Search

Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15–20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.

The aged brain is associated with an inevitable decline in cognitive function and increased vulnerability to neurodegenerative disorders. Multiple molecular hallmarks have been associated with the aging nervous system through transcriptomics and proteomic studies. Recently, epitranscriptomic analysis has highlighted the role of RNA chemical modification in various biological processes. In particular, N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNAs, has been functionally linked to multiple aspects of RNA metabolism with the roles of m6A in processes such as learning and memory, leading to our current investigation of how the m6A-transcriptomic landscape is shaped during aging. Using the inbred C57BL/6 line, we compared the m6A-transcriptomic profiles from the hippocampi of young (3-month-old) and aged (20-month-old) mice. Methylated RNA immunoprecipitation (MeRIP)-sequencing analysis revealed hyper- and hypomethylation in 426 and 102 genes, respectively, in the aged hippocampus (fold change1.5, false discovery rate 0.05). By correlating the methylation changes to their steady-state transcript levels in the RNA-Seq data, we found a significant concordance between m6A and transcript levels in both directions. Notably, the myelin regulator gene Gpr17 was downregulated in the aged hippocampus concomitant with reduced m6A levels in its 3'UTR. Using reporter constructs and mutagenesis analysis, we demonstrated that the putative m6A sites in the 3'UTR of Gpr17 are important for mRNA translation but not for regulating transcript stability. Overall, the positive correlation between m6A and the transcript expression levels indicates a co-transcriptional regulation of m6A with gene expression changes that occur in the aged mouse hippocampus.

BackgroundRNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. To date, most studies have focused on m6A, while other RNA modifications, including 5hmC, remain poorly characterised. The interplay between different RNA modifications that may occur in specific cellular contexts remains similarly unexplored.ResultsWe profiled m6A and 5hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. We observed that decreased expression of m6A and 5hmC writers, METTL3 and TET-enzymes respectively, facilitated monocyte-to-macrophage differentiation. Despite a global trend of m6A and 5hmC loss during macrophage differentiation, enrichment of m6A and/or 5hmC on specific categories of transcripts essential for macrophage differentiation positively correlated with their expression and translation. m6A and 5hmC mark and are associated with the expression of transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discovered the coexistence of m6A and 5hmC marking alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of transcripts with key roles in macrophage biology. In specific examples, RNA 5hmC controls the decay of transcripts independently of m6A.ConclusionsThis study: i) uncovers m6A, 5hmC and their writer enzymes as regulators of monocyte and macrophage gene expression programs and ii) provides a comprehensive dataset to interrogate the role of RNA modifications in a plastic system. Altogether, this work sheds light on the role of RNA modifications as central regulators of effector cells in innate immunity.

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type- or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (mRNA-Seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning, we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role for chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing.

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment., SCOPUS: ar.j, info:eu-repo/semantics/published

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.

Monocytes play a crucial role in maintaining homeostasis and mediating a successful innate immune response. They also act as central players in diverse pathological conditions, thus making them an attractive therapeutic target. Within the bone marrow, monocytes arise from a committed precursor termed Common Monocyte Progenitor (cMoP). However, molecular mechanisms that regulate the differentiation of cMoP to various monocytic subsets remain unclear. Herein, we purified murine myeloid precursors for deep poly-A-enriched RNA sequencing to understand the role of alternative splicing in the development and differentiation of monocytes under homeostasis. Our analyses revealed intron retention to be the major alternative splicing mechanism involved in the monocyte differentiation cascade, especially in the differentiation of Ly6Chi monocytes to Ly6Clo monocytes. Furthermore, we found that the intron retention of key genes involved in the differentiation of murine Ly6Chi to Ly6Clo monocytes was also conserved in humans. Our data highlight the unique role of intron retention in the regulation of the monocytic differentiation pathway.

The liver has complex interconnecting blood vessel and biliary networks; however, how the vascular and biliary network form and regulate each other and liver function are not well-understood. We aimed to examine the role of Heg in mammalian liver development and functional maintenance.Global (HegHeg deficiency in liver ECs led to a sparse liver vascular and biliary network. This network paucity does not compromise liver function under baseline conditions but did alter liver zonation. Molecular analysis revealed that endothelial Heg deficiency decreased expression of Wnt ligands/agonists including Wnt2, Wnt9b, and Rspo3 in ECs, which limits Axin2 mediated canonical Wnt signaling and the expression of cytochrome P450 enzymes in hepatocytes. Under chemical-induced stressed conditions, Heg-deficiency in liver ECs protected mice from drug-induced liver injuries.Our study found that endothelial Heg is essential for the 3-D patterning of the liver vascular and indirectly regulates biliary networks and proper liver zonation via its regulation of Wnt ligand production in liver endothelial cells. The endothelial Heg-initiated changes of the liver metabolic zonation and metabolic enzyme expression in hepatocytes was functionally relevant to xenobiotic metabolism and drug induced liver toxicity.

During ontogeny, macrophages develop from CD115+precursors, including erythro-myeloid progenitors (EMP). EMP arise in the embryonic yolk sac, the primary site of early haematopoiesis. In adults, CD115+bone marrow-derived monocytes represent essential macrophage precursors. Herein, we identify a CD115-macrophage precursor within the adult bone marrow that is unrelated to the classical monocyte lineage but rather shares transcriptomic and functional characteristics of embryonic EMP. These EMPROR (forErythroMyeloidPrecursor) cells are capable of efficiently generating macrophages in disease settings. During early development, EMPROR cells were largely absent from the yolk sac but were instead found at the embryonic-maternal interface in the uterine wall. Unexpectedly, the latter site contains robust haematopoietic activity and harbours defined embryonic haematopoietic progenitor cells, including classical CD115+EMP. Our data suggest the existence of an alternative pathway of macrophage generation in the adult. Further, we uncover a hitherto unknown site of earliest blood cell development.

Background and Purpose: Cerebral cavernous malformation (CCM) is a common cerebrovascular disease. CCMs are major causes of stroke, cerebral hemorrhage, and neurological deficits in young individuals. Loss-of-function mutations in CCM1 , CCM2 , and CCM3 have been identified to cause CCM in humans. Ccm2-like ( Ccm2l ) is a paralog of Ccm2 and is predominantly expressed in endothelial cells (ECs). CCM2L (CCM2-like) competes with CCM2 for binding to CCM1 and has been shown to have an antagonistic function to that of CCM2 during vascular development. The role of CCM2L in CCM pathogenesis is unknown. Methods: We isolated brain ECs from the inducible-CCM mouse models for gene expression analysis. Micro-computed tomography imaging was used to analyze CCM lesion burden from the genetic cross of Ccm2l knockout mice ( Ccm2l −/− ) with Ccm1 or Ccm2 -deficient mice to determine the role of Ccm2l in CCM pathogenesis. Genetic crosses with Map3k3 fl/fl mice were used to determine the role of Map3k3 in Ccm2l -facilitated CCM formation. Results: We demonstrated increased Ccm2l expression in brain ECs of Ccm2 -deficient mice. Analysis of RNA-seq data from CCM patient samples revealed a trend of increased CCM2L expression and its positive correlation with Kruppel-like factor 2/4 (KLF2/4 ) expression. Micro-computed tomography revealed that the deletion of Ccm2l in Ccm2 -deficient mice increased CCM lesion volume compared with that of controls but had no effect on lesion numbers. Correlating to the increased lesion burden, Klf2/4 mRNA expressions in brain ECs were significantly increased in double knockouts ( Ccm2 - and Ccm2l -deficient mice) compared with that of controls ( Ccm2 deficient). Hemizygous deletion of Map3k3 in ECs relieved CCM lesion burden in the double knockouts. These results suggest that CCM2L regulates the Map3k3-KLF signaling pathway in CCM pathogenesis. Conclusions: Loss of CCM2L aggravates CCM lesion formation in the Ccm2 -deficient mouse model through increased Map3k3-KLF signaling. Our data suggest that increased Ccm2l expression is a compensatory mechanism in CCM pathogenesis.

Various types of mutation and editing (M/E) events in microRNAs (miRNAs) can change the stabilities of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles can contain many mutated and edited miRNAs. Systematic detection of miRNA mutation and editing sites from the huge volume of sRNA HTS profiles is computationally difficult, as high sensitivity and low false positive rate (FPR) are both required. We propose a novel method (named MiRME) for an accurate and fast detection of miRNA M/E sites using a progressive sequence alignment approach which refines sensitivity and improves FPR step-by-step. From 70 sRNA HTS profiles with over 1.3 billion reads, MiRME has detected thousands of statistically significant M/E sites, including 3'-editing sites, 57 A-to-I editing sites (of which 32 are novel), as well as some putative non-canonical editing sites. We demonstrated that a few non-canonical editing sites were not resulted from mutations in genome by integrating the analysis of genome HTS profiles of two human cell lines, suggesting the existence of new editing types to further diversify the functions of miRNAs. Compared with six existing studies or methods, MiRME has shown much superior performance for the identification and visualization of the M/E sites of miRNAs from the ever-increasing sRNA HTS profiles.

Neuroblastoma is the most common solid tumor during early childhood. One of the key features of neuroblastoma is extensive tumor-driven angiogenesis due to hypoxia. However, the mechanism through which neuroblastoma cells drive angiogenesis is poorly understood. Here we show that the long noncoding RNA MALAT1 was upregulated in human neuroblastoma cell lines under hypoxic conditions. Conditioned media from neuroblastoma cells transfected with small interfering RNAs (siRNA) targeting MALAT1, compared with conditioned media from neuroblastoma cells transfected with control siRNAs, induced significantly less endothelial cell migration, invasion and vasculature formation. Microarray-based differential gene expression analysis showed that one of the genes most significantly down-regulated following MALAT1 suppression in human neuroblastoma cells under hypoxic conditions was fibroblast growth factor 2 (FGF2). RT-PCR and immunoblot analyses confirmed that MALAT1 suppression reduced FGF2 expression, and Enzyme-Linked Immunosorbent Assays revealed that transfection with MALAT1 siRNAs reduced FGF2 protein secretion from neuroblastoma cells. Importantly, addition of recombinant FGF2 protein to the cell culture media reversed the effects of MALAT1 siRNA on vasculature formation. Taken together, our data suggest that up-regulation of MALAT1 expression in human neuroblastoma cells under hypoxic conditions increases FGF2 expression and promotes vasculature formation, and therefore plays an important role in tumor-driven angiogenesis.

N-Myc gene amplification occurs in one quarter of human neuroblastoma tissues, and is a marker for poor patient prognosis. We performed RNA sequencing experiments, and identified 5 transcripts, including RP1NB1, which were most considerably differentially expressed between N-Myc gene amplified and nonamplified human neuroblastoma cell lines. Affymetrix microarray studies revealed that DEPD was one of the few genes considerably downregulated in neuroblastoma cells after RP1NB1 depletion. Chromatin immunoprecipitation assays showed that knocking down RP1NB1 expression reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the DEPD gene promoter. Luciferase assays demonstrated that knocking down RP1NB1 decreased DEPD gene promoter activity. Depletion of RP1NB1 or DEPD with two independent siRNAs or shRNAs significantly reduced ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, N-Myc protein stabilization, neuroblastoma cell proliferation and survival. Clonogenic assays showed that knocking down RP1NB1 with doxycycline completely abolished colony formation capacity of neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNAs. Importantly, treatment with doxycycline in mice xenografted with neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNA led to tumor eradication. In human neuroblastoma tissues from 600 neuroblastoma patients, high levels of RP1NB1 gene expression correlated with DEPD gene expression and poor patient prognosis. In conclusion, this study identifies the novel long noncoding RNA RP1NB1 as an important regulator of N-Myc protein stability and neuroblastoma tumorigenesis. Citation Format: Andrew E. Tee, Pei Y. Liu, Giorgio Milazzo, Kate M. Hannan, Jesper Maag, Nenad Bartonicek, Renhua Song, Chen C. Jiang, Xu D. Zhang, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Jinyan Li, Jo Vandesompele, John S. Mattick, Pieter Mestdagh, Giovanni Perini, Ross D. Hannan, Marcel E. Dinger, Tao Liu. Eradication of neuroblastoma by suppressing the expression of a single noncoding RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2453.

© 2014 Song et al. Background: Altered expression profiles of microRNAs (miRNAs) are linked to many diseases including lung cancer. miRNA expression profiling is reproducible and miRNAs are very stable. These characteristics of miRNAs make them ideal biomarker candidates. Method: This work is aimed to detect 2-and 3-miRNA groups, together with specific expression ranges of these miRNAs, to form simple linear discriminant rules for biomarker identification and biological interpretation. Our method is based on a novel committee of decision trees to derive 2-and 3-miRNA 100%-frequency rules. This method is applied to a data set of lung miRNA expression profiles of 61 squamous cell carcinoma (SCC) samples and 10 normal tissue samples. A distance separation technique is used to select the most reliable rules which are then evaluated on a large independent data set. Results: We obtained four 2-miRNA and three 3-miRNA top-ranked rules. One important rule is that: If the expression level of miR-98 is above 7.356 and the expression level of miR-205 is below 9.601 (log2 quantile normalized MirVan miRNA Bioarray signals), then the sample is normal rather than cancerous with specificity and sensitivity both 100%. The classification performance of our best miRNA rules remarkably outperformed that by randomly selected miRNA rules. Our data analysis also showed that miR-98 and miR-205 have two common predicted target genes FZD3 and RPS6KA3, which are actually genes associated with carcinoma according to the Online Mendelian Inheritance in Man (OMIM) database. We also found that most of the chromosomal loci of these miRNAs have a high frequency of genomic alteration in lung cancer. On the independent data set (with balanced controls), the three miRNAs miR-126, miR-205 and miR-182 from our best rule can separate the two classes of samples at the accuracy of 84.49%, sensitivity of 91.40% and specificity of 77.14%. Conclusion: Our results indicate that rule discovery followed by distance separation is a powerful computational method to identify reliable miRNA biomarkers. The visualization of the rules and the clear separation between the normal and cancer samples by our rules will help biology experts for their analysis and biological interpretation.

Co-regulations of miRNAs have been much less studied than the research on regulations between miRNAs and their target genes, although these two problems are equally important for understanding the entire mechanisms of complex post-transcriptional regulations. The difficulty to construct a miRNA–miRNA co-regulation network lies in how to determine reliable miRNA pairs from various resources of data related to the same disease such as expression levels, gene ontology (GO) databases, and protein–protein interactions. Here we take a novel integrative approach to the discovery of miRNA–miRNA co-regulation networks. This approach can progressively refine the various types of data and the computational analysis results. Applied to three lung cancer miRNA expression data sets of different subtypes, our method has identified a miRNA–miRNA co-regulation network and co-regulating functional modules common to lung cancer. An example of these functional modules consists of genes SMAD2, ACVR1B, ACVR2A and ACVR2B. This module is synergistically regulated by let-7a/b/c/f, is enriched in the same GO category, and has a close proximity in the protein interaction network. We also find that the co-regulation network is scale free and that lung cancer related miRNAs have more synergism in the network. According to our literature survey and database validation, many of these results are biologically meaningful for understanding the mechanism of the complex post-transcriptional regulations in lung cancer.

N-Myc gene amplification occurs in one quarter of human neuroblastoma tissues, and is a marker for poor patient prognosis. We performed RNA sequencing experiments, and identified 5 transcripts, including RP1NB1, which were most considerably differentially expressed between N-Myc gene amplified and nonamplified human neuroblastoma cell lines. Affymetrix microarray studies revealed that DEPD was one of the few genes considerably downregulated in neuroblastoma cells after RP1NB1 depletion. Chromatin immunoprecipitation assays showed that knocking down RP1NB1 expression reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the DEPD gene promoter. Luciferase assays demonstrated that knocking down RP1NB1 decreased DEPD gene promoter activity. Depletion of RP1NB1 or DEPD with two independent siRNAs or shRNAs significantly reduced ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, N-Myc protein stabilization, neuroblastoma cell proliferation and survival. Clonogenic assays showed that knocking down RP1NB1 with doxycycline completely abolished colony formation capacity of neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNAs. Importantly, treatment with doxycycline in mice xenografted with neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNA led to tumor eradication. In human neuroblastoma tissues from 600 neuroblastoma patients, high levels of RP1NB1 gene expression correlated with DEPD gene expression and poor patient prognosis. In conclusion, this study identifies the novel long noncoding RNA RP1NB1 as an important regulator of N-Myc protein stability and neuroblastoma tumorigenesis. Citation Format: Andrew E. Tee, Pei Y. Liu, Giorgio Milazzo, Kate M. Hannan, Jesper Maag, Nenad Bartonicek, Renhua Song, Chen C. Jiang, Xu D. Zhang, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Jinyan Li, Jo Vandesompele, John S. Mattick, Pieter Mestdagh, Giovanni Perini, Ross D. Hannan, Marcel E. Dinger, Tao Liu. Eradication of neuroblastoma by suppressing the expression of a single noncoding RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2453.

Neuroblastoma is the most common solid tumour during early childhood, and accounts for 15% of all childhood cancer death. One of the key features of neuroblastoma is aggressive progression due to tumour-driven angiogenesis. However, the mechanism through which neuroblastoma cells drive angiogenesis is unclear. Here we showed that the long noncoding RNA MALAT1 was over-expressed in human neuroblastoma cells under hypoxic condition, the condition which triggers pro-angiogenic switch and angiogenesis. In vitro angiogenesis assays demonstrated that conditioned medium from neuroblastoma cells transfected with MALAT1 siRNAs, compared with conditioned medium from neuroblastoma cells transfected with control siRNAs, induced considerably less endothelial cell migration, invasion and vascular sprouting under hypoxic condition. Affymetrix microarray differential gene expression study showed that one of the genes most significantly down-regulated by MALAT1 siRNAs in human neuroblastoma cells under hypoxic condition, was fibroblast growth factor (FGF). RT-PCR and immunoblot analyses confirmed that MALAT1 siRNAs reduced FGF mRNA and protein expression, and in vitro angiogenesis assays demonstrated that forced over-expression of FGF in neuroblastoma cells blocked MALAT1 siRNA-mediated reduction in endothelial cell migration, invasion and vascular sprouting. Taken together, our data suggest that over-expression of MALAT1 in human neuroblastoma cells plays an important role hypoxia-driven angiogenesis by up-regulating FGF mRNA expression. Citation Format: Andrew E. Tee, Peiyan Liu, Jesper Maag, Renhua Song, Jinyan Li, Belamy B. Cheung, Michelle Haber, Murray D. Norris, Glenn M. Marshall, Marcel Dinger, Tao Liu. The long noncoding RNA MALAT1 promotes hypoxia-driven angiogenesis by upregulating pro-angiogenic gene expression in neuroblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 146. doi:10.1158/1538-7445.AM2015-146

A convenient synthesis of 5-fluorofuran-2-carboxylic acid has been achieved in two steps and 56% total yield. Fluorodenitration of commercially available benzyl 5-nitrofuran-2-carboxylate utilizing potassium fluoride and catalytic tetraphenylphosphonium bromide in sulfolane at 140 °C for 2 h furnished benzyl 5-fluorofuran-2-carboxylate. Hydrogenolysis of benzyl 5-fluorofuran-2-carboxylate gave the title compound.

© 2015 Song et al.; licensee BioMed Central Ltd. Background: Intensive research based on the inverse expression relationship has been undertaken to discover the miRNA-mRNA regulatory modules involved in the infection of Hepatitis C virus (HCV), the leading cause of chronic liver diseases. However, biological studies in other fields have found that inverse expression relationship is not the only regulatory relationship between miRNAs and their targets, and some miRNAs can positively regulate a mRNA by binding at the 5' UTR of the mRNA.Results: This work focuses on the detection of both inverse and positive regulatory relationships from a paired miRNA and mRNA expression data set of HCV patients through a 'change-to-change' method which can derive connected discriminatory rules. Our study uncovered many novel miRNA-mRNA regulatory modules. In particular, it was revealed that GFRA2 is positively regulated by miR-557, miR-765 and miR-17-3p that probably bind at different locations of the 5' UTR of this mRNA. The expression relationship between GFRA2 and any of these three miRNAs has not been studied before, although separate research for this gene and these miRNAs have all drawn conclusions linked to hepatocellular carcinoma. This suggests that the binding of mRNA GFRA2 with miR-557, miR-765, or miR-17-3p, or their combinations, is worthy of further investigation by experimentation. We also report another mRNA QKI which has a strong inverse expression relationship with miR-129 and miR-493-3p which may bind at the 3' UTR of QKI with a perfect sequence match. Furthermore, the interaction between hsa-miR-129-5p (previous ID: hsa-miR-129) and QKI is supported with CLIP-Seq data from starBase. Our method can be easily extended for the expression data analysis of other diseases.Conclusion: Our rule discovery method is useful for integrating binding information and expression profile for identifying HCV miRNA-mRNA regulatory modules and can be applied to the study of the expression profiles of other complex human diseases.

© 2015 Liu et al. Motivation: Deciphering gene interaction networks (GINs) from time-course gene expression (TCGx) data is highly valuable to understand gene behaviors (e.g., activation, inhibition, time-lagged causality) at the system level. Existing methods usually use a global or local proximity measure to infer GINs from a single dataset. As the noise contained in a single data set is hardly self-resolved, the results are sometimes not reliable. Also, these proximity measurements cannot handle the co-existence of the various in vivo positive, negative and time-lagged gene interactions. Methods and results: We propose to infer reliable GINs from multiple TCGx datasets using a novel conserved subsequential pattern of gene expression. A subsequential pattern is a maximal subset of genes sharing positive, negative or time-lagged correlations of one expression template on their own subsets of time points. Based on these patterns, a GIN can be built from each of the datasets. It is assumed that reliable gene interactions would be detected repeatedly. We thus use conserved gene pairs from the individual GINs of the multiple TCGx datasets to construct a reliable GIN for a species. We apply our method on six TCGx datasets related to yeast cell cycle, and validate the reliable GINs using protein interaction networks, biopathways and transcription factor-gene regulations. We also compare the reliable GINs with those GINs reconstructed by a global proximity measure Pearson correlation coefficient method from single datasets. It has been demonstrated that our reliable GINs achieve much better prediction performance especially with much higher precision. The functional enrichment analysis also suggests that gene sets in a reliable GIN are more functionally significant. Our method is especially useful to decipher GINs from multiple TCGx datasets related to less studied organisms where little knowledge is available except gene expression data.