The Long Noncoding RNA DNM3OS Is a Reservoir of FibromiRs with Major Functions in Lung Fibroblast Response to TGF-ß and Pulmonary Fibrosis.

Rationale: Given the paucity of effective treatments for idiopathic pulmonary fibrosis (IPF), new insights into the deleterious mechanisms controlling lung fibroblast activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies. TGF-ß (transforming growth factor-ß) is the main profibrotic factor, but its inhibition is associated with severe side effects because of its pleiotropic role. Objectives: To determine if downstream noncoding effectors of TGF-ß in fibroblasts may represent new effective therapeutic targets whose modulation may be well tolerated. Methods: We investigated the whole noncoding fraction of TGF-ß-stimulated lung fibroblast transcriptome to identify new genomic determinants of lung fibroblast differentiation into myofibroblasts. Differential expression of the long noncoding RNA (lncRNA) DNM3OS (dynamin 3 opposite strand) and its associated microRNAs (miRNAs) was validated in a murine model of pulmonary fibrosis and in IPF tissue samples. Distinct and complementary antisense oligonucleotide-based strategies aiming at interfering with DNM3OS were used to elucidate the role of DNM3OS and its associated miRNAs in IPF pathogenesis. Measurements and Main Results: We identified DNM3OS as a fibroblast-specific critical downstream effector of TGF-ß-induced lung myofibroblast activation. Mechanistically, DNM3OS regulates this process in trans by giving rise to three distinct profibrotic mature miRNAs (i.e., miR-199a-5p/3p and miR-214-3p), which influence SMAD and non-SMAD components of TGF-ß signaling in a multifaceted way. In vivo, we showed that interfering with DNM3OS function not only prevents lung fibrosis but also improves established pulmonary fibrosis. Conclusions: Pharmacological approaches aiming at interfering with the lncRNA DNM3OS may represent new effective therapeutic strategies in IPF.

PIWI-interacting RNAs as novel regulators of pancreatic beta cell function.

AIMS/HYPOTHESIS: P-element induced Wimpy testis (PIWI)-interacting RNAs (piRNAs) are small non-coding RNAs that interact with PIWI proteins and guide them to silence transposable elements. They are abundantly expressed in germline cells and play key roles in spermatogenesis. There is mounting evidence that piRNAs are also present in somatic cells, where they may accomplish additional regulatory tasks. The aim of this study was to identify the piRNAs expressed in pancreatic islets and to determine whether they are involved in the control of beta cell activities. METHODS: piRNA profiling of rat pancreatic islets was performed by microarray analysis. The functions of piRNAs were investigated by silencing the two main Piwi genes or by modulating the level of selected piRNAs in islet cells. RESULTS: We detected about 18,000 piRNAs in rat pancreatic islets, many of which were differentially expressed throughout islet postnatal development. Moreover, we identified changes in the level of several piRNAs in the islets of Goto-Kakizaki rats, a well-established animal model of type 2 diabetes. Silencing of Piwil2 or Piwil4 genes in adult rat islets caused a reduction in the level of several piRNAs and resulted in defective insulin secretion and increased resistance of the cells to cytokine-induced cell death. Furthermore, overexpression in the islets of control animals of two piRNAs that are upregulated in diabetic rats led to a selective defect in glucose-induced insulin release. CONCLUSIONS/INTERPRETATION: Our results provide evidence for a role of PIWI proteins and their associated piRNAs in the control of beta cell functions, and suggest a possible involvement in the development of type 2 diabetes. DATA AVAILABILITY: Data have been deposited in Gene Expression Omnibus repository under the accession number GSE93792. Data can be accessed via the following link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ojklueugdzehpkv&acc=GSE93792.

MicroRNA-375/SEC23A as biomarkers of the in vitro efficacy of vandetanib.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib.Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: Reproducibility and predictivity results from an inter-laboratory study.

The SENS-IS test protocol for the in vitro detection of sensitizers is based on a reconstructed human skin model (Episkin) as the test system and on the analysis of the expression of a large panel of genes. Its excellent performance was initially demonstrated with a limited set of test chemicals. Further studies (described here) were organized to confirm these preliminary results and to obtain a detailed statistical analysis of the predictive capacity of the assay. A ring-study was thus organized and performed within three laboratories, using a test set of 19 blind coded chemicals. Data analysis indicated that the assay is robust, easily transferable and offers high predictivity and excellent within- and between-laboratories reproducibility. To further evaluate the predictivity of the test protocol according to Cooper statistics a comprehensive test set of 150 chemicals was then analyzed. Again, data analysis confirmed the excellent capacity of the SENS-IS assay for predicting both hazard and potency characteristics, confirming that this assay should be considered as a serious alternative to the available in vivo sensitization tests.

miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFß exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFß signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.

"Seed-Milarity" confers to hsa-miR-210 and hsa-miR-147b similar functional activity.

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.

Spt6 levels are modulated by PAAF1 and proteasome to regulate the HIV-1 LTR.

BACKGROUND: Tat-mediated activation of the HIV-1 promoter depends upon a proteasome-associated factor, PAAF1, which dissociates 26S proteasome to produce 19S RP that is essential for transcriptional elongation. The effect of PAAF1 on proteasome activity could also potentially shield certain factors from proteolysis, which may be implicated in the transcriptional co-activator activity of PAAF1 towards the LTR. RESULTS: Here, we show that Spt6 is targeted by proteasome in the absence of PAAF1. PAAF1 interacts with the N-terminus of Spt6, suggesting that PAAF1 protects Spt6 from proteolysis. Depletion of either PAAF1 or Spt6 reduced histone occupancy at the HIV-1 promoter, and induced the synthesis of aberrant transcripts. Ectopic Spt6 expression or treatment with proteasome inhibitor partially rescued the transcription defect associated with loss of PAAF1. Transcriptional profiling followed by ChIP identified a subset of cellular genes that are regulated in a similar fashion to HIV-1 by Spt6 and/or PAAF1, including many that are involved in cancer, such as BRCA1 and BARD1. CONCLUSION: These results show that intracellular levels of Spt6 are fine-tuned by PAAF1 and proteasome, which is required for HIV-1 transcription and extends to cellular genes implicated in cancer.