[Show abstract][Hide abstract] ABSTRACT: The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.
Full-text · Article · Mar 2013 · Cell Death & Disease
[Show abstract][Hide abstract] ABSTRACT: Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5'-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a "minimal" 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families.
[Show abstract][Hide abstract] ABSTRACT: Congenital erythropoietic porphyria (CEP) is due to a deficiency in the enzymatic activity of uroporphyrinogen III synthase (UROS); such a deficiency leads to porphyrin accumulation and results in skin lesions and hemolytic anemia. CEP is a candidate for retrolentivirus-mediated gene therapy, but recent reports of insertional leukemogenesis underscore the need for safer methods. The discovery of induced pluripotent stem cells (iPSCs) has opened up new horizons in gene therapy because it might overcome the difficulty of obtaining sufficient amounts of autologous hematopoietic stem cells for transplantation and the risk of genotoxicity. In this study, we isolated keratinocytes from a CEP-affected individual and generated iPSCs with two excisable lentiviral vectors. Gene correction of CEP-derived iPSCs was obtained by lentiviral transduction of a therapeutic vector containing UROS cDNA under the control of an erythroid-specific promoter shielded by insulators. One iPSC clone, free of reprogramming genes, was obtained with a single proviral integration of the therapeutic vector in a genomic safe region. Metabolic correction of erythroblasts derived from iPSC clones was demonstrated by the disappearance of fluorocytes. This study reports the feasibility of porphyria gene therapy with the use of iPSCs.
Full-text · Article · Jul 2012 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: We used K562 cells sensitive or generated resistant to imatinib or nilotinib to investigate their response to mycophenolic acid (MPA). MPA induced DNA damage leading to cell death with a minor contribution of apoptosis, as revealed by annexin V labeling (up to 25%). In contrast, cell cycle arrest and positive staining for senescence-associated β-galactosidase activity were detected for a large cell population (80%). MPA-induced cell death was potentialized by the inhibition of autophagy and this is associated to the upregulation of apoptosis. In contrast, senescence was neither decreased nor abrogated in autophagy deficient K562 cells. Primary CD34 cells from CML patients sensitive or resistant to imatinib or nilotinib respond to MPA although apoptosis is mainly detected. These results show that MPA is an interesting tool to overcome resistance in vitro and in vivo mainly in the evolved phase of the disease.
[Show abstract][Hide abstract] ABSTRACT: The term 'thyroid tumors of uncertain malignant potential' (TT-UMP) was coined by surgical pathologists to define well-differentiated tumors (WDT) showing inconclusive morphological evidence of malignancy or benignity. We have analyzed the expression of microRNA (miRNA) in a training set of 42 WDT of different histological subtypes: seven follicular tumors of UMP (FT-UMP), six WDT-UMP, seven follicular thyroid adenomas (FTA), 11 conventional papillary thyroid carcinomas (C-PTC), five follicular variants of PTC (FV-PTC), and six follicular thyroid carcinomas (FTC), which led to the identification of about 40 deregulated miRNAs. A subset of these altered miRNAs was independently validated by qRT-PCR, which included 18 supplementary TT-UMP (eight WDT-UMP and ten FT-UMP). Supervised clustering techniques were used to predict the first 42 samples. Based on the four possible outcomes (FTA, C-PTC, FV-PTC, and FTC), about 80% of FTA and C-PTC and 50% of FV-PTC and FTC samples were correctly assigned. Analysis of the independent set of 18 WDT-UMP by quantitative RT-PCR for the selection of the six most discriminating miRNAs was unable to separate FT-UMP from WDT-UMP, suggesting that the miRNA signature is insufficient in characterizing these two clinical entities. We conclude that considering FT-UMP and WDT-UMP as distinct and specific clinical entities may improve the diagnosis of WDT of the thyroid gland. In this context, a small set of miRNAs (i.e. miR-7, miR-146a, miR-146b, miR-200b, miR-221, and miR-222) appears to be useful, though not sufficient per se, in distinguishing TT-UMP from other WDT of the thyroid gland.
Full-text · Article · Jul 2011 · Endocrine Related Cancer
[Show abstract][Hide abstract] ABSTRACT: Multiciliated cells lining the surface of some vertebrate epithelia are essential for various physiological processes, such as airway cleansing. However, the mechanisms governing motile cilia biosynthesis remain poorly elucidated. We identify miR-449 microRNAs as evolutionarily conserved key regulators of vertebrate multiciliogenesis. In human airway epithelium and Xenopus laevis embryonic epidermis, miR-449 microRNAs strongly accumulated in multiciliated cells. In both models, we show that miR-449 microRNAs promote centriole multiplication and multiciliogenesis by directly repressing the Delta/Notch pathway. We established Notch1 and its ligand Delta-like 1(DLL1) as miR-449 bona fide targets. Human DLL1 and NOTCH1 protein levels were lower in multiciliated cells than in surrounding cells, decreased after miR-449 overexpression and increased after miR-449 inhibition. In frog, miR-449 silencing led to increased Dll1 expression. Consistently, overexpression of Dll1 mRNA lacking miR-449 target sites repressed multiciliogenesis, whereas both Dll1 and Notch1 knockdown rescued multiciliogenesis in miR-449-deficient cells. Antisense-mediated protection of miR-449-binding sites of endogenous human Notch1 or frog Dll1 strongly repressed multiciliogenesis. Our results unravel a conserved mechanism whereby Notch signalling must undergo miR-449-mediated inhibition to permit differentiation of ciliated cell progenitors.
Full-text · Article · Jun 2011 · Nature Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Extensive sequencing efforts, combined with ad hoc bioinformatics developments, have now led to the identification of 1222 distinct miRNAs in human (derived from 1368 distinct genomic loci) and of many miRNAs in other multicellular organisms. The present chapter is aimed at describing a general experimental strategy to identify specific miRNA expression profiles and to highlight the functional networks operating between them and their mRNA targets, including several miRNAs deregulated in cystic fibrosis and during differentiation of airway epithelial cells.
Full-text · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.
Full-text · Article · Oct 2010 · Cell death and differentiation
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) act at the posttranscriptional level to control gene expression in virtually every biological process, including oncogenesis. Here, we report the identification of a set of miRNAs that are differentially regulated in childhood adrenocortical tumors (ACT), including miR-99a and miR-100. Functional analysis of these miRNAs in ACT cell lines showed that they coordinately regulate expression of the insulin-like growth factor-mammalian target of rapamycin (mTOR)-raptor signaling pathway through binding sites in their 3'-untranslated regions. In these cells, the active Ser(2448)-phosphorylated form of mTOR is present only in mitotic cells in association with the mitotic spindle and midbody in the G(2)-M phases of the cell cycle. Pharmacologic inhibition of mTOR signaling by everolimus greatly reduces tumor cell growth in vitro and in vivo. Our results reveal a novel mechanism of regulation of mTOR signaling by miRNAs, and they lay the groundwork for clinical evaluation of drugs inhibiting the mTOR pathway for treatment of adrenocortical cancer.
[Show abstract][Hide abstract] ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
Full-text · Article · Jul 2009 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: Epithelial-mesenchymal interactions are critical in regulating many aspects of vertebrate embryo development, and for the maintenance of homeostatic equilibrium in adult tissues. The interactions between epithelium and mesenchyme are believed to be mediated by paracrine signals such as cytokines and extracellular matrix components secreted from fibroblasts that affect adjacent epithelia. In this study, we sought to identify the repertoire of microRNAs (miRNAs) in normal lung human fibroblasts and their potential regulation by the cytokines TNF-α, IL-1β and TGF-β.
[Show abstract][Hide abstract] ABSTRACT: Liver ischemia-reperfusion injury occurring in orthotopic liver transplantation (OLT) may be responsible for early graft failure. Molecular mechanisms underlying initial poor graft function (IPGF) have been poorly documented in human. The purpose of this study was to identify the major transcriptional alterations occurring in human livers during OLT. Twenty-one RNA extracts derived from liver transplant biopsies taken after graft reperfusion were compared with 7 RNA derived from normal control livers. Three hundred seventy-one genes were significantly modulated and classified in molecular pathways relevant to liver metabolism, inflammatory response, cell proliferation and liver protection. Grafts were then subdivided into two groups based on their peak levels of serum aspartate amino transferase within 72 h after OLT (group 1, non-IPGF: 14 patients; group 2, IPGF: 7 patients). The two corresponding data sets were compared using a supervised prediction method. A new set of genes able to correctly classify 71% of the patients was defined. These genes were functionally associated with oxidative stress, inflammation and inhibition of cell proliferation. This study provides a comprehensive picture of the transcriptional events associated with human OLT and IPGF. We anticipate that such alterations provide a framework for the elucidation of the molecular mechanisms leading to IPGF.
Full-text · Article · Jun 2008 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: Different sensitivities to profibrotic compounds such as bleomycin are observed among mouse strains.
To identify genetic factors contributing to the outcome of lung injury.
Physiological comparison of C57BL/6 (sensitive) and BALB/c (resistant) mice challenged by intratracheal bleomycin instillation revealed several early differences: global gene expression profiles were thus established from lungs derived from the two strains, in the absence of any bleomycin administration.
Expression of 25 genes differed between the two strains. Among them, two molecules, not previously associated with pulmonary fibrosis, were identified. The first corresponded to dipeptidyl-peptidase I (DPPI), a cysteine peptidase (also known as cathepsin C) essential for the activation of serine proteinases produced by immune/inflammatory cells. The second corresponded to tissue inhibitor of matrix metalloproteinase-3, which also inhibits members of the ADAM (a disintegrin and metalloproteinase) family, such as the tumor necrosis factor-converting enzyme. In functional studies performed in the bleomycin-induced lung fibrosis model, the level of expression of these two genes was closely correlated with specific early events associated with lung fibrosis, namely activation of polymorphonuclear neutrophil-derived serine proteases and tumor necrosis factor-alpha-dependent inflammatory syndrome. Surprisingly, genetic deletion of DPPI in the context of a C57BL/6 genetic background did not protect against bleomycin-mediated fibrosis, suggesting additional function(s) for this key enzyme.
This study highlights the importance of the early inflammatory events that follow bleomycin instillation in the development of lung fibrosis, and describes for the first time the roles that DPPI and tissue inhibitor of matrix metalloproteinase-3 may play in this process.
No preview · Article · Jan 2008 · American Journal of Respiratory and Critical Care Medicine
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas aeruginosa is a gram-negative bacilli frequently encountered in human pathology. This pathogen is involved in a large number of nosocomial infections and chronic diseases. Herein we investigated the effects of polyunsaturated fatty acids (PUFA) in chronic Pseudomonas aeruginosa lung infection. C57BL/6 mice were fed for 5 wk with specifically designed diets with high contents in either omega-3 (omega-3) or omega-6 PUFA and compared to a control diet. P. aeruginosa included in agarose beads was then instilled intratracheally, and the animals were studied for 7 days. On the 4th day, the mice fed with the omega-3 diet had a higher lean body mass gain and a lower omega-6:omega-3 ratio of fatty acids extracted from the lung tissue compared with the other groups (P < 0.05). The omega-3 group had the lowest mortality. Distal alveolar fluid clearance (DAFC) as well as the inflammatory response and the cellular recruitment were higher in the omega-3 group on the 4th day. The effect on DAFC was independent of alpha-epithelial Na(+) channels (alpha-ENaC), beta-ENaC, and alpha(1)-Na-K-ATPase mRNA expressions, which were not altered by the different diets. In conclusion, a diet enriched in omega-3 PUFA can change lung membrane composition and improve survival in chronic pneumonia. This effect on survival is probably multifactorial involving the increased DAFC capacity as well as the optimization of the initial inflammatory response. This work suggests that a better control of the omega-6/omega-3 PUFA balance may represent an interesting target in the prevention and/or control of P. aeruginosa infection in patients.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host
cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery
in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication
both in peripheral blood mononuclear cells from HIV-1–infected donors and in latently infected cells. In turn, HIV-1 actively
suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient
viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement
of the miRNA-silencing pathway in HIV-1 replication and latency.
[Show abstract][Hide abstract] ABSTRACT: A comparative analysis of the structure of the melanin-concentrating hormone (MCH) precursor reveals that this sequence has been subjected to a higher selection pressure in mammals than in teleosts, suggesting that the structural constraints have not been the same throughout the vertebrate lineage. In contrast, the MCH peptide sequence has been very well conserved in all species. A sensitive and reproducible eel skin assay was developed and allowed us to define the structural features needed for a full MCH bioactivity. It was shown that the minimal structure carrying the critical residues was the same in fishes and in mammals. A pharmacological approach confirmed that MCH receptor activation decreased the cAMP levels in the fish skin, but this effect appeared to be independent from a Galphai protein. We propose that one of the intracellular signaling pathways of the MCH receptor in fish skin is the activation of one or several cellular phosphodiesterases.
[Show abstract][Hide abstract] ABSTRACT: The melanin-concentrating hormone (MCH) gene encodes two proteins, pro-MCH and MCH-gene-overprinted polypeptide (MGOP), produced through alternative splicing of the primary transcript. Our initial purpose was to characterize the MGOP-immunoreactive material. First, MGOP mRNA was clearly found in rat and mouse hypothalami but Western blot analysis failed to unambiguously identify MGOP in protein extracts. Immunohistochemical experiments with wild-type and MCH gene-null mice demonstrated genuine expression of MGOP confined to the MCH-containing neurons in the lateral hypothalamus area and the presence of an 'MGOP-like' antigen in periventricular nucleus and arcuate nucleus neurons and their area of projection. This suggested a colocalization in somatostatin (SRIF) hypophysiotropic neurons. Further characterization, using SRIF gene-null mice and Western blot analysis with recombinant proteins, revealed that the MGOP-like product was pro-SRIF1-64. The role of pro-SRIF1-64 on fetal hypothalamic neurons was evaluated and a strong tonic inhibitory effect on SRIF secretion was found. These results (i) indicate that MGOP expression is restricted to the MCH neurons in the lateral hypothalamus and that MGOP-like immunoreactivity outside this system corresponds to pro-SRIF1-64, and (ii) provide the first evidence for a negative feedback regulation by pro-SRIF1-64 on SRIF secretion, suggesting new mechanisms by which the pro-region of a neuropeptide precursor may control the regulated secretion of a neuropeptide derived from the same precursor.
No preview · Article · Mar 2004 · European Journal of Neuroscience