Implication of miR-612 and miR-1976 in the regulation of TP53 and CD40 and their relationship in the response to specific weight-loss diets

Background: Non-coding RNAs (i.e., miRNAs) play a role in the development of obesity and related comorbidities and the regulation of body weight. Objective: To identify candidate miRNA biomarkers throughout omics approaches in order to predict the response to specific weight-loss dietary treatments. Design: Genomic DNA and cDNA isolated from white blood cells of a subset from the RESMENA nutritional intervention study (Low-responders (LR) vs High-responders (HR)) was hybridized in Infinium Human Methylation450 BeadChip and in Illumina Human HT-12 v4 gene expression BeadChips arrays respectively. A bioinformatic prediction of putative target sites of selected miRNAs was performed by applying miRBase algorithms. HEK-293T cells were co-transfected with expression vectors containing the 3'-UTR of candidate genes to validate the binding of miRNAs to its target sites. Results: 134 miRNAs were differentially methylated between HR and LR in the methylation array, whereas 44 miRNAs were differentially expressed between both groups in the expression array. Specifically, miR-1237, miR-1976, miR-642, miR-636, miR-612 and miR-193B were simultaneously hypomethylated and overexpressed in HR. miR-612 and miR-1976 showed greatest differences in methylation and expression levels, respectively. The bioinformatic prediction revealed that TP53 was a putative target gene of miR-612 and CD40 of miR-1976. Moreover, TP53 was downregulated in the expression array when comparing HR vs LR expression levels adjusted by sex, diet, age and baseline weight, and CD40 showed a statistical trend. Furthermore, gene expression levels of TP53 and CD40 in white blood cells, when measured by qPCR, were also downregulated in HR. Finally, miR-612 and miR-1976 potently repressed TP53 and CD40 respectively by targeting its 3'-UTR regions. Conclusion: miR-612 and miR-1976 levels could be prospective biomarkers of response to specific weight-loss diets and might regulate the gene expression of TP53 and CD40

miRNAs as predictive biomarkers of obesity and weight loss: epigenetic regulation and target gene identification

Epigenetics refers to molecular factors and processes around the DNA that regulate genome activity without nucleotide sequence modification. One of these epigenetic factors are miRNAs, a class of small (17-25 nts in length) single stranded non coding RNAs that post transcriptionally regulate gene expression in animals, plants and unicellular eukaryotes. Furthermore, miRNAs coding regions may also be regulated by other epigenetic mechanisms, such as DNA methylation. Therefore, a complex interactive epigenetic regulation may occur onto DNA, ultimately controlling gene transcription and translation. Consequently, it has been demonstrated that epigenetics can be involved in the development of obesity, inflammation and metabolic disturbances (type 2 diabetes, hypercholesterolemia, hypertension, or cardiovascular disease). Furthermore, in the era of personalized nutrition , miRNAs have emerged as promising tools for the prediction, screening, diagnosis and prognosis of obesity and related comorbidities. In this sense, the present research work has applied different omics approaches such as methylation/expression microarrays and deep sequencing technologies in order to address the following objectives: 1) to demonstrate the role of miRNA epigenetic regulation in the early stages of childhood obesity; 2) to identify miRNA-type biomarkers for weight loss prediction; and 3) to verify the regulation of selected miRNAs over their predicted target genes. In the first chapter we performed a methylation microarray in an infant population and identified that DNA methylation levels in miR-1203, miR-412 and miR-216A coding regions significantly correlated with body mass index standard deviation score (BMI SDS). Moreover, these methylation levels were able to explain up to 40% of the variation of BMI SDS in childhood obesity. In chapter 2, after combining methylation and expression microarrays data, we observed that miR-612 and miR-1976 were hypomethylated and overexpressed in high responders to a weight loss intervention. Additionally, after construction of specific expression vectors containing the particular 3 -UTR of each gene, we demonstrated that miR-612 and miR-1976 targeted TP53 and CD40 genes respectively. Furthermore, miR-1976 was able to down-regulate CD40 expression levels when transfected into mature human adipocytes. Lastly, in chapter 3, we noticed that miR-548q and miR-1185-1 also differed in their expression levels between high and low responders. Overexpression miRNA mimic experiments and luciferase reporter vectors showed that both miRNAs were implicated in GSK3B mRNA expression regulation, by a direct binding of the miRNA over the 3 UTR of GSK3B in the case of miR-1185-1, and by an indirect regulation in the case of miR-548q. Overall, the results exhibited in this thesis add new insights into the importance of miRNAs in the management of diseases, especially of obesity and inflammation related illnesses; presenting different miRNAs involved in obesity development as prognostic biomarkers with potential utility in the personalization of weight loss treatments

miRNAs as predictive biomarkers of obesity and weight loss: epigenetic regulation and target gene identification

Epigenetics refers to molecular factors and processes around the DNA that regulate genome activity without nucleotide sequence modification. One of these epigenetic factors are miRNAs, a class of small (17-25 nts in length) single stranded non coding RNAs that post transcriptionally regulate gene expression in animals, plants and unicellular eukaryotes. Furthermore, miRNAs coding regions may also be regulated by other epigenetic mechanisms, such as DNA methylation. Therefore, a complex interactive epigenetic regulation may occur onto DNA, ultimately controlling gene transcription and translation. Consequently, it has been demonstrated that epigenetics can be involved in the development of obesity, inflammation and metabolic disturbances (type 2 diabetes, hypercholesterolemia, hypertension, or cardiovascular disease). Furthermore, in the era of personalized nutrition , miRNAs have emerged as promising tools for the prediction, screening, diagnosis and prognosis of obesity and related comorbidities. In this sense, the present research work has applied different omics approaches such as methylation/expression microarrays and deep sequencing technologies in order to address the following objectives: 1) to demonstrate the role of miRNA epigenetic regulation in the early stages of childhood obesity; 2) to identify miRNA-type biomarkers for weight loss prediction; and 3) to verify the regulation of selected miRNAs over their predicted target genes. In the first chapter we performed a methylation microarray in an infant population and identified that DNA methylation levels in miR-1203, miR-412 and miR-216A coding regions significantly correlated with body mass index standard deviation score (BMI SDS). Moreover, these methylation levels were able to explain up to 40% of the variation of BMI SDS in childhood obesity. In chapter 2, after combining methylation and expression microarrays data, we observed that miR-612 and miR-1976 were hypomethylated and overexpressed in high responders to a weight loss intervention. Additionally, after construction of specific expression vectors containing the particular 3 -UTR of each gene, we demonstrated that miR-612 and miR-1976 targeted TP53 and CD40 genes respectively. Furthermore, miR-1976 was able to down-regulate CD40 expression levels when transfected into mature human adipocytes. Lastly, in chapter 3, we noticed that miR-548q and miR-1185-1 also differed in their expression levels between high and low responders. Overexpression miRNA mimic experiments and luciferase reporter vectors showed that both miRNAs were implicated in GSK3B mRNA expression regulation, by a direct binding of the miRNA over the 3 UTR of GSK3B in the case of miR-1185-1, and by an indirect regulation in the case of miR-548q. Overall, the results exhibited in this thesis add new insights into the importance of miRNAs in the management of diseases, especially of obesity and inflammation related illnesses; presenting different miRNAs involved in obesity development as prognostic biomarkers with potential utility in the personalization of weight loss treatments

Implication of miR-612 and miR-1976 in the regulation of TP53 and CD40 and their relationship in the response to specific weight-loss diets

Background: Non-coding RNAs (i.e., miRNAs) play a role in the development of obesity and related comorbidities and the regulation of body weight. Objective: To identify candidate miRNA biomarkers throughout omics approaches in order to predict the response to specific weight-loss dietary treatments. Design: Genomic DNA and cDNA isolated from white blood cells of a subset from the RESMENA nutritional intervention study (Low-responders (LR) vs High-responders (HR)) was hybridized in Infinium Human Methylation450 BeadChip and in Illumina Human HT-12 v4 gene expression BeadChips arrays respectively. A bioinformatic prediction of putative target sites of selected miRNAs was performed by applying miRBase algorithms. HEK-293T cells were co-transfected with expression vectors containing the 3'-UTR of candidate genes to validate the binding of miRNAs to its target sites. Results: 134 miRNAs were differentially methylated between HR and LR in the methylation array, whereas 44 miRNAs were differentially expressed between both groups in the expression array. Specifically, miR-1237, miR-1976, miR-642, miR-636, miR-612 and miR-193B were simultaneously hypomethylated and overexpressed in HR. miR-612 and miR-1976 showed greatest differences in methylation and expression levels, respectively. The bioinformatic prediction revealed that TP53 was a putative target gene of miR-612 and CD40 of miR-1976. Moreover, TP53 was downregulated in the expression array when comparing HR vs LR expression levels adjusted by sex, diet, age and baseline weight, and CD40 showed a statistical trend. Furthermore, gene expression levels of TP53 and CD40 in white blood cells, when measured by qPCR, were also downregulated in HR. Finally, miR-612 and miR-1976 potently repressed TP53 and CD40 respectively by targeting its 3'-UTR regions. Conclusion: miR-612 and miR-1976 levels could be prospective biomarkers of response to specific weight-loss diets and might regulate the gene expression of TP53 and CD40

Alteraciones genéticas en las neoplasias hematológicas de origen linfoide implicaciones en la práctica clínica

La mejora de las técnicas de citogenética convencional, el desarrollo de la citogenética molecular y la aplicación de técnicas de biología molecular al análisis genético ha conducido a una verdadera revolución en el conocimiento de los procesos implicados en el desarrollo y progresión de las neoplasias linfoides. De esta manera, se han caracterizado gran parte de las alteraciones presentes en las células malignas estableciendo cuáles son los genes implicados en el proceso transformativo. Esto tiene importantes consecuencias en el manejo clínico de este tipo de enfermedades y permite un diagnóstico más exacto a través de una sistematización de las distintas entidades basada en sus características biológicas. Por otra parte, la introducción de nuevas técnicas de análisis, como la PCR en tiempo real, posibilitará la monitorización cuantitativa de la enfermedad permitiendo valorar la respuesta a los distintos tratamientos y estableciendo valores predictivos de recaídas. En el futuro, todo este conocimiento permitirá el establecimiento de terapias genotipo-específicas y el desarrollo de nuevos fármacos dirigidos a la alteración causante del proceso maligno y con menores efectos colaterales indeseables.The improvement of the conventional cytogenetic techniques, the development of molecular cytogenetics and the application of techniques of molecular biology to genetic analysis have led to an authentic revolution in the knowledge of the processes implied in the development and progression of lymphoid neoplasias. In this way, a great part of the alterations present in malign cells have been characterised, and the genes involved in the transformative process have been established. This has important consequences for the clinical handling of this type of disease and makes possible a more exact diagnosis through a systematisation of the different entities based on their biological characteristics. On the other hand, the introduction of new techniques of analysis, such as real time PCR, will make it possible to monitor the disease quantitatively, making it possible to evaluate response to the different treatments and to establish predictive values for relapses. In the future, all of this knowledge will make it possible to establish genotype-specific therapies and to develop new medicines aimed at the alteration responsible for the malignant process and with less undesired collateral effects

Alteraciones genéticas en las neoplasias hematológicas de origen linfoide implicaciones en la práctica clínica

La mejora de las técnicas de citogenética convencional, el desarrollo de la citogenética molecular y la aplicación de técnicas de biología molecular al análisis genético ha conducido a una verdadera revolución en el conocimiento de los procesos implicados en el desarrollo y progresión de las neoplasias linfoides. De esta manera, se han caracterizado gran parte de las alteraciones presentes en las células malignas estableciendo cuáles son los genes implicados en el proceso transformativo. Esto tiene importantes consecuencias en el manejo clínico de este tipo de enfermedades y permite un diagnóstico más exacto a través de una sistematización de las distintas entidades basada en sus características biológicas. Por otra parte, la introducción de nuevas técnicas de análisis, como la PCR en tiempo real, posibilitará la monitorización cuantitativa de la enfermedad permitiendo valorar la respuesta a los distintos tratamientos y estableciendo valores predictivos de recaídas. En el futuro, todo este conocimiento permitirá el establecimiento de terapias genotipo-específicas y el desarrollo de nuevos fármacos dirigidos a la alteración causante del proceso maligno y con menores efectos colaterales indeseables.The improvement of the conventional cytogenetic techniques, the development of molecular cytogenetics and the application of techniques of molecular biology to genetic analysis have led to an authentic revolution in the knowledge of the processes implied in the development and progression of lymphoid neoplasias. In this way, a great part of the alterations present in malign cells have been characterised, and the genes involved in the transformative process have been established. This has important consequences for the clinical handling of this type of disease and makes possible a more exact diagnosis through a systematisation of the different entities based on their biological characteristics. On the other hand, the introduction of new techniques of analysis, such as real time PCR, will make it possible to monitor the disease quantitatively, making it possible to evaluate response to the different treatments and to establish predictive values for relapses. In the future, all of this knowledge will make it possible to establish genotype-specific therapies and to develop new medicines aimed at the alteration responsible for the malignant process and with less undesired collateral effects

Expression of endothelial NOX5 alters the integrity of the blood-brain barrier and causes loss of memory in aging mice

Blood-Brain barrier (BBB) disruption is a hallmark of central nervous system (CNS) dysfunction, and oxidative stress is one of the molecular mechanisms that may underlie this process. NADPH oxidases (NOX) are involved in oxidative stress-mediated vascular dysfunction and participate in the pathophysiology of its target organs. The NADPH oxidase 5 (NOX5) isoform is absent in rodents, and although little is known about the role it may play in disrupting the BBB, it has recently been implicated in experimental stroke. Our aim was to investigate the role of NADPH oxidase 5 (NOX5) in promoting vascular alterations and to identify its impact on the cognitive status of aged mice. No differences were detected in the arterial blood pressure or body weight between knock-in mice expressing endothelial NOX5 and the control mice. The Morris water maze test showed memory impairments in the aged knock-in mice expressing NOX5 compared with their control littermates. For assessing the BBB integrity, we studied the protein expression of two tight junction (TJ) proteins: Zonula occludens-1 (ZO-1) and occludin. Compared to the control animals, Aged NOX5 mice exhibited reduced levels of both proteins, demonstrating an alteration of the BBB integrity. Our data indicate that vascular NOX5 may favor behavioral changes with aging through oxidative stress-mediated BBB breakdown

Venetoclax improves CD20 immunotherapy in a mouse model of MYC/BCL2 double-expressor diffuse large B-cell lymphoma

BackgroundApproximately one-third of diffuse large B cell lymphoma (DLBCL) patients exhibit co-expression of MYC and BCL2 (double-expressor lymphoma, DEL) and have a dismal prognosis. Targeted inhibition of the anti-apoptotic protein BCL2 with venetoclax (ABT-199) has been approved in multiple B-cell malignancies and is currently being investigated in clinical trials for DLBCL. Whether BCL2 anti-apoptotic function represents a multifaceted vulnerability for DEL-DLBCL, affecting both lymphoma B cells and T cells within the tumor microenvironment, remains to be elucidated.MethodsHere, we present novel genetically engineered mice that preclinically recapitulate DEL-DLBCL lymphomagenesis, and evaluate their sensitivity ex vivo and in vivo to the promising combination of venetoclax with anti-CD20-based standard immunotherapy.ResultsVenetoclax treatment demonstrated specific killing of MYC+/BCL2(+) lymphoma cells by licensing their intrinsically primed apoptosis, and showed previously unrecognized immunomodulatory activity by specifically enriching antigen-activated effector CD8 T cells infiltrating the tumors. Whereas DEL-DLBCL mice were refractory to venetoclax alone, inhibition of BCL2 significantly extended overall survival of mice that were simultaneously treated with a murine surrogate for anti-CD20 rituximab.ConclusionsThese results suggest that the combination of anti-CD20-based immunotherapy and BCL2 inhibition leads to cooperative immunomodulatory effects and improved preclinical responses, which may offer promising therapeutic opportunities for DEL-DLBCL patients