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Abstract Lung cancer (LC) mortality rates are still increasing globally. As survival is linked to stage, there is a need to identify markers for earlier LC diagnosis and individualized treatment. The whole blood transcriptome of LC patients represents a source of potential LC biomarkers. We compared expression of > 60,000 genes in whole blood specimens taken from LC cases at diagnosis (n = 128) and controls (n = 62) using genome-wide RNA sequencing, and identified 14 candidate genes associated with LC. High expression of ANXA3, ARG1 and HP was strongly associated with lower survival in late-stage LC cases (hazard ratios (HRs) = 2.81, 2.16 and 2.54, respectively). We validated these markers in two independent population-based studies with pre-diagnostic whole blood specimens taken up to eight years prior to LC diagnosis (n = 163 cases, 184 matched controls). ANXA3 and ARG1 expression was strongly associated with LC in these specimens, especially with late-stage LC within two years of diagnosis (odds ratios (ORs) = 3.47 and 5.00, respectively). Additionally, blood CD4 T cells, NK cells and neutrophils were associated with LC at diagnosis and improved LC discriminative ability beyond candidate genes. Our results indicate that in whole blood, increased expression levels of ANXA3, ARG1 and HP are diagnostic and prognostic markers of late-stage LC.

Accurate investigations of neural circuitry require specific genetic access to individual circuit elements, i.e., the myriad neuronal cell-types in the brain. However, native promoters cannot achieve this because while most genes are expressed in the brain, few are expressed in a single neuronal cell-type. We recently used enhancers, the subcomponents of the transcriptional apparatus which tell promoters when and where to express, combined with heterologous minimal promoters to increase specificity of transgene expression, an approach we call Enhancer-Driven Gene Expression (EDGE). As we discuss, EDGE is a marked improvement in specificity over native promoters, but still requires careful anatomical analysis to avoid off-target effects. In this study we present a more complete set of genomic markers from the mouse brain and characterize a novel EDGE viral vector capable of specifically driving expression in distinct subtypes of hippocampal neurons, even though it can express in other cell-types elsewhere. The advent of cell-type specific viral tools in wild-type animals provides a powerful strategy for neural circuit investigation and holds promise for studies using animal models for which transgenic tools are not available.

ABSTRACTPhenotypic plasticity of metabolism and growth are essential for adaptation to new environmental conditions, such as those experienced during domestication. Epigenetic regulation plays a key role in this process but the underlying mechanisms are poorly understood, especially in the case of hydroxymethylation. Using reduced representation 5-hydroxymethylcytosine profiling, we compared the liver hydroxymethylomes in full-sib Nile tilapia with distinct growth rates (3.8-fold difference) and demonstrated that DNA hydroxymethylation is strongly associated with phenotypic divergence of somatic growth during the early stages of domestication. The 2677 differentially hydroxymethylated cytosines between fast- and slow-growing fish were enriched within gene bodies (79%), indicating a pertinent role in transcriptional regulation. Moreover, they were found in genes involved in biological processes related to skeletal system and muscle structure development, and there was a positive association between somatic growth and 5hmC levels in genes coding for growth factors, kinases and receptors linked to myogenesis. Single nucleotide polymorphism analysis revealed no genetic differentiation between fast- and slow-growing fish. In addition to unveiling a new link between DNA hydroxymethylation and epigenetic regulation of growth in fish during the initial stages of domestication, this study suggests that epimarkers may be applied in selective breeding programmes for superior phenotypes.

Abstract Background Context-dependent transcription factor (TF) binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

Abstract Background Transcription factor binding to DNA requires both an appropriate binding element and suitably open chromatin, which together help to define regulatory elements within the genome. Current methods of identifying regulatory elements, such as promoters or enhancers, typically rely on sequence conservation, existing gene annotations or specific marks, such as histone modifications and p300 binding methods, each of which has its own biases. Results Herein we show that an approach based on clustering of transcription factor peaks from high-throughput sequencing coupled with chromatin immunoprecipitation (Chip-Seq) can be used to evaluate markers for regulatory elements. We used 67 data sets for 54 unique transcription factors distributed over two cell lines to create regulatory element clusters. By integrating the clusters from our approach with histone modifications and data for open chromatin, we identified general methylation of lysine 4 on histone H3 (H3K4me) as the most specific marker for transcription factor clusters. Clusters mapping to annotated genes showed distinct patterns in cluster composition related to gene expression and histone modifications. Clusters mapping to intergenic regions fall into two groups either directly involved in transcription, including miRNAs and long noncoding RNAs, or facilitating transcription by long-range interactions. The latter clusters were specifically enriched with H3K4me1, but less with acetylation of lysine 27 on histone 3 or p300 binding. Conclusion By integrating genomewide data of transcription factor binding and chromatin structure and using our data-driven approach, we pinpointed the chromatin marks that best explain transcription factor association with different regulatory elements. Our results also indicate that a modest selection of transcription factors may be sufficient to map most regulatory elements in the human genome.

Abstract Background Despite experiments showing that the number of microRNA (miRNA) target sites is critical for miRNA targeting, most existing methods focus on identifying individual miRNA target sites and do not model contributions of multiple target sites to miRNA regulation. To address this possible fault, we developed a miRNA target prediction model that recognizes the individual characteristics of functional binding sites and the global characteristics of miRNA-targeted mRNAs. Results Benchmark experiments showed that this two-step model generally had a higher overall performance than other established miRNA target prediction algorithms and that the model was especially suited to identify true miRNA targets among genes that all contain conserved target sites. Conclusions This improved performance could partly be explained by the model not relying on conservation when predicting targets. The critical factors for the model's performance, however, were mRNA-level features that characterized the number and strength of individual target sites within the mRNA. The model is available for online predictions or as pre-computed predictions on the human genome http://tare.medisin.ntnu.no/mirna_target.

Abstract The mechanisms underlying the fast genome evolution that occurs during animal domestication are poorly understood. Here, we present a genome-wide epigenetic dataset that quantifies DNA hydroxymethylation at single nucleotide resolution among full-sib Nile tilapia (Oreochromis niloticus) with distinct growth performance. In total, we obtained 355 million, 75 bp reads from 5 large- and 5 small-sized fish on an Illumina NextSeq500 platform. We identified several growth-related genes to be differentially hydroxymethylated, especially within gene bodies and promoters. Previously, we proposed that DNA hydroxymethylation greatly affects the earliest responses to adaptation and potentially drives genome evolution through its targeted enrichment and elevated nucleotide transversion rates. This dataset can be analysed in various contexts (e.g., epigenetics, evolution and growth) and compared to other epigenomic datasets in the future, namely DNA methylation and histone modifications. With forthcoming advancements in genome research, this hydroxymethylation dataset will also contribute to better understand the epigenetic regulation of key genomic features, such as cis-regulatory and transposable elements.

Lung cancer (LC) incidence is increasing globally and altered levels of microRNAs (miRNAs) in blood may contribute to identification of individuals with LC. We identified miRNAs differentially expressed in peripheral blood at LC diagnosis and evaluated, in pre‐diagnostic blood specimens, how long before diagnosis expression changes in such candidate miRNAs could be detected. We identified upregulated candidate miRNAs in plasma specimens from a hospital‐based study sample of 128 patients with confirmed LC and 62 individuals with suspected but confirmed negative LC (FalsePos). We then evaluated the expression of candidate miRNAs in pre‐diagnostic plasma or serum specimens of 360 future LC cases and 375 matched controls. There were 1663 miRNAs detected in diagnostic specimens, nine of which met our criteria for candidate miRNAs. Higher expression of three candidates, miR‐320b, 320c, and 320d, was associated with poor survival, independent of LC stage and subtype. Moreover, miR‐320c and miR‐320d expression was higher in pre‐diagnostic specimens collected within 2 years of LC diagnosis. Our results indicated that elevated levels of miR‐320c and miR‐320d may be early indications of imminent and advanced LC.

Abstract Background Gene expression measurements from breast cancer (BrCa) tumors are established clinical predictive tools to identify tumor subtypes, identify patients showing poor/good prognosis, and identify patients likely to have disease recurrence. However, diverse breast cancer datasets in conjunction with diagnostic clinical arrays show little overlap in the sets of genes identified. One approach to identify a set of consistently dysregulated candidate genes in these tumors is to employ meta-analysis of multiple independent microarray datasets. This allows one to compare expression data from a diverse collection of breast tumor array datasets generated on either cDNA or oligonucleotide arrays. Results We gathered expression data from 9 published microarray studies examining estrogen receptor positive (ER+) and estrogen receptor negative (ER-) BrCa tumor cases from the Oncomine database. We performed a meta-analysis and identified genes that were universally up or down regulated with respect to ER+ versus ER- tumor status. We surveyed both the proximal promoter and 3' untranslated regions (3'UTR) of our top-ranking genes in each expression group to test whether common sequence elements may contribute to the observed expression patterns. Utilizing a combination of known transcription factor binding sites (TFBS), evolutionarily conserved mammalian promoter and 3'UTR motifs, and microRNA (miRNA) seed sequences, we identified numerous motifs that were disproportionately represented between the two gene classes suggesting a common regulatory network for the observed gene expression patterns. Conclusion Some of the genes we identified distinguish key transcripts previously seen in array studies, while others are newly defined. Many of the genes identified as overexpressed in ER- tumors were previously identified as expression markers for neoplastic transformation in multiple human cancers. Moreover, our motif analysis identified a collection of specific cis-acting target sites which may collectively play a role in the differential gene expression patterns observed in ER+ versus ER- breast cancer tumors. Importantly, the gene sets and associated DNA motifs provide a starting point with which to explore the mechanistic basis for the observed expression patterns in breast tumors.

Abstract Background Protein remote homology detection is a central problem in computational biology. Most recent methods train support vector machines to discriminate between related and unrelated sequences and these studies have introduced several types of kernels. One successful approach is to base a kernel on shared occurrences of discrete sequence motifs. Still, many protein sequences fail to be classified correctly for a lack of a suitable set of motifs for these sequences. Results We introduce the GPkernel, which is a motif kernel based on discrete sequence motifs where the motifs are evolved using genetic programming. All proteins can be grouped according to evolutionary relations and structure, and the method uses this inherent structure to create groups of motifs that discriminate between different families of evolutionary origin. When tested on two SCOP benchmarks, the superfamily and fold recognition problems, the GPkernel gives significantly better results compared to related methods of remote homology detection. Conclusion The GPkernel gives particularly good results on the more difficult fold recognition problem compared to the other methods. This is mainly because the method creates motif sets that describe similarities among subgroups of both the related and unrelated proteins. This rich set of motifs give a better description of the similarities and differences between different folds than do previous motif-based methods.

Breast milk from people with overweight/obesity may differ in composition compared with that from normal-weight people. Exercise training can modify breast milk composition in rodent models, with a beneficial impact demonstrated on the offspring’s metabolism, but whether these findings translate to humans is unclear. This trial aims to determine the effect of an exercise intervention on breast milk composition and whether an exercise-induced modification of breast milk impacts the infants’ growth and body composition. Effect of Exercise Training on Breastmilk Composition is a randomised, controlled trial with two parallel groups, one exercise group and one control group, with a 1:1 allocation. We will include a minimum of 62 exclusively breastfeeding participants, 6 weeks postpartum. The exercise intervention lasts 8 weeks and comprises 25 supervised endurance exercise sessions with moderate or high intensity. The primary outcome measure is the change in the relative concentration of the human milk oligosaccharide 3′sialyllactose in breast milk from baseline at 6 weeks postpartum to the end of the intervention period. Secondary outcomes include breast milk concentrations of other metabolites, cytokines, hormones and microRNA, maternal health outcomes, infant growth, infant gut microbiome and infant circulating microRNA. Maternal and infant outcomes will be measured before, during and after the intervention period, with a follow-up of the infants until they are 24 months old. Trial registration number NCT05488964.

Colorectal cancer (CRC) is one of the most common cancer types worldwide. Chemotherapy is toxic to normal cells, and combinatory treatment with natural well-tolerated products is being explored. Some omega-3 polyunsaturated fatty acids (n-3 PUFAs) and marine fish oils have anti-cancer effects on CRC cells. The salmon oil OmeGo (Hofseth BioCare) contains a spectrum of fatty acids, including the n-3 PUFAs docosahexaenoic acid (DHA) and eicosahexaenoic acid (EPA). We explored a potential anti-cancer effect of OmeGo on the four CRC cell lines DLD-1, HCT-8, LS411N, and LS513, alone and in combination with the chemotherapeutic agent 5-Fluorouracil (5-FU). Screening indicated a time- and dose-dependent effect of OmeGo on the viability of the DLD-1 and LS513 CRC cell lines. Treatment with 5-FU and OmeGo (IC20–IC30) alone indicated a significant reduction in viability. A combinatory treatment with OmeGo and 5-FU resulted in a further reduction in viability in DLD-1 and LS513 cells. Treatment of CRC cells with DHA + EPA in a concentration corresponding to the content in OmeGo alone or combined with 5-FU significantly reduced viability of all four CRC cell lines tested. The lowest concentration of OmeGo reduced viability to a higher degree both alone and in combination with 5-FU compared to the corresponding concentrations of DHA + EPA in three of the cell lines. Results suggest that a combination of OmeGo and 5-FU could have a potential as an alternative anti-cancer therapy for patients with CRC.

Background: The developing fetus is particularly vulnerable to the effects of endocrine disrupting chemicals (EDCs). Molecular fingerprints of EDCs can be identified via microRNA (miRNA) expression profiles and may be etiologically implicated in the developmental origin of disease (DOHaD). Methods/design: This pilot study includes pregnant women at high risk (smoking at conception), and low risk (non-smoking at conception) for SGA birth (birthweight

Background: The developing fetus is particularly vulnerable to the effects of endocrine disrupting chemicals (EDCs). Molecular fingerprints of EDCs can be identified via microRNA (miRNA) expression profiles and may be etiologically implicated in the developmental origin of disease (DOHaD). Methods/design: This pilot study includes pregnant women at high risk (smoking at conception), and low risk (non-smoking at conception) for SGA birth (birthweight

Gunn Hildrestrand, Veslemøy Rolseth, and Nicolas Kunath et al. examine mice lacking the NEIL1 and NEIL2 DNA glycosylases involved in base excision repair. Their results suggest that loss of both NEIL1 and NEIL2 dysregulates genes relevant to synaptic function and modulates behavior in mice.

Abstract Proper regulation of the cell cycle is necessary for normal growth and development of all organisms. Conversely, altered cell cycle regulation often underlies proliferative diseases such as cancer. Long non-coding RNAs (lncRNAs) are recognized as important regulators of gene expression and are often found dysregulated in diseases, including cancers. However, identifying lncRNAs with cell cycle functions is challenging due to their often low and cell-type specific expression. We present a highly effective method that analyses changes in promoter activity, transcription, and RNA levels for identifying genes enriched for cell cycle functions. Specifically, by combining RNA sequencing with ChIP sequencing through the cell cycle of synchronized human keratinocytes, we identified 1009 genes with cell cycle-dependent expression and correlated changes in RNA polymerase II occupancy or promoter activity as measured by histone 3 lysine 4 trimethylation (H3K4me3). These genes were highly enriched for genes with known cell cycle functions and included 57 lncRNAs. We selected four of these lncRNAs—SNHG26, EMSLR, ZFAS1, and EPB41L4A-AS1—for further experimental validation and found that knockdown of each of the four lncRNAs affected cell cycle phase distributions and reduced proliferation in multiple cell lines. These results show that many genes with cell cycle functions have concomitant cell-cycle dependent changes in promoter activity, transcription, and RNA levels and support that our multi-omics method is well suited for identifying lncRNAs involved in the cell cycle.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

Abstract Active smoking has been linked to modulated gene expression in blood. However, there is a need for a more thorough understanding of how quantitative measures of smoking exposure relate to differentially expressed genes (DEGs) in whole-blood among ever smokers. This study analysed microarray-based gene expression profiles from whole-blood samples according to smoking status and quantitative measures of smoking exposure among cancer-free women (n = 1708) in the Norwegian Women and Cancer postgenome cohort. When compared with never smokers and former smokers, current smokers had 911 and 1082 DEGs, respectively and their biological functions could indicate systemic impacts of smoking. LRRN3 was associated with smoking status with the lowest FDR-adjusted p-value. When never smokers and all former smokers were compared, no DEGs were observed, but LRRN3 was differentially expressed when never smokers were compared with former smokers who quit smoking ≤ 10 years ago. Further, LRRN3 was positively associated with smoking intensity, pack-years, and comprehensive smoking index score among current smokers; and negatively associated with time since cessation among former smokers. Consequently, LRRN3 expression in whole-blood is a molecular signal of smoking exposure that could supplant self-reported smoking data in further research targeting blood-based markers related to the health effects of smoking.

The historically recent domestication of fishes has been essential to meet the protein demands of a growing human population. Selection for traits of interest during domestication is a complex process whose epigenetic basis is poorly understood. Cytosine hydroxymethylation is increasingly recognized as an important DNA modification involved in epigenetic regulation. In the present study, we investigated if hydroxymethylation plays a role in fish domestication and demonstrated for the first time at a genome-wide level and single nucleotide resolution that the muscle hydroxymethylome changes after a single generation of Nile tilapia (Oreochromis niloticus, Linnaeus) domestication. The overall decrease in hydroxymethylcytosine levels was accompanied by the downregulation of 2015 genes in fish reared in captivity compared to their wild progenitors. In contrast, several myogenic and metabolic genes that can affect growth potential were upregulated. There were 126 differentially hydroxymethylated cytosines between groups, which were not due to genetic variation; they were associated with genes involved in immune-, growth- and neuronal-related pathways. Taken together, our data unveil a new role for DNA hydroxymethylation in epigenetic regulation of fish domestication with impact in aquaculture and implications in artificial selection, environmental adaptation and genome evolution.

How genome stability is maintained at regions of active transcription is currently not entirely clear. Here, the authors reveal an association between base excision repair factors and transcription elongation to modulate DNA repair.

Breast milk is the primary source of nutrition and hydration for the newborn infant but also plays an important role in the child’s first immune defense. Additionally, several breast milk factors have been implicated in immune-related health outcomes later in life, including immunoglobulins, cytokines, chemokines, growth factors and, more recently, non-coding RNA (ncRNA) species. In this systematic review, we provide a comprehensive summary of the current literature on endogenous ncRNAs found in human breast milk. Thirty (30) relevant studies were identified and, whilst the majority studies focused on microRNAs (miRNAs), there is evidence that breast milk contains high quantities of RNA which also include long-coding RNAs, circular RNAs, as well as other short RNAs and fragmented tRNA and rRNAs. Among studies investigating miRNAs, miR-148a-3p, miR-30a/d-5p, miR-22-3p, miR-146b-5p, miR-200a/c-3p, and the 5p end of the let-7 miRNAs were commonly reported among the top 10 miRNAs in the cell, lipid, and skim milk fractions of breast milk. Methodological difference and small sample sizes limit the possibility of conclusively identifying which maternal and infant characteristics affect the miRNA profile. The highly expressed miRNAs were generally reported to be similar across lactational stage, milk fraction, maternal and infant characteristics, or infant growth and health. All the same, individual studies identify potential differences in miRNA expression levels which should be confirmed by future studies. Stability, uptake, and physiological functions of miRNAs were also considered in several studies. Breast milk miRNAs are relatively resistant to a range of harsh conditions and uptake experiments suggest that extracellular vesicles containing miRNAs and circular RNAs can be taken up by intestinal epithelial cells. Although the evidence regarding the functional effect of breast milk miRNAs is limited, the predicted functions range from metabolic and biosynthetic processes to signaling pathways, cellular adhesion, communication, growth, and differentiation. Finally, this systematic review highlights some of the methodological challenges and knowledge gaps which can help direct future research in this field. In particular, it is important to further investigate the bioavailability of miRNAs in different milk fractions, and to characterize other ncRNAs which are largely unstudied.Systematic Review RegistrationPROSPERO https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=138989, identifier CRD42020138989.

Elevated low-density lipoprotein cholesterol (LDL-C) is a treatable, heritable risk factor for cardiovascular disease. Genome-wide association studies (GWASs) have identified 157 variants associated with lipid levels but are not well suited to assess the impact of rare and low-frequency variants. To determine whether rare or low-frequency coding variants are associated with LDL-C, we exome sequenced 2,005 individuals, including 554 individuals selected for extreme LDL-C (>98(th) or

Despite the progress in our understanding of genes essential for stem cell regulation and development, little is known about the factors secreted by stem cells and their effect on tissue regeneration. In particular, the factors secreted by human CD34+ cells remain to be elucidated. We have approached this challenge by performing a cytokine/growth factor microarray analysis of secreted soluble factors in medium conditioned by adherent human CD34+ cells. Thirty-two abundantly secreted factors have been identified, all of which are associated with cell proliferation, survival, tissue repair, and wound healing. The cultured CD34+ cells expressed known stem cell genes such as Nanog, Oct4, Sox2, c-kit, and HoxB4. The conditioned medium containing the secreted factors prevented cell death in liver cells exposed to liver toxin in vitro via inhibition of the caspase-3 signaling pathway. More importantly, in vivo studies using animal models of liver damage demonstrated that injection of the conditioned medium could repair damaged liver tissue (significant reduction in the necroinflammatory activity), as well as enable the animals to survive. Thus, we demonstrate that medium conditioned by human CD34+ cells has the potential for therapeutic repair of damaged tissue in vivo.

UnlabelledHepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation.ConclusionA novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.

Abstract Background MicroRNAs (miRNA) and other small RNAs are frequently dysregulated in cancer and are promising biomarkers for colon cancer. Here we profile human, virus and bacteria small RNAs in normal and tumor tissue from early stage colon cancer and correlate the expression with clinical parameters. Methods Small RNAs from colon cancer tissue and adjacent normal mucosa of 48 patients were sequenced using Illumina high-throughput sequencing. Clinical parameters were correlated with the small RNA expression data using linear models. We performed a meta-analysis by comparing publicly available small RNA sequencing datasets with our original sequencing data to confirm the main findings. Results We identified 331 differentially expressed miRNAs between tumor and normal samples. We found that the major changes in miRNA expression between left and right colon are due to miRNAs located within the Hox-developmental genes, including miR-10b, miR-196b and miR-615. Further, we identified new miRNAs associated with microsatellite instability (MSI), including miR-335, miR-26 and miR-625. We performed a meta-analysis on all publicly available miRNA-seq datasets and identified 117 common miRNAs that were differentially expressed between tumor and normal tissue. The miRNAs miR-135b and miR-31 were the most significant upregulated miRNA in tumor across all datasets. The miRNA miR-133a was the most strongly downregulated miRNA in our dataset and also showed consistent downregulation in the other datasets. The miRNAs associated with MSI and tumor location in our data showed similar changes in the other datasets. Finally, we show that small RNAs from Epstein-Barr virus and Fusobacterium nucleatum are differentially expressed between tumor and normal adjacent tissue. Conclusions Small RNA profiling in colon cancer tissue revealed novel RNAs associated with MSI and tumor location. We show that Fusobacterium nucleatum are detectable at the RNA-level in colon tissue, and that both Fusobacterium nucleatum and Epstein-Barr virus separate tumor and normal tissue.

Upon functional loss of insulin producing islet β-cells, some patients with diabetes become dependent on life-long insulin supplementation therapy. Bioengineering surrogate insulin producing cells is an alternative replacement strategy. We have developed a novel approach using short-activating RNA oligonucleotides to differentiate adult human CD34(+) cells into insulin-secreting cells. By transfecting RNA to increase transcript levels of the master regulator of insulin biosynthesis, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), several pancreatic endodermal genes were upregulated during the differentiation procedure. These included Pancreatic and duodenal homeobox gene-1 (PDX1), Neurogenin 3, NeuroD, and NK6 homeobox 1 (NKx6-1). Differentiated CD34(+) cells also expressed glucokinase, glucagon-like peptide 1 receptor (GLP1R), sulfonylurea receptor-1 (SUR1) and phogrin-all essential for glucose sensitivity and insulin secretion. The differentiated cells appropriately processed C-peptide and insulin in response to increasing glucose stimulation as shown by enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting analysis, western blotting, and immunofluorescence staining. We provide a new approach using short-activating RNA in developing insulin producing surrogate cells for treating diabetes.Molecular Therapy - Nucleic Acids (2013) 2, e97; doi:10.1038/mtna.2013.23; advance online publication 4 June 2013.

Exploiting the properties of stem cells by microRNA (miRNA) profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC), the targets corresponding to many of these miRNA have not yet been fully elucidated. By miRNA profiling in a subpopulation of CD34+ cells isolated from peripheral blood, we have identified eight clusters of miRNA that were differentially expressed. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3' compensatory site in the 3'UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Our studies suggest that miR-181a* may be important in controlling the expression level of Nanog in a subpopulation of CD34+ cells.

It is now recognized that small noncoding RNA sequences have the ability to mediate transcriptional activation of specific target genes in human cells. Using bioinformatics analysis and functional screening, we screened short-activating RNA (saRNA) oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors, Kruppel-like factor 4 (KLF4) and c-MYC. We identified KLF4 and c-MYC promoter-targeted saRNA sequences that consistently induced increases in their respective levels of nascent mRNA and protein expression in a time- and dose-dependent manner, as compared with scrambled sequence control oligonucleotides. The functional consequences of saRNA-induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis, which revealed significant increases in mRNA levels of their respective downstream pathway genes. Notably, the microarray profile after saRNA-mediated induction of endogenous KLF4 and c-MYC showed similar gene expression patterns for stem cell- and cell cycle-related genes as compared with lentiviral vector-mediated overexpression of exogenous KLF4 and c-MYC transgenes, while divergent gene expression patterns common to viral vector-mediated transgene delivery were also noted. The use of promoter-targeted saRNAs for the activation of pluripotency reprogramming factors could have broad implications for stem cell research.

Background: The developing fetus is particularly vulnerable to the effects of endocrine disrupting chemicals (EDCs). Molecular fingerprints of EDCs can be identified via microRNA (miRNA) expression profiles and may be etiologically implicated in the developmental origin of disease (DOHaD). Methods/design: This pilot study includes pregnant women at high risk (smoking at conception), and low risk (non-smoking at conception) for SGA birth (birthweight

Summary: Telomerase biogenesis is a complex process where several steps remain poorly understood. Single-strand-selective uracil-DNA glycosylase (SMUG1) associates with the DKC1-containing H/ACA ribonucleoprotein complex, which is essential for telomerase biogenesis. Herein, we show that SMUG1 interacts with the telomeric RNA component (hTERC) and is required for co-transcriptional processing of the nascent transcript into mature hTERC. We demonstrate that SMUG1 regulates the presence of base modifications in hTERC, in a region between the CR4/CR5 domain and the H box. Increased levels of hTERC base modifications are accompanied by reduced DKC1 binding. Loss of SMUG1 leads to an imbalance between mature hTERC and its processing intermediates, leading to the accumulation of 3′-polyadenylated and 3′-extended intermediates that are degraded in an EXOSC10-independent RNA degradation pathway. Consequently, SMUG1-deprived cells exhibit telomerase deficiency, leading to impaired bone marrow proliferation in Smug1-knockout mice. : Kroustallaki et al. show that the single-strand-selective uracil-DNA glycosylase (SMUG1) functions in telomere maintenance, by removing modified bases from telomeric DNA and also by regulating modified bases in the telomerase RNA component (hTERC). SMUG1-knockout cells accumulate hTERC containing modified bases that interfere with binding of DKC1. Consequently, SMUG1-knockout cells and mice exhibit telomere maintenance defects. Keywords: SMUG1, telomere attrition, TERC, modified bases, RNA processing

Cancer patient-derived xenografts (PDXs) better preserve tumor characteristics and microenvironment than traditional cancer cell line derived xenografts and are becoming a valuable model in translational cancer research and personalized medicine. We have established a PDX model for colorectal cancer (CRC) in CIEA NOG mice with a 50% engraftment rate. Tumor fragments from patients with CRC (n = 5) were engrafted in four mice per tumor (n = 20). Mice with established PDXs received a liquid diet enriched with fish oil or placebo, and fatty acid profiling was performed to measure fatty acid content in whole blood. Moreover, a biobank consisting of tissue and blood samples from patients was established. Histology, immunohistochemistry and in situ hybridization procedures were used for staining of tumor and xenograft tissue slides. Results demonstrate that key histological characteristics of the patients’ tumors were retained in the established PDXs, and the liquid diets were consumed as intended by the mice. Some of the older mice developed lymphomas that originated from human Ki67+, CD45+, and EBV+ lymphoid cells. We present a detailed description of the process and methodology, as well as possible issues that may arise, to refine the method and improve PDX engraftment rate for future studies. The established PDX model for CRC can be used for exploring different cancer treatment regimes, and liquid diets enriched with fish oil may be successfully delivered to the mice through the drinking flasks.