Network-Based Integration of Multi-Omics Data Identifies the Determinants of miR-491-5p Effects.

The identification of miRNAs' targets and associated regulatory networks might allow the definition of new strategies using drugs whose association mimics a given miRNA's effects. Based on this assumption we devised a multi-omics approach to precisely characterize miRNAs' effects. We combined miR-491-5p target affinity purification, RNA microarray, and mass spectrometry to perform an integrated analysis in ovarian cancer cell lines. We thus constructed an interaction network that highlighted highly connected hubs being either direct or indirect targets of miR-491-5p effects: the already known EGFR and BCL2L1 but also EP300, CTNNB1 and several small-GTPases. By using different combinations of specific inhibitors of these hubs, we could greatly enhance their respective cytotoxicity and mimic the miR-491-5p-induced phenotype. Our methodology thus constitutes an interesting strategy to comprehensively study the effects of a given miRNA. Moreover, we identified targets for which pharmacological inhibitors are already available for a clinical use or in clinical trials. This study might thus enable innovative therapeutic options for ovarian cancer, which remains the leading cause of death from gynecological malignancies in developed countries.

PATL2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice.

The genetic causes of oocyte meiotic deficiency (OMD), a form of primary infertility characterised by the production of immature oocytes, remain largely unexplored. Using whole exome sequencing, we found that 26% of a cohort of 23 subjects with OMD harboured the same homozygous nonsense pathogenic mutation in PATL2, a gene encoding a putative RNA-binding protein. Using Patl2 knockout mice, we confirmed that PATL2 deficiency disturbs oocyte maturation, since oocytes and zygotes exhibit morphological and developmental defects, respectively. PATL2's amphibian orthologue is involved in the regulation of oocyte mRNA as a partner of CPEB However, Patl2's expression profile throughout oocyte development in mice, alongside colocalisation experiments with Cpeb1, Msy2 and Ddx6 (three oocyte RNA regulators) suggest an original role for Patl2 in mammals. Accordingly, transcriptomic analysis of oocytes from WT and Patl2-/- animals demonstrated that in the absence of Patl2, expression levels of a select number of highly relevant genes involved in oocyte maturation and early embryonic development are deregulated. In conclusion, PATL2 is a novel actor of mammalian oocyte maturation whose invalidation causes OMD in humans.

The Yin and Yang of Microrna Assay Methods.

OBJECTIVE: microRNA assessments in biological samples can be performed by different methods that mainly rely on hybridization process, qPCR or RNA sequencing. With the aim to detect and validate microRNA biomarkers in tumor samples, we challenged the consistency of the quantitative results obtained with the different methods. METHODS: We measured microRNA concentrations in several biological samples such as cultured tumor cells or tumor tissues (frozen tissues or FFPE samples) using different microRNA assay methods, in particular hybridization to AffymetrixTM arrays, qPCR and digital droplet qPCR (BioradTM) based on Taqman microRNA assays (Life TechnologiesTM). We also compared our results to other data that have been obtained with different technical approaches and available in the literature. RESULTS: We found poor consistency for the microRNA amounts measured in the samples assayed by the different methods. Both technical platforms and microRNA assays protocols may be responsible for the observed inconsistencies. CONCLUSION: When assaying microRNAs for clinical purpose or fundamental researches it seems necessary to keep in mind the specific pitfalls of all the microRNA detection methods such as those we disclose here. Obviously, valid inter sample comparisons and meaningful multicenter studies can only be obtained when microRNA assessments are strictly performed with identical technical approaches and reagents.

Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy.

Circulating miRNAs are promising biomarkers in oncology but have not yet been implemented in the clinic given the lack of concordance across studies. In order to increase the cross-studies reliability, we attempted to reduce and to control the circulating miRNA expression variability between patients. First, to maximize profiling signals and to reduce miRNA expression variability, three isolation kits were compared and the NucleoSpin(®) kit provided higher miRNA concentrations than the other widely used kits. Second, to control inter-sample variability during the profiling step, the exogenous miRNAs normalization method commonly used for RT-qPCR validation step was adapted to microarray experiments. Importantly, exogenous miRNAs presented two-fold lower inter-sample variability than the widely used endogenous miR-16-5p reflecting that the latter is subject to both biological and technical variability. Although Caenorhabditis elegans miRNAs isolation yields were heterogeneous, they correlated to each other and to their geometrical mean across samples. The normalization based on the geometrical mean of three exogenous miRNAs increased the correlation up-to 0.97 between the microarrays and individual RT-qPCR steps of circulating miRNAs expression. Overall, this new strategy open new avenue to identify reliable circulating miRNA signatures for translation into clinical practice.

The Yin and Yang of microRNA Assay Methods.

OBJECTIVE: microRNA assessments in biological samples can be performed by different methods that mainly rely on hybridization process, qPCR or RNA sequencing. With the aim to detect and validate microRNA biomarkers in tumor samples, we challenged the consistency of the quantitative results obtained with the different methods. METHODS: We measured microRNA concentrations in several biological samples such as cultured tumor cells or tumor tissues (frozen tissues or FFPE samples) using different microRNA assay methods, in particular hybridization to AffymetrixTM arrays, qPCR and digital droplet qPCR (BioradTM) based on Taqman microRNA assays (Life TechnologiesTM). We also compared our results to other data that have been obtained with different technical approaches and available in the literature. RESULTS: We found poor consistency for the microRNA amounts measured in the samples assayed by the different methods. Both technical platforms and microRNA assays protocols may be responsible for the observed inconsistencies. CONCLUSION: When assaying microRNAs for clinical purpose or fundamental researches it seems necessary to keep in mind the specific pitfalls of all the microRNA detection methods such as those we disclose here. Obviously, valid inter sample comparisons and meaningful multicenter studies can only be obtained when microRNA assessments are strictly performed with identical technical approaches and reagents.

Exosomal MicroRNAs in Tumoral U87 MG Versus Normal Astrocyte Cells.

Brain glial tumors, and particularly glioblastomas, are tumors with a very poor prognosis. Currently, the parameters that control aggressiveness, migration, or chemoresistance are not well known. In this tumor context, microRNAs are thought to be essential actors of tumorigenesis as they are able to control the expression of numerous genes. microRNAs are not only active in controlling tumor cell pathways, they are also secreted by cells, inside microvesicles called exosomes, and may play specific roles outside the tumor cells in the tumor microenvironment. We analyzed the microRNA content of exosomes produced in vitro by normal glial cells (astrocytes) and tumor glial cells (U87 MG) using Affymetrix microarrays. It appears that the exosome microRNA profiles are qualitatively quite similar. Nevertheless, their quantitative profiles are different and may be potentially taken as an opportunity to carry out assays of diagnostic interest. We submitted the cultured cells to several stresses such as oxygen deprivation or treatments with chemical drugs (GW4869 or 5-Aza-2'- deoxycitidine) to assess the impact of the cellular microRNA profile modifications on the exosome microRNA profiles. We found that modifications of the cellular microRNA content are not strictly mirrored in exosomes. On the basis of these results, we propose that the way microRNAs are released in exosomes is probably the result of a combination of different excretion mechanisms or constraints that concur in a controlled regulation of the exosome microRNA secretion.

Additional clues for a protective role of vitamin D in neurodegenerative diseases: 1,25-dihydroxyvitamin D3 triggers an anti-inflammatory response in brain pericytes.

Epidemiological and experimental studies suggest that 1,25-dihydroxyvitamin D3 (1,25D) plays a neuroprotective role in neurodegenerative diseases including Alzheimer's disease. Most of the experimental data regarding the genes regulated by this hormone in brain cells have been obtained with neuron and glial cells. Pericytes play a critical role in brain function that encompasses their classical function in blood-brain barrier control and maintenance. However, the gene response of brain pericyte to 1,25D remains to be investigated. Analyses of the transcriptomic response of human brain pericytes to 1,25D demonstrate that human brain pericytes in culture respond to 1,25D by regulating genes involved in the control of neuroinflammation. In addition, pericytes respond to the pro-inflammatory cytokines tumor necrosis factor-α and Interferon-γ by inducing the expression of the CYP27B1 gene which is involved in 1,25D synthesis. Taken together, these results suggest that neuroinflammation could trigger the synthesis of 1,25D by brain pericytes, which in turn respond to the hormone by a global anti-inflammatory response. These findings identify brain pericytes as a novel 1,25D-responsive cell type and provide additional evidence for the potential value of vitamin D in the prevention or therapy of Alzheimer's disease and other neurodegenerative/neuropsychiatric diseases associated with an inflammatory component.

Hypoxia-induced expression of VE-cadherin and filamin B in glioma cell cultures and pseudopalisade structures.

Most of our knowledge regarding glioma cell biology comes from cell culture experiments. For many years the standards for glioma cell culture were the use of cell lines cultured in the presence of serum and 20 % O2. However, in vivo, normoxia in many brain areas is in close to 3 % O2. Hence, in cell culture, the experimental value referred as the norm is hyperoxic compared to any brain physiological value. Likewise, cells in vivo are not usually exposed to serum, and low-passaged glioma neurosphere cultures maintained in serum-free medium is emerging as a new standard. A consequence of changing the experimental normoxic standard from 20 % O2 to the more brain physiological value of 3 % O2, is that a 3 % O2 normoxic reference point enabled a more rigorous characterization of the level of regulation of genes by hypoxia. Among the glioma hypoxia-regulated genes characterized using this approach we found VE-cadherin that is required for blood vessel formation, and filamin B a gene involved in endothelial cell motility. Both VE-cadherin and filamin B were found expressed in pseudopalisades, a glioblastoma pathognomonic structure made of hypoxic migrating cancer cells. These results provide additional clues on the role played by hypoxia in the acquisition of endothelial traits by glioma cells and on the functional links existing between pseudopalisades, hypoxia, and tumor progression.

The transcriptomic response of mixed neuron-glial cell cultures to 1,25-dihydroxyvitamin d3 includes genes limiting the progression of neurodegenerative diseases.

Seasonal or chronic vitamin D deficiency and/or insufficiency is highly prevalent in the human population. Receptors for 1,25-dihydroxyvitamin D3, the hormonal metabolite of vitamin D, are found throughout the brain. To provide further information on the role of this hormone on brain function, we analyzed the transcriptomic profiles of mixed neuron-glial cell cultures in response to 1,25-dihydroxyvitamin D3. 1,25-dihydroxyvitamin D3 treatment increases the mRNA levels of 27 genes by at least 1.9 fold. Among them, 17 genes were related to neurodegenerative and psychiatric diseases, or brain morphogenesis. Notably, 10 of these genes encode proteins potentially limiting the progression of Alzheimer's disease. These data provide support for a role of 1,25-dihydroxyvitamin D3 in brain disease prevention. The possible consequences of circannual or chronic vitamin D insufficiencies on a tissue with a low regenerative potential such as the brain should be considered.

Computational identification of transcriptionally co-regulated genes, validation with the four ANT isoform genes.

BACKGROUND: The analysis of gene promoters is essential to understand the mechanisms of transcriptional regulation required under the effects of physiological processes, nutritional intake or pathologies. In higher eukaryotes, transcriptional regulation implies the recruitment of a set of regulatory proteins that bind on combinations of nucleotide motifs. We developed a computational analysis of promoter nucleotide sequences, to identify co-regulated genes by combining several programs that allowed us to build regulatory models and perform a crossed analysis on several databases. This strategy was tested on a set of four human genes encoding isoforms 1 to 4 of the mitochondrial ADP/ATP carrier ANT. Each isoform has a specific tissue expression profile linked to its role in cellular bioenergetics. RESULTS: From their promoter sequence and from the phylogenetic evolution of these ANT genes in mammals, we constructed combinations of specific regulatory elements. These models were screened using the full human genome and databases of promoter sequences from human and several other mammalian species. For each of transcriptionally regulated ANT1, 2 and 4 genes, a set of co-regulated genes was identified and their over-expression was verified in microarray databases. CONCLUSIONS: Most of the identified genes encode proteins with a cellular function and specificity in agreement with those of the corresponding ANT isoform. Our in silico study shows that the tissue specific gene expression is mainly driven by promoter regulatory sequences located up to about a thousand base pairs upstream the transcription start site. Moreover, this computational strategy on the study of regulatory pathways should provide, along with transcriptomics and metabolomics, data to construct cellular metabolic networks.

MicroRNAs: molecular features and role in cancer.

microRNAs (miRNAs) are small noncoding endogenously produced RNAs that play key roles in controlling the expression of many cellular proteins. Once they are recruited and incorporated into a ribonucleoprotein complex miRISC, they can target specific mRNAs in a miRNA sequence-dependent process and interfere in the translation into proteins of the targeted mRNAs via several mechanisms. Consequently, miRNAs can regulate many cellular pathways and processes. Dysregulation of their physiological roles may largely contribute to disease. In particular, in cancer, miRNAs can be involved in the deregulation of the expression of important genes that play key roles in tumorigenesis, tumor development, and angiogenesis and have oncogenic or tumor suppressor roles. This review focuses on the biogenesis and maturation of miRNAs, their mechanisms of gene regulation, and the way their expression is deregulated in cancer. The involvement of miRNAs in several oncogenic pathways such as angiogenesis and apoptosis, and in the inter-cellular dialog mediated by miRNA-loaded exosomes as well as the development of new therapeutical strategies based on miRNAs will be discussed.

MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes.

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.

The heterogeneity of meningioma revealed by multiparameter analysis: infiltrative and non-infiltrative clinical phenotypes.

Tumor invasion or infiltration of adjacent tissues is the source of clinical challenges in diagnosis as well as prevention and treatment. Among brain tumors, infiltration of the adjacent tissues with diverse pleiotropic mechanisms is frequently encountered in benign meningiomas. We assessed whether a multiparametric analysis of meningiomas based on data from both clinical observations and molecular analyses could provide a consistent and accurate appraisal of invasive and infiltrative phenotypes and help determine the diagnosis of these tumors. Tissue analyses of 37 meningiomas combined enzyme-linked immunosorbent assay (ELISA) and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) assays of two different protein biomarkers (thrombospondin 1 and a phosphorylated form of vimentin) as well as gene expression analyses with oligonucleotide micro-arrays. Up to four different clinical and molecular parameters were then examined for tumor classification. From this study, we were able to cluster 36 out of the 37 tumors into two different subsets corresponding to infiltrative/invasive and non-infiltrative tumors. In addition, meningiomas that invade brain and those that infiltrate the neighboring skull bone exhibited no distinguishable molecular features. Our multi-parameter analysis that combines clinical data, transcriptomic and molecular assays clearly reveals the heterogeneity of meningiomas and distinguishes the intrinsically infiltrative/invasive tumors from the non-infiltrative meningiomas.