y métodos: a partir de muestras congeladas de biopsias de adenocarcinoma de pulmón, se ha secuenciado ARN total del tejido tumoral y sano adyacente de ocho pacientes intervenidos en el Hospital Regional de Málaga. Se han analizado con un flujo de trabajo bioinformático específico que cuantifica y proporciona la expresión diferencial de los transposones cuando se compara el tejido sano y tumoral de cada paciente. Resultados: en este trabajo prospectivo, el nivel de transposición global no cambia entre el pulmón sano y el adenocarcinoma. Se han identificado siete transposones con expresión diferencial significativa: cinco se sobreexpresan en las células del adenocarcinoma y los otros dos se sobreexpresan en las células sanas del pulmón. Todos los que son de la clase de retrovirus endógenos humanos (HERV) tienen un gran potencial como biomarcador al reprogramarse de la misma forma en todos los pacientes. Conclusión: el nivel de transposición en el cáncer de pulmón no está desregulado, sino reprogramado, y los transposones afectados por la reprogramación podrían considerarse biomarcadores en potencia

HYPOTHESIS AND OBJECTIVE: transposon reprogramming can be related to cancer development. The aim of this study is to determine the role of transposons in lung cancer and evaluate the posible role of transposon as a diagnostic and pronostic biomarkers in lung cáncer. MATERIAL AND METHODS: total RNA from lung adenocarcinome was sequenced. We analized RNA from tumor and adyacent healthy tissue from eight patients. By using a specific software the differential expression of the transposons in healthy and tumor tissue was analyzed in each patient. RESULTS: this prospective study shows that in our population, the overall transposition level does not change between the healthy and lung adenocarcinoma tissue. Seven transposons with significative differential expression have been found. Five were upregulated in adenocarcinoma cells, and the other two were upregulated in healthy lung cells. Those that belong to the human endogenous retroviruses (HERV) class show a high biomarker potential since they are reprogrammed in the same way in all patients. CONCLUSION: the transposition level in lung cancer is not deregulated but reprogrammed. Transposons affected by the reprogramming may be considered as potential biomarkers

La influencia del tabaquismo en los elementos genéticos móviles / The influence of smoking habit on mobile genetic elements

Se sabe que el tabaco produce alteraciones en distintas partes del organismo que conllevan la aparición del cáncer. Los retrotransposones parecen estar implicados tanto en el inicio como en la evolución de esta enfermedad. Se ha visto que también contribuyen a que determinados tratamientos contra el cáncer, como la inmunoterapia, dejen de ser eficaces. En este trabajo analizamos cómo se ven afectados los elementos genéticos móviles por los distintos compuestos presentes en el tabaco (AU)

It is known that smoking causes alterations in different parts of the body that entail appearance of cancer. The retrotransposons seem to be involved both at the onset and in the evolution of this disease. It has also been seen that they contribute to the fact that certain treatments against cancer, such as immunotherapy, stop being effective. In this work, we analyze how the mobile genetic elements are affected by the different compounds present in tobacco (AU)