miR-1185-1 and miR-548q Are Biomarkers of Response to Weight Loss and Regulate the Expression of GSK3B

The aim of the present investigation was to identify putative miRNAs involved in the response to weight loss. Reverse-transcribed RNA isolated from white blood cells (WBCs) of a subpopulation from the Reduction of the Metabolic Syndrome in Navarra-Spain (RESMENA-S) study (low-responders (LR) and high-responders (HR)) was hybridized in a gene expression microarray. Moreover, miRNAs were sequenced by miRNA-Seq. It was found that miR-548q and miR-1185-1 were overexpressed in HR, both in the microarray and in the miRNA-Seq. A bioinformatic prediction of putative target genes of the selected miRNAs found that GSK3B, a putative target for miR-548q and miR-1185-1, was downregulated in HR. Particular 3′-UTR binding regions of GSK3B were cloned downstream of the firefly luciferase gene. HEK-293T cells were co-transfected with either 0.25 μg of empty pmiR-GLO or pmiR-GLO-548q-3′-UTR/pmiR-GLO-1185-1-3′-UTR, and 7.5 pmol of miR-548q/miR-1185-1 mimics, demonstrating that miR-1185-1 bound to the 3′-UTR region of GSK3B. THP-1 cells were transfected with either 20/40 nM of miR-548q/miR-1185-1 mimics, evidencing that miR-1185-1inhibited the expression of the gene when transfected at doses of 20/40 nM, whereas miR-548q inhibited GSK3B expression at a dose of 40 nM. As a conclusion, miR-548q and miR-1185-1 levels in WBCs are biomarkers of response to weight-loss diets and could be involved in the regulation of the proinflammatory gene GSK3B

Validation of miR-612 and its target gene <i>TP53</i>.

<p>A) Correlation between miR-612 methylation levels by microarray and EpiTyper. B) Gene expression of <i>TP53</i> in HR and LR in the microarray. C) Validation of <i>TP53</i> expression profile in HR and LR white blood cells by qPCR, adjusted for age, sex, diet and baseline body weight. * p < 0.05 from ANCOVA test. D) Luciferase activity assay of pmiR-GLO-<i>TP53</i>-3’-UTR after co-transfection with miR-612. Normalized luciferase activity is presented as the mean ± SEM of three separate triplicate experiments. *** p < 0.001 from Student t-test.</p

Identification of miR-612 and miR-1976 in the methylation and expression microarrays.

<p>A) Volcano plot of miRNAs differentially methylated between HR and LR. B) Volcano plot of miRNAs differentially expressed between HR and LR.</p

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

<div><p>Background</p><p>Non-coding RNAs (i.e., miRNAs) play a role in the development of obesity and related comorbidities and the regulation of body weight.</p><p>Objective</p><p>To identify candidate miRNA biomarkers throughout omics approaches in order to predict the response to specific weight-loss dietary treatments.</p><p>Design</p><p>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.</p><p>Results</p><p>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 <i>TP53</i> was a putative target gene of miR-612 and <i>CD40</i> of miR-1976. Moreover, <i>TP53</i> was downregulated in the expression array when comparing HR vs LR expression levels adjusted by sex, diet, age and baseline weight, and <i>CD40</i> showed a statistical trend. Furthermore, gene expression levels of <i>TP53</i> and <i>CD40</i> in white blood cells, when measured by qPCR, were also downregulated in HR. Finally, miR-612 and miR-1976 potently repressed <i>TP53</i> and <i>CD40</i> respectively by targeting its 3’-UTR regions.</p><p>Conclusion</p><p>miR-612 and miR-1976 levels could be prospective biomarkers of response to specific weight-loss diets and might regulate the gene expression of <i>TP53</i> and <i>CD40</i>.</p></div

Validation of miR-1976 and its target gene <i>CD40</i>.

<p>A) miR-612 expression levels by qPCR in HR and LR, adjusted for age, sex, diet and baseline body weight. * p < 0.05 from ANCOVA test. B) Pearson’s correlation between miR-1976 expression levels in the microarray and by qPCR, adjusted by sex, age, baseline body weight and diet. C) Gene expression of <i>CD40</i> in HR and LR in the microarray. t p = 0.069. D) Pearson’s correlations between miR-1976 expression levels and its target gene (<i>CD40</i>) in the microarray, adjusted by sex, age, baseline body weight and diet. E) Validation of <i>CD40</i> expression profile in HR and LR white blood cells by qPCR adjusted for age, sex, diet and baseline body weight. * p < 0.05 from ANCOVA test. F) Luciferase activity assays of pmiR-GLO-<i>CD40</i>-3’-UTR after co-transfection with miR-1976. Normalized luciferase activity is presented as the mean ± SEM of three separate triplicate experiments. * p < 0.05 from Student t-test.</p

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