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

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