Three of these miRNAs  were down-regulated in HCC, and their up-regulated gene targets are primarily associated with aberrant cell proliferation that involves DNA replication, transcription and nucleotide metabolism. The other three miRNAs  were up-regulated in HCC, and their down-regulated gene targets are primarily involved in metabolism and immune system processes. We further found evidence for a coordinated miRNA-induced regulation of important cellular processes, a finding to be considered when designing therapeutic applications based on miRNAs.To date, studies of the roles of microRNAs (miRNAs) in hepatocellular carcinoma (HCC) have either focused on specific individual miRNAs and a small number of suspected targets or simply reported a list of differentially expressed miRNAs based on expression profiling. Here, we seek a more in-depth understanding of the roles of miRNAs and their targets in HCC by integrating the miRNA and messenger RNA (mRNA) expression profiles of tumorous and adjacent non-tumorous liver tissues of 100 HCC patients. We assessed the levels of 829 mature miRNAs, of which 32 were significantly differentially expressed. Statistical analysis indicates that six of these miRNAs regulate a significant proportion of their  Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwideMicroRNAs (miRNAs) are a class of short, non-coding RNAs that play important roles in gene expression regulation. They control gene expression by binding to the 3′UTR of mRNAs, leading to their degradation or translational repression. There is accumulating evidence suggesting that miRNAs play an important role in carcinogenesis, either as tumor suppressors or onco-miRsmiR-21, miR-221, miR-222and miR-224687PTENSMAD4CDKN1B/p27, and CDKN1C/p57iR-122miR-199aPAK4/Raf/MEK/ERK pathwayNumerous miRNAs are associated with HCC. Of these, six were consistently reported in several studies to be differentially expressed in tumorous compared to non-tumorous tissues of HCC patients  > 1.5 and FDR < 0.05) between the tumorous and adjacent non-tumorous liver of HCC patients . Six of in silico prediction of likely gene targets of miRNAs. As miRNAs modulate gene expression through mRNA degradation or translational repression, we first examined a negative correlation in expression between miRNA and their gene targets.To identify the relevant gene targets of these differentially expressed miRNAs in HCC, we employed an integrated approach, which combines miRNA with gene expression profiles as well as miR-93 and PTPRN2 is shown in in silico targets predicted by the MiRanda programin silico predicted target, the enrichment score is increased. Otherwise, the enrichment score is decreased. An example of an enrichment plot for miR-93 is shown in in silico targets predicted by MirTarget2miR-21, miR-26a, miR-93, miR-122, miR-130a, and miR-221) were statistically significant against both MiRanda and MirTarget2  that is up-regulated and a pair of miRNAs (miR-26a and miR-130a) that is down-regulated in the tumors of HCC patients are correlated , suggesting that most of these miRNAs are likely regulated independently in HCC patients.We first examined if any of the six abovementioned miRNAs are coordinately dys-regulated in the tumors of HCC patients using Spearman’s correlation. As evident in To elucidate the pathways that are likely to be affected by the relevant gene targets of the three up-regulated and three down-regulated miRNAs, we first examined the differentially expressed gene targets of these differentially expressed miRNAs. Fewer differentially expressed gene targets were identified for down-regulated miRNAs than for up-regulated miRNAs . The lismiR-21, miR-93, and miR-221) coordinately target a set of pathways that is distinct from all three down-regulated miRNAs , which also coordinately target a set of pathways  were found to coordinately target primarily metabolism pathways, particularly the amino acid, carbohydrate, and lipid metabolism pathways, as well as the immune system  primarily target Genetic Information Processing, in particular, DNA replication and repair, transcription, cell growth, and nucleotide metabolism . We found that the network of genes in the metabolism pathways, dys-regulated by the three up-regulated miRNAs, is primarily centered on nodal molecules. IL6 and CXCL8 are nodal molecules of metabolism pathways, both of which are targets of miR-93  program . Similarly, miR-221 was significantly correlated with age (FDR: 0.13) and hepatitis infection (FDR: 0.15). More specifically, HBV-positive patients showed a significantly higher miR-221 expression compared to HBV-negative patients. Next, we associated the differentially expressed targets of the six miRNAs with various clinical characteristics. While only a single differentially expressed gene target of down-regulated miR-122 was found to be significantly associated with stage of HCC, 13, 2, and 5 down-regulated gene targets of up-regulated miRNAs were found to be significantly associated with stage of cancer, tumor grade, and encapsulation. No miRNA was significantly associated with relapse status using the Cox proportional hazards model  levels  and 5. Ads model , althougds model .We employed IPA to obtain a glimpse of the major cancer-related pathways dys-regulated by these genes associated with various clinical characteristics. Interestingly, greater than 75% of the genes dys-regulated by up-regulated miRNAs that are associated with a lower stage of cancer, tumor grade, or encapsulation were found to be in cancer or related pathways .Through profiling, miRNAs have been shown to be differentially expressed in the tumors of HCC patients  of these six miRNAs were previously reported to be differentially expressed in HCC in several studies and to dys-regulate important cancer pathways122223miR-93 with miR-221 and miR-26a with miR-130a), which are moderately correlated in expression  of these six miRNAs were down-regulated while the other three miRNAs  were up-regulated in the tumors of HCC  were enriched in metabolism and immune system processes  were enriched in genetic information processing pathways including cell proliferation, DNA replication, and metabolism, as well as RNA transcription. These observations suggest built-in redundancy of different gene targets regulated by different miRNAs targeting the same set of pathways. This redundancy may pose challenges for any single miRNA to be used as a target for cancer therapy.Notably, although the majority of gene targets of the six miRNAs were unique, pathway enrichment analysis revealed that relevant gene targets of all six up-regulated miRNAs  have previously been implicated in the regulation of lipid metabolism29303133The pathways targeted by these six miRNAs represent the various hallmarks of cancer. The predicted gene targets of the three up-regulated miRNAs reside in the metabolism pathway, which has recently generated great interest2627miR-26a, miR-122, and miR-130a) target cell-cycle processes while the up-regulated miRNAs  target apoptosis. These observations are consistent with previous literature which reported that miR-26amiR-122miR-221miR-21In addition to being a nodal molecule for inflammation and immune response, TP53, together with TP73, were identified as key molecules involved in the cell-cycle progression and apoptosis pathways. Targets of all six miRNAs  modulatemiR-26a, miR-122, and miR-130a) target cell proliferation and genetic information processing including DNA replication, transcription, and nucleotide metabolism  may perturb the AKT axis, leading to the disruption of cell death.As mentioned earlier, the three down-regulated miRNAs  Tissue Repository with prior written informed consent. The NCCS Institutional Review Board (NCC IRB) (2008/440/B) and the SingHealth Central Institutional Review Board (CIRB) (2013/455/B) approved this study. All analyses were performed in accordance with relevant guidelines and regulations. The demographics and clinical information of these 100 HCC patients are summarized in We sent samples to Miltenyi Biotec, CA, for gene and miRNA expression microarray profiling. Briefly, total RNA isolation for tumorous and adjacent non-tumorous tissue samples was performed using the standard RNA extraction protocols (Trizol). We assessed total RNA integrity using the Agilent 2100 Bioanalyzer platform (Agilent Technologies), and only samples with RNA integrity number (RIN) >6 were profiled for gene and miRNA expressionFor gene expression profiling, we used 1 μg of each total RNA sample as starting material. We amplified and labeled total RNA samples using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer’s protocol. We measured cRNA yields and dye-incorporation rates with the ND-1000 Spectrophotometer (NanoDrop Technologies). We labeled non-tumorous samples with Cy3 and tumorous samples with Cy5. We performed hybridization according to Agilent’s 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, we combined and hybridized 825 ng of the corresponding Cy3- and Cy5-labeled fragmented cRNA overnight  to Agilent Whole Human Genome Oligo Microarrays 4 × 44K (G4112F). We washed microarrays with 6x SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37 °C) containing 0.005% N-lauroylsarcosine for 1 min. The second washing step was performed with acetonitrile for 30 seconds. Finally, we detected fluorescence signals using Agilent’s DNA microarray scanner (Agilent Technologies).For miRNA profiling, we labeled samples according to the undisclosed miRXplore™ user manual. We hybridized fluorescent-labeled samples to miRXplore™ microarrays using the a-Hyb™ hybridization station. We labeled non-tumorous samples with Hy3 and tumorous samples with Hy5. Finally, we detected fluorescence intensity signals using an Agilent laser scanner (Agilent Technologies).http://www.ncbi.nlm.nih.gov/geo/) through these GEO IDs: GSE62007 (miRNA profiles), GSE62043 (mRNA profiles), and GSE62044 (combined).All microarray expression profiles are publicly available on Gene Expression Omnibus For every differentially expressed miRNA, we calculated the negative Spearman’s correlation coefficient between the miRNA and all genes in expression across tumorous and adjacent non-tumorous tissues. We ranked genes by their negative correlation with the miRNA. Next, we downloadedFinally, based on this ranking metric, we identified biological themes for each miRNA using GSEA′s pre-ranked function. The KEGG (excluding human disease) gene sets from MSigDB were employed in our analyses. Then we summarized enrichment results by consolidating pathways to pathway groups and pathway groups to categories based on the KEGG orthology conceptual hierarchy, implemented in the Bioconductor keggorthology package (version 2.1.0). We ranked pathway groups by their total number of underlying pathways, and we considered up- and down-regulated miRNAs separately. For ties, we ranked groups with at least one pathway enriched in all three up-regulated miRNAs, down-regulated respectively, and then more significant FDRs higher. We rearranged categories according to the highest-ranking pathway group in up-regulated miRNAs.We collected genes for each pathway group across three up-regulated miRNAs and three down-regulated respectively. We only considered differentially expressed genes of the GSEA core enrichment set. IPA was employed to infer regulatory networks and functional annotation for differentially expressed genes associated with selected pathways in each category in MiRNAs and their relevant MiRanda predicted targets with significant differential expression  were associated with clinical features, namely gender, age, hepatitis infection status, liver cirrhosis, stage, grade, size, relapse status, encapsulation, and Alpha Fetoprotein (AFP) levels. We evaluated association through the log-rank test and Cox’s proportional hazards model (relapse status) or the Kruskal-Wallis rank sum test . We obtained the genes’ functional annotation through IPA.How to cite this article: Thurnherr, T. et al. Differentially Expressed miRNAs in Hepatocellular Carcinoma Target Genes in the Genetic Information Processing and Metabolism Pathways. Sci. Rep.6, 20065; doi: 10.1038/srep20065 (2016).


Drosophila ovary.In this study, Senti et al investigate how cytoplasmic post-transcriptional silencing influences transcriptional silencing in the nucleus. They show that Piwi-bound piRNA populations depend almost exclusively on prior piRNA-guided transcript slicing, thus providing further insight into the regulation of piRNA biogenesis in the developing  Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways.PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing  The invasion of eukaryotic genomes by transposable elements (TEs) is an ancient genetic conflict that triggered the evolution of efficient defense systems on the host side . In fungThree core principles characterize the piRNA pathway: First, piRNAs are processed from single-stranded precursor RNAs, which are derived from hundreds of genomic piRNA source loci. The predominant piRNA precursors are long noncoding RNAs (often enriched in TE sequences) and genic mRNAs where piRNA production is largely restricted to 3′ untranslated regions (UTRs) .In choosing a genetic perturbation system, we considered that TE expression as well as piRNA populations differ substantially between rt cells . We therrt cells . Using trt cells B. Moreovnanos promoter-driven GFP-Piwi transgene)  D. This aTo examine the impact of Aub/Ago3-bound secondary piRNAs on primary piRNA biogenesis on a global scale, we compared the levels and identities of germline Piwi-bound piRNAs from ovaries depleted for Aub, Ago3, or Aub/Ago3 together with those from control ovaries.The failure to load Piwi with a piRNA prevents Piwi's nuclear accumulation and leads to its degradation . We therflamenco . The population of mRNA-derived piRNAs is very diverse. In contrast to somatic cells, genic piRNAs in the germline are derived from most of the mRNA molecule and are not restricted to 3′ UTR portions , tRNAs, or rRNAs are unchanged or even increased in ovaries depleted for secondary piRNAs B. This pCR8 mRNA . DepletiCR8 mRNA F. We proCR8 mRNA . In suppportions . Althougportions , we coulTaken together, our results demonstrate that Aub/Ago3-mediated cleavage is the major trigger for primary piRNA biogenesis fueling germline Piwi but that alternative entry routes that feed into primary piRNA biogenesis exist.20A, which is transcribed independently of Piwi and Rhino  are unchanged or even increased in ovaries depleted for Aub, Ago3, or both together  reveals a striking correlation between the remaining secondary piRNAs and primary biogenesis initiation sites  chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) from control ovaries and ovaries with germline-specific piRNA pathway perturbations. We restricted the analysis to TE promoter regions, which—if active—display a pronounced Pol II peak that coincides with the transcription start site (TSS), as evidenced by a 5′ Cap-seq analysis .Burdock exhibits strong transcriptional increases upon loss of Aub or Ago3  , which coincides with increased GATE transcription  A,B are ocription B.n = 13), a Piwi/Aub-dominant group (n = 5), and a Piwi/Aub/Ago3 group (n = 7) . All but one of these are transcriptionally derepressed in Piwi-depleted ovaries, confirming the central role of Piwi in orchestrating transcriptional silencing C–E. Almo (n = 7) C–E.To test whether transcriptional derepression generally correlates with reduced levels of Piwi-bound piRNAs, we considered the recent finding that the absolute number of Piwi-bound piRNAs antisense to the nascent target transcript—in particular to TSS-proximal regions—defines Piwi's transcriptional silencing capacity . We deteIn control ovaries, Pol II occupancy for TEs belonging to the three groups was similar overall F, top, bTaken together, cytoplasmic Aub/Ago3-mediated post-transcriptional silencing feeds back onto transcriptional silencing of target TEs via defining the pool of nuclear Piwi-bound piRNAs. However, different TEs display different sensitivities toward loss of Aub or Ago3. We hypothesized that this is caused by the variable dependencies of the respective secondary piRNA populations on the presence of Aub and/or Ago3.TEs of the Piwi/Aub/Ago3 group are expected to engage in an obligatory heterotypic ping-pong cycle that requires both Aub and Ago3 A. Loss orover, burdock, and the I element in developing ovaries. For rover and the I element, we also visualized TE-encoded proteins by immunofluorescence . In contrast, cytoplasmic rover RNA and rover Gag protein only accumulate in nurse cells and the oocytes of ovaries depleted for Aub or Ago3 but not for Piwi . In the case of burdock, loss of Piwi partially impairs the ping-pong cycle . Consequently, burdock RNA accumulates in the cytoplasm and the developing oocyte but to a lower extent than in ovaries depleted for Aub or Ago3 .To examine this in more detail, we performed single-molecule RNA-FISH for for Piwi E. In Piwinto Aub B,E. SimiTaken together, TEs belonging to the Piwi/Aub/Ago3 group display a strict heterotypic ping-pong cycle that consumes TE transcripts in the cytoplasm and defines the Piwi-bound piRNA pool for additional transcriptional silencing.The existence of a Piwi/Aub-dominant group of TEs whose transcriptional repression requires Aub but not Ago3 predicts that secondary piRNA biogenesis for these elements switches from heterotypic ping-pong to the homotypic Aub/Aub cycle in the absence of Ago3 . Indeed,TEs in the Piwi/Aub-dominant group also display higher levels of steady-state RNA in Aub-depleted versus Piwi-depleted ovaries .For a detailed analysis, we selected Taken together, TEs of the Piwi-dominant group are very robustly wired into secondary piRNA biogenesis. Only the simultaneous loss of Aub and Ago3 reveals that, also for this group, secondary piRNA populations are the major specificity signal for the generation of Piwi-bound piRNAs in the germline.D. melanogaster genome. To test this, we determined the approximate sequence divergence of all TEs in the genome. We determined the amount of piRNAs mapping with zero versus three mismatches to the TE consensus sequence (which resembles the active element) in control ovaries. This shows that piRNAs matching TEs from the Piwi/Aub-dominant and the Piwi/Aub/Ago3 groups match consensus sequences closely, reflecting evolutionarily young TEs. In contrast, piRNAs derived from many Piwi-dominant TEs are much more diverged, suggesting that these are evolutionarily older TEs . We speculate that older TEs have more insertions in piRNA-producing loci, making them more robust toward genetic perturbations in the ping-pong cycle.We speculated that the robustness of Piwi-dominant TEs is based on their much longer evolutionary history within the white1118 background using CRISPR/Cas9  versus nonself (TE RNA) transcripts. Here we show that transcript slicing guided by secondary piRNAs is the major specificity signal that defines the population of secondary as well as primary piRNAs in the get RNAs , this seget RNAs . In the The adaptive nature of secondary piRNA biogenesis (ping-pong cycle) is considered to be a central feature of the piRNA pathway . Our finmdg3 and burdock), TE sequences have not yet been hardwired into major piRNA clusters (which are maintained Piwi-independently) but that stand-alone insertions  serve as the major sources of antisense transcripts to continuously fuel ping-pong.As Piwi also specifies TE insertions as piRNA source loci via the Rhino–Deadlock–Cutoff complex , sense aDrosophila germline—where nurse cells supply all RNA and protein to the growing oocyte through cytoplasmic bridges—highlight the biological significance of the post-transcriptional silencing branch. This is particularly evident in Piwi-depleted ovaries and for those TEs  where loss of Piwi does not impair the ping-pong cycle. Transcripts and proteins from these TEs—despite maximal transcriptional derepression—do not or only very mildly accumulate in the oocyte, preventing efficient TE transposition. In contrast to this, we observed maximal TE RNA and protein accumulation in ovaries lacking Aub and Ago3 together, where transcriptional and post-transcriptional silencing are both impaired.The syncytial architecture of the female D. melanogaster genome more recently and are more sensitive to pathway perturbations. In contrast, many Piwi-dominant TEs are present as older insertions in the genome and might have populated many piRNA source loci, thereby exhibiting robust ping-pong patterns. Alternatively, the different absolute levels of sense/antisense precursor transcripts impinge on ping-pong robustness.What underlies the different dependencies of the various TEs on Aub and Ago3 in terms of maintaining a ping-pong cycle is mostly unclear. Our analysis of TE sequence divergence suggests that TEs of the Piwi/Aub-dominant and Piwi/Aub/Ago3 groups have colonized the Our data attest a central role to transcript slicing in specifying the diverse primary and secondary piRNA populations in the ovarian germline . Much leDrosophila ovarian somatic cells is steered to piRNA cluster transcripts and a subset of cellular mRNAs is a central, unresolved question. Our data indicate that alternative, slicer-independent ways to initiate piRNA biogenesis  (How primary piRNA biogenesis in cursors) F do exiscursors) . This unDrosophila ovarian germline and synthesize several previously unconnected findings into a coherent model of TE repression. In the broader context, it is remarkable that small RNA-guided transcript slicing is repeatedly being used during evolution to initiate small RNA biogenesis in various pathways of plants, fungi, and animals . All aub/ago3 double GLKD experiments used aub-sh (attP40) and ago3-sh (attP2), except those that included the germline Piwi transgene (attP40), where aub-sh (attP33) was used with ago3-sh (attP2). The novel piwi, aub, and ago3 alleles were isolated in a white1118 background isogenic for the second and third chromosomes using two independent gRNAs per gene . Frameshift alleles induced by independent gRNAs were analyzed in trans to avoid potential “off-target” effects. GFP-tagged Piwi and Aub BAC transgenes rescued the otherwise sterile piwi and aub transheterozygous combinations to full fertility.The germline GFP-Piwi cDNA transgene (inserted into attP40)  was drivonstruct , GFP-Nuponstruct , MTD-Galand ago3 . We estaPrimer sequences used for RT-qPCR are listed in Supplemental Table S3 (primer and gRNA sequences).rover Gag, mdg3 Gag, I-element ORF1, and HeT-A ORF  were raised by Eurogentec.The following mouse monoclonal antibodies against His-tagged fragments of Piwi, Aub, and Ago3 (amino acids 1–150) were generated by the Max F. Perutz Laboratories Monoclonal Antibody Facility: Piwi, 8C2-E4 (immunoprecipitation and immunofluorescence); Aub, 8A3-D7 ; and Ago3, 5H12-G12 (immunoprecipitation and Western blotting) and 7B4-C2 (immunofluorescence). Rabbit anti-Piwi and anti-Armi were used for Western blotting. Rabbit antisera against His-tagged Rpb3  and agaiCAL Fluor Red 590-labeled Stellaris oligo probes were used to detect the various TE transcripts . For each probe set, 48 oligos were computationally filtered to map only to the cognate TEs. RNA-FISH was performed as in nanos-driven GFP-Piwi levels are much lower than for GFP-Piwi (BAC). Imaging of these required confocal settings that inadvertently also excited DAPI at a low level, leading to a background signal in the GFP channel; this was also observed for the w1118 strain without germline GFP-Piwi.Confocal stacks of DAPI and GFP-Piwi fluorescence of stage 7 egg chambers were acquired (Zeiss LSM780) with identical acquisition parameters for all samples. A script written in the Definians software suite performed the unbiased analysis of confocal stacks: Small somatic or large nurse cell nuclei were detected and classified using DAPI. Nuclear GFP-Piwi fluorescence intensity was measured in two dimensions at the Z-section showing the largest nuclear circumference for each nucleus for all somatic and nurse cell nuclei. For GFP-Piwi (BAC rescue construct), the ratio of nurse cell GFP signal and somatic follicle cell GFP signal was calculated, averaged for the same genotype, and set to 100% in controls. For germline GFP-Piwi, the average intensity of all nurse cell nuclei per egg chamber was calculated, and the genotype average was set to 100% in controls. Note that D. melanogaster TE entries from Repbase (http://www.girinst.org/repbase) with those from BDGP_embl version 9.41. Pairwise BLAST (bl2seq) was used to identify identical or highly similar entries, from which only one (Berkeley Drosophila Genome Project [BDGP] nomenclature) was retained. Unique entries from either database were added. The D. mel aurora entry was replaced with the D. sim ninja entry, as the latter is more related to the insertions in the D. melanogaster reference genome.A FASTA sequence file with 122 TEs  was compiled by merging all white GLKD ovaries was sheared to average 400 base pairs (bp), size-selected to 200–600 bp, cloned, and sequenced (paired-end 100 nt). RNA Pol II ChIPs were performed in biological triplicates with rabbit anti-Rbp3 according to For DNA-seq, genomic DNA isolated from Piwi, Aub, Ago3, and GFP-Piwi immunoprecipitations from ovary lysates were carried out as in DNA-seq, Pol II ChIP-seq, and RNA-seq reads were trimmed to bases 5–45, and paired-end RNA-seq reads were split into forward and reverse reads and mapped separately. After normalization using genome-unique mappers, Pol II ChIP-seq and RNA-seq reads were mapped to the TE consensus file with BWA, allowing up to three mismatches and two mappings per TE (LTR coverage). To calculate the TE-specific numerical read counts, we generated a TE consensus file in which all 40mer reads mapping to multiple TEs were masked, and reads falling into masked regions were omitted from analysis. TE promoter mapping reads of RNA Pol II ChIP libraries  were averaged . Analysis of Cap-seq data was done as described .The approximate TE copy numbers in the used strains were calculated by dividing length-normalized TE matching read counts by average genic read counts.HeT-A, TART, and TAHRE promoters, 500 bp centered on the Cap-seq peaks observed in Piwi depletion were used .TE promoter sequences were defined as follows: LTR sequences were used for LTR elements. The first 500 bp of LINEs and DNA elements were used. For For each genotype, Piwi , Aub, and Ago3 immunoprecipitation libraries were normalized relative to a total small RNA-seq library  using the most asymmetrically distributed piRNA species. Similarly, for control ovaries, the ppm-normalized total Piwi immunoprecipitation library was used to normalize germline GFP-Piwi immunoprecipitation and soma-specific Piwi immunoprecipitation libraries (Piwi-IP from ovaries with Piwi depleted in germline) , <0.01 (**), and <0.001 (***).TE promoter Pol II occupancy and germline Piwi piRNA levels from different genotypes were compared by one-tailed Wilcoxon matched pairs signed rank test. Levels of germline GFP-Piwi piRNA of different groups of TEs from control ovaries were tested by unpaired, one-tailed Illumina deep sequencing data sets have been deposited at NCBI Gene Expression Omnibus (GSE71775).


KRAS initiate pancreatic cancer. There are, however, two other Ras family members, Nras and Hras, which can be activated in the presence of oncogenic Kras. The role of these wild-type Ras proteins in cancer remains unclear, as their disruption has been shown to enhance or inhibit tumorigenesis depending upon the context. As pancreatic cancer is critically dependent upon Ras signaling, we tested and now report that loss of Hras increases tumor load and reduces survival in an oncogenic Kras-driven pancreatic adenocarcinoma mouse model. These effects were traced to the earliest stages of pancreatic cancer, suggesting that wild-type Hras may suppress tumor initiation. In normal cells, activated Ras can suppress proliferation through p53-dependent mechanisms. We find that the tumor suppressive effects of Hras are nullified in a homozygous mutant p53 background. As such, loss of wild-type Hras fosters the earliest stages of pancreatic cancer in a p53-dependent manner.Oncogenic, activating mutations in  The vast majority of these individuals will succumb to this disease, with a five-year survival rate of only 6.7%  promotes the conversion of Ras from an inactive, GDP-bound state to an active, GTP-bound state. Ras-GTP binds to and activates effector proteins that engage the MAPK, PI3K, RalGEF, and other signaling pathways involved in cellular growth and survival. GTPase-activating proteins (GAPs) then enhance hydrolysis of GTP and revert Ras back to an inactive state [KRAS gene inactivate the endogenous or GAP-stimulated GTPase activity of the encoded protein, resulting in constitutively GTP-bound and activated Kras [Kras gene, induce early stage pancreatic cancer that can progress to frank PDAC [KRAS are thought to be the driver mutations in PDAC.The somatic mutations detected in the ted Kras . In addited Kras , these mted Kras  and, wheank PDAC . As suchS-nitrosylation [Although the involvement of the oncogenic mutant form of Kras in pancreatic cancer is well described, the remaining wild-type Ras isoforms may also be activated. The first hints in this regard were the findings that targeting the negative regulator RasGAP to the plasma membrane  or reducsylation  or expresylation . Moreovesylation .Kras or Hras in lung and skin tumors of mice are often accompanied by loss of the reciprocal wild-type allele [RAS gene has been reported in human cancers [Kras [Hras or Nras, develop more Kras mutation-positive lung tumors [Nras was also observed in a mouse model of thymic lymphomas driven by oncogenic Nras [Wild-type Ras proteins can have divergent effects depending on the setting. On one hand, they can inhibit tumorigenesis. Specifically, oncogenic Ras can induce a senescent growth arrest when expressed in normal cells  and inhie allele –15. Simi cancers . Moreoveg tumors . A tumornic Nras . Thus, wHras or Nras develop fewer carcinogen-induced Hras mutation-positive skin tumors [KRAS mutation-positive human cancer cell lines. Conversely, stimulation of wild-type Ras proteins by EGF promotes proliferation of such cells [Wild-type Ras proteins can also foster tumorigenesis. Specifically, mice lacking one or both alleles of wild-type n tumors . Knockinn tumors , 19, or n tumors  or SOS [ch cells . Thus, pKRAS mutations, namely pancreatic. Thus, we assessed the consequence of disrupting the endogenous, wild-type Hras gene on the development of oncogenic Kras-driven tumorigenesis in the pancreas of mice. We report that loss of wild-type Hras promotes tumorigenesis in this model, suggesting a tumor suppressive role for wild-type Hras at the early stages of pancreatic cancer.Given that wild-type Ras proteins can be activated downstream of oncogenic Kras, yet give rise to opposite effects on tumorigenesis depending on the context, it is important to ascertain their role in the cancer most frequently associated with KrasLSL-G12D/+, Trp53LSL-R172/+, and Pdx-1-Cretg/+ mice were obtained from Jackson Labs and as a kind gift from David Kirsch. Hras-/- mice were a kind gift from the NCI and Eugenio Santos. KrasLSL-G12D/+;Hras+/- and Pdx-1-Cretg/+;Hras+/- mice were bred to generate Hras+/+ and Hras-/- KC  littermates. KrasLSL-G12D/+;Trp53LSL-R172/+;Hras+/+ and Pdx-1-Cretg/+;Hras+/+ mice were bred to generate Hras+/+ KPC  mice. KrasLSL-G12D/+;Trp53LSL-R172/+;Hras-/- and Pdx-1-Cretg/+;Hras-/- mice were bred to generate Hras-/- KPC mice. KrasLSL-G12D/+;Trp53LSL-R172/+;Hras+/+ and Pdx-1-Cretg/+;Trp53LSL-R172/+;Hras+/+ mice were bred to generate Hras+/+ KPPC  mice. KrasLSL-G12D/+;Trp53LSL-R172/+;Hras-/- and Pdx-1-Cretg/+;Trp53LSL-R172/+;Hras-/- mice were bred to generate Hras-/- KPPC mice. The described mice were monitored for health and weight three times per week and were euthanized at either the indicated fixed time points or upon reaching a moribundity endpoint. Moribundity endpoints were defined as weight loss exceeding 15% of total body weight, indications of abdominal ascites or swelling, or signs of pain or distress. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. A protocol for this study was specifically approved by the Institutional Animal Care and Use Committee at Duke University (protocol A279-13-11).Hras+/+ and three Hras-/- KPC mice was minced in collagenase V (Sigma-Aldrich) for 30 minutes at 37°C, and resultant cells were cultured in Dulbecco's Modified Eagle's Media (DMEM) + 10% FBS for at least 4 passages.Pancreatic tumor tissue from three The percentage of normal acinar area was quantified at 8 and 36 week time-points in KC mice in a blinded study of 4–5 randomly selected high-power fields of H & E-stained pancreatic section per mouse. The amount of acinar area per section was expressed as a percentage of the total area of tissue in the field, determined using Adobe Photoshop freehand selection tool.H&E stained sections were blindly reviewed by pathologists  at 4, 8, and 36 week time points. Each lobule of the pancreas from each section was examined, and the highest grade lesion (ADM or PanIN) from each lobule was identified, and the percent of lobules with the highest grade of each type of lesion was determined based upon the total number of lobules counted.KC mice were snap frozen in liquid nitrogen and 8 uM-thick sections were cut using a cryotome. The samples were fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton-X-100. Samples were blocked in normal donkey serum then incubated with 1:100 dilution of the primary antibodies Goat Anti-Amylase C-20 and sc-12821 Rabbit Anti-CK-19 H-60 (Santa Cruz Biotechnology Inc.) overnight at 4°C. Samples were then incubated with the secondary antibodies Alexa fluor 488 Donkey anti-rabbit IgG (Invitrogen A-21206) or Alexa-fluor 594 Donkey anti-goat IgG (Invitrogen A-11058) at a 1:500 dilution for 1 hour at room temperature, then mounted with ProLong Gold antifade reagent with DAPI (Life Technologies P36931). Total number of lesions with cells co-staining for amylase and CK-19 were quantified per high power field in merged images from a Zeiss Axio Imager widefield fluorescence microscope in a blinded fashion.Pancreatic specimens from et al [Snap-frozen pancreatic samples were stained as described by Debacq-Chainiaux et al  and 5 raHeat-induced antigen retrieval was performed on formalin-fixed, paraffin-embedded sections, followed by staining with an anti-Ki67 primary antibody at a 1:50 dilution (Dako M7249), an anti-CC3 primary antibody at a 1:100 dilution , or anti-p16 primary antibody  at a 1:100 dilution. Peroxidase-based detection was performed using Vectastain Elite ABC Kit (Vector labs). The total number of Ki67-positive staining cells per field in each of 5 randomly selected, high-power fields was counted in a blinded fashion, as well as the number of lesions with at least one positive-staining cell and quantified as a percentage of the total number of lesions. For CC3 and p16, 5 randomly selected, high-power fields were quantified in a blinded fashion using Image J software. Color thresholding was used to determine the total amount of CC3 and p16 staining per high-power field, and the freehand selection tool was used to select only the lesions, where color thresholding was applied to calculate the percentage of positive staining area from only the lesions.Hras-/- KPC cell lines were stably infected with retroviruses derived from pBabePuro (vector control) or pBabePuroFLAG-Hras encoding wild-type mouse Hras cDNA, and selected with puromycin, as previously described [Early passage (within 5 passages from adaption to culture) escribed . Stable escribed .Kras alleles as previously described [DNA was isolated from pancreatic tissue, facial papillomas, or vulvar tumors and amplified by PCR to detect the wild-type and recombined escribed .t-test was used to compare the amount of normal acinar tissue remaining in the pancreata in Hras+/+ and Hras-/-KC and KPPC mice, and to compare levels of Ki67, p16, ADM, SA-β-gal, and CC3 staining in each cohort. A 2-sided unpaired t-test was also used to compare the levels of each type of graded lesions at the specified time-points and the number of facial papillomas between cohorts. These results were then confirmed by Mann-Whitney test using the same software. Kaplan-Meier survival curves were generated for each of the KPC and KPPC cohorts, as well as for the development of facial and vulvar papillomas over time in the KPC cohort, and P-values were calculated using the log-rank (Mantel-Cox) test.Statistical analyses were performed using GraphPad Prism v5 (GraphPad Software). A 2-sided, unpaired Kras-driven pancreatic tumorigenesis in vivo, mice were generated with either homozygous null Hras-/- [Hras+/+ alleles in a KrasLSL-G12D/+;Pdx-1-Cretg/+ (KC) background [Ras genes, is the only one that is not embryonic lethal in a background like that of KC mice which encodes only one functional Kras allele [Kras in the developing pancreas of these mice results in the development of pre-invasive pancreatic intraepithelial neoplasias (PanINs) that progress at a low frequency to PDAC through a series of histological changes that resemble those seen in human patients [Hras+/+ and Hras-/- KC mice were generated and then euthanized at 36 weeks of age, a time when a spectrum of PanIN lesions are easily detected [Hras-/- KC mice  lacked any lesion (Hras-/- KC mice (Hras-/- versus Hras+/+ acinar cells when LSL-KrasG12D was activated by Ad-Cre (not shown). However, we independently and directly validated the increase in ADM lesions in vivo in the Hras-/- KC mice by quantitating the number of cells co-staining for immunoflourescent markers of acinar (amylase) and ductal (CK-19) cells  cells . Quantif KC mice . There w KC mice , suggestHras+/+ KC mice  background were euthanized at a fixed time point of 14 days of age, and the amount of normal acinar tissue remaining in the pancreas was assessed . Collectively, these results suggest that the loss of Hras enhances the earliest stages of pancreatic tumorigenesis, perhaps even initiation. Taking this concept one step further, perhaps epigenetic differences in levels of Ras proteins may influence whether an oncogenic mutation in KRAS leads to pancreatic tumorigenesis. As KPC mice had a reduced lifespan in the Hras-/- background, such early effects of wild-type Ras proteins may have lasting repercussions.We report that a homozygous null e cancer , 17, 18.ng event . We atteKras, increasing the chances that an oncogenic mutation in Kras will initiate tumorigenesis. More specifically, normal cells can be sensitive to high levels of Ras signaling, resulting in senescence rather than proliferation, which can inhibit tumor formation [Kras have been reported to be selectively lost at two to six weeks of age, apparently a consequence of high Kras signaling [Hras+/+ and Hras-/- genotypes in a homozygous mutant p53 background of pancreatic cancer. Admittedly, there was no difference in β-galactosidase staining, a common marker for senescence, between Hras+/+ and Hras-/-KC mice at 8-weeks of age, and endogenous Hras was not detected by immunoblot in the pancreatic tissue at this time point. A number of other mechanisms are most certainly possible. For example, loss of Hras may reduce apoptosis or increase proliferation. However, as with the case of senescence markers, there was no difference in Ki67, cleaved caspase 3, or TUNEL staining between Hras+/+ and Hras-/-KC mice at 8-weeks of age, arguing either against this model or pointing towards an effect at an earlier time point. Alternatively, rather than the loss of Hras reducing the amplitude of Ras signaling, it is also possible that this loss changes the spectrum of this signaling. More specifically, Ras isoforms exhibit differences in their subcellular localizations, which may result in engagement of effector proteins in different locations within the cells [Hras-/-KC mice.While it remains to be determined how wild-type Hras suppresses early pancreatic tumorigenesis, a number of mechanisms are possible. One possibility is that the loss of Hras may reduce the pool of active Ras to a level below that which triggers a growth arrest in response to activation of oncogenic ormation , 33. In ignaling . As suchignaling . In agrehe cells . Loss ofhe cells . The losNras has been shown to promote oncogenic Kras-driven lung tumorigenesis, yet inhibit oncogenic Hras-driven skin tumorigenesis [Nras were found to be more tumorigenic than one allele in a mouse model of CMML and JMML [KRAS mutation-positive cancer cell lines isolated from late stage disease [We fully recognize that the tumor-suppressive effect of wild-type Hras may very well be context dependent. Indeed, loss of igenesis . Similarand JMML . The neg disease –10, 19. S1 FigKras allele or the LSL-KrasG12D allele following Cre-excision  shows successful recombination in the pancreata (panc) and facial papillomas (pap), but not the negative control tails. DNA was isolated from the tissues of 9-month old Hras+/+ and Hras-/- mice.Representative PCR amplification using primers specific for the wild-type (PDF)Click here for additional data file.S2 FigA) Ki67 as a marker for proliferation, (B) CC3 as a marker for apoptosis, (C) p16 and, (D) SA-β-gal as markers for senescence. For each, the % of positive-staining lesions was quantified in 10 random high-power fields from 5 mice of each cohort. (bar: mean ± S.E.M.). ns = not significant. Line = 50 μM.Representative stained sections and quantification of % positive-staining lesions for ((PDF)Click here for additional data file.


P = 0.041, Kaplan-Meier analysis). Through gain- and loss-of function experiments, we demonstrated that miR-573 inhibits PCa cell migration, invasion and TGF-β1-induced epithelial-mesenchymal transition (EMT) in vitro and lung metastasis in vivo. Mechanistically, miR573 directly targets the fibroblast growth factor receptor 1 (FGFR1) gene. Knockdown of FGFR1 phenocopies the effects of miR-573 expression on PCa cell invasion, whereas overexpression of FGFR1 partially attenuates the functions of miR-573. Consequently, miR-573 modulates the activation of FGFR1-downstream signaling in response to fibroblast growth factor 2 (FGF2). Importantly, we showed that GATA3 directly increases miR-573 expression, and thus down-regulates FGFR1 expression, EMT and invasion of PCa cells in a miR-573-dependent manner, supporting the involvement of GATA3, miR-573 and FGFR1 in controlling the EMT process during PCa metastasis. Altogether, our findings demonstrate a novel mechanism by which miR-573 modulates EMT and metastasis of PCa cells, and suggest miR-573 as a potential biomarker and/or therapeutic target for PCa management.The metastastic cascade is a complex process that is regulated at multiple levels in prostate cancer (PCa). Recent evidence suggests that microRNAs (miRNAs) are involved in PCa metastasis and hold great promise as therapeutic targets. In this study, we found that miR-573 expression is significantly lower in metastatic tissues than matched primary PCa. Its downregulation is correlated with high Gleason score and cancer-related mortality of PCa patients ( Metastasis is often responsible for the recurrence, poor prognosis and death of prostate cancer (PCa). Thus, a better understanding the molecular mechanisms involved in PCa metastasis will contribute to the identification of novel prognostic biomarkers and therapeutic targets . MicroRNEpithelial-mesenchymal transition (EMT), characterized by the loss of epithelial characteristics and acquisition of a mesenchymal phenotype, plays a critical role in PCa progression and metastasis . ReporteIn this study, our data highlight the role of miR-573 in inhibiting PCa metastasis through targeting FGFR1 and therefore disrupting EMT process. In addition, miR-573 itself is a transcriptional target of GATA3, which is thus able to regulate FGFR1 expression and EMT in a miR-573-dependent manner. This GATA3-miR-573-FGFR1 axis may represent a central pathway that controls EMT during PCa progression and thus hold great promise for therapeutic intervention in the future.To identify metastasis-related miRNAs, we comparatively analyzed the expression of 20 miRNAs in matched pairs of primary and metastatic tissues from three PCa patients. Of note, all of these miRNAs were previously reported to be highly dysregulated in PCa but their exact roles remained unclear –10. Sevein vitro gain- and loss-of-function analyses in VCaP and PC3 prostate cancer cell lines transfected with miR-573 mimics and antagomir, respectively. Matrigel-coated (for invasion) or -uncoated (for migration) transwell assays showed that ectopic miR-573 expression significantly inhibited VCaP and PC3 cell migration . Notably, mice transplanted with control VCaP cells displayed prominent lung metastasis (n = 56.5 ± 5.7), whereas mice bearing VCaP/miR-573 mimics tumors showed significantly less metastasis   and control mimics (962.6 ± 72.8 mm3) mice were hardly affected . Collectively, our data suggest that miR-573 significantly suppresses PCa invasion and metastasis.We next tested whether miR-573 could inhibit metastasis ) Figure . The cord Figure , which wAs EMT is an important cellular process that augments cell motility and leads to tumor metastasis , we wondTo understand the molecular mechanism by which miR-573 inhibits PCa EMT, invasion and metastasis, we analyzed putative targets of miR-573 using two bioinformatics tools, TargetScan  and miRWNext, we investigated the effects of FGFR1 on the invasive capacity in miR-573–transduced VCaP and 22RV1 cells. As shown in Figure In light of the critical role of FGFR1 in tumorigenesis and metastasis , especiaIn an attempt to address how miR-573 became downregulated in PCa, we hypothesized that an unknown transcriptional factor might play a role in the regulation of miR-573. The miRBase Sequences database was firstly utilized to identify the human mature miR-573 sequence. Sequence analysis with TESS and TFSEARCH databases was subsequently performed and we identified two conserved GATA3 motifs in the miR-573 promoter Figure . GATA3 hTo determine whether GATA3-mediated suppression of PCa metastasis involves miR-573, we transfected VCaP-GATA3 cells with miR-573 antagomir. Our data showed that GATA3 overexpression inhibited invasion of PCa cells, but concomitant inhibition of miR-573 abrogated these effects of GATA3 Figure . In addiWe also characterized the expression of GATA3, miR-573 and FGFR1 during TGF-β1-induced EMT in VCaP and 22RV1 cells. After TGF-β treatment for 72 h, the expression of GATA3 was significantly downregulated Figure , but FGFp = 0.044), but not with age, PSA level, or tumor grade.To further understand the clinical relevance of the above findings in human PCa, the expression of miR-573 was examined in 65 PCa cases. As shown in Table p = 0.041 Figure Next, we compared cancer-related survival rates between patients with high or low miR-573 levels in univariate models. Kaplan-Meier survival analysis showed that the group of patients who had higher miR-573 levels had a less rate of mortality (p = 0.029). Accordingly, there was a tendency for association between GATA3 expression and high miR-573 level in PCa cases, although it did not reach a significant difference (p = 0.075).We next determined whether there was an association between the expression levels of miR-573 and its targets in clinical PCa tissues. Significantly, as shown in Table Accumulating evidence suggests a causal role of miRNAs in the metastasis of human PCa due to their capacity in coordinately suppressing multiple target genes . PreviouFurther, our study identified FGFR1 as a key downstream target of miR-573. As previously reported, FGFR1 is highly expressed in various malignancies and its overexpression induces a series of cellular and molecular changes associated with EMT in bladder cancer and PCa , 17. OurFurther, our data showed that miR-573 is a potential downstream effector of GATA3, which has been shown to be a key factor in preventing PCa progression in the castration-resistant PTEN-deficient model, through antagonizing Akt signalling . GATA3 wIn summary, our study demonstrated prostate tumorigenesis caused by miR-573 down-regulation, which links FGFR1-mediated oncogenic signaling pathways with GATA3 dysregulation in PCa. Therefore, miR-573 might be a potential biomarker and targeting this novel GATA3/miR-573/FGFR1 axis will be a promising strategy for the treatment of a subset of metastatic PCa.n = 12), primary localized PCa (n = 65) and metastatic tissues (n = 15) were collected and embedded in paraffin for histological diagnosis and immunohistolocical study. None of the localized PCa patients received preoperative radiation or androgen deprivation therapy. Of note, matched pairs of primary and metastatic tissues were obtained from three PCa patients. The tissue morphology was validated by two pathologists (B.H. and W.T.). Detailed clinical and pathological profile were obtained from medical records and maintained on a secure relational database. This study was approved by the Institutional Review Board at the School of Medicine of Shandong University. Clinicopathological characters of localized PCa patients included in this study are included in Table Benign Prostate Hyperplasia BPH, n = 2, primarhttp://microrna.sanger.ac.uk/sequences/, University of Manchester, UK). The sequence of the miR-573 promoter was retrieved from the ISB lab . Transcriptional factor binding sites were predicted by the Transcription Element Search System  and the TFSEARCH system .The mature human miR-573 sequence was retrieved from the miRBase Sequence Database  cells were obtained from the American Type Culture Collection  and cultured by following the manufacturer's instructions. Recombinant human FGF basic (FGF2) was purchased from R&D Systems . 5-Aza-2′-deoxycytidine (5-Aza-dC) and Trichostatin A (TSA) were obtained from Sigma-Aldrich .Indicated siRNA, vectors and miR-573 mimics/antagomir transfections were carried out using Lipofectamine 2000 according to the manufacturer's protocol and each sequence is listed in The wild-type and mutants FGFR1 3′-UTR were generated on the miR-573 target recognition sites (seed sequences) as shown in ® Green I  according to the manufacturer's instructions. Melting analysis of the PCR products was conducted to validate the amplification of the specific product. The universal small nuclear RNA U6 was used as an endogenous control for miRNAs.Paraffin sections 10 mm thick were cut, dewaxed, rehydrated and lightly stained with hematoxylin. Tumor tissues were examined and microdissected under a dissecting microscope . miRNA extraction was performed with a miRNeasy FFPE kit , which enabled copurification of total RNA, including miRNA, from formalin-fixed paraffin-embedded tissue sections. Expression of miR-573 was measured using SYBR Primescript miRNA RT-PCR kit and SYBRIHC was performed as previously described . ImmunohAfter transfection with the indicated miRNA, siRNA or plasmids, migration and invasion assays were performed as previously described .Predicted targets for miRNAs and their target sites were analyzed using TargetScan , and miRDetails were described previously  and the Male Balb/c athymic nude mice at 6 weeks of age were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Scientific Investigation Board of Medical School of Shandong University.6 cells per mouse). Metastatic lesions in the lungs were counted macroscopically 12–14 weeks after injection.For measurement of tumor invasion and metastasis, 10 of these mice were injected subcutaneously with VCaP cells infected with adenoviruses expressing miR-573-EGFP and 10 mice were injected with VCaP cells infected with adenoviruses expressing control-EGFP  with 95% confidence intervals (CI) were calculated. A P-value of 0.05 was considered significant.Statistical analyses were carried out using the Statistical Package for Social Sciences, version 19.0 . Two-sided Student's


CDK4 and STAT3. We further showed that expression of miR-506-3p, but not miR-124-3p, is dramatically upregulated in differentiated neuroblastoma cells, suggesting the important role of endogenous miR-506-3p in differentiation and tumorigenesis. Overall, our functional screen on microRNAs provided the first comprehensive analysis on the involvements of microRNA species in neuroblastoma cell differentiation and identified novel differentiation-inducing microRNAs. Further investigations are certainly warranted to fully characterize the function of the identified microRNAs in order to eventually benefit neuroblastoma therapy.Neuroblastoma, the most common extracranial solid tumor of childhood, arises from neural crest cell precursors that fail to differentiate. Inducing cell differentiation is an important therapeutic strategy for neuroblastoma. We developed a direct functional high-content screen to identify differentiation-inducing microRNAs, in order to develop microRNA-based differentiation therapy for neuroblastoma. We discovered novel microRNAs, and more strikingly, three microRNA seed families that induce neuroblastoma cell differentiation. In addition, we showed that microRNA seed families were overrepresented in the identified group of fourteen differentiation-inducing microRNAs, suggesting that microRNA seed families are functionally more important in neuroblastoma differentiation than microRNAs with unique sequences. We further investigated the differentiation-inducing function of the microRNA-506-3p/microRNA-124-3p seed family, which was the most potent inducer of differentiation. We showed that the differentiation-inducing function of microRNA-506-3p/microRNA-124-3p is mediated, at least partially, by down-regulating expression of their targets  Neuroblastoma is the most common solid tumor of infancy and the most common extracranial solid tumor of childhood, accounting for more than 7% of childhood cancers and 15% of cancer-related childhood deaths . Neuroblin vitro [High-throughput screening approaches significantly facilitate the discovery of novel anti-cancer drugs and drug targets. More recently, high-content screens (HCSs) based on automated cell imaging have been developed. However, current HCSs generally either use genetic engineered cell lines expressing fluorescent signals or involve staining of fixed cells , 10, whiin vitro -14. WhilmiRNAs are endogenously expressed small RNAs that play a critical role in tumorigenesis -19. The in vitro [Neurite outgrowth is well recognized as a morphological hallmark of neuroblastoma cell differentiation in vitro -14. Thisin vitro -35, inhip<0.0001), supporting the reliability of the screen. As shown in Figure p=0.16), which informs us to use this model  in BE(2)-C cells. Figure We next focused on characterizing the differentiation-inducing function of the top 5 microRNAs that are most potent in inducing neurite outgrowth Figure . We show-RA) (42.6±3.7%), a differentiation-agent currently used to treat neuroblastoma patients [To further examine the potential pathophysiological relevance of endogenous miR-506-3p and miR-124-3p in regulating neuroblastoma differentiation, we measured their expression levels in differentiated BE(2)-C cells. Figure ) 42.6±3.%, a diffFrom the screen, we also identified miRNAs that significantly reduce cell survival but do not induce differentiation . This inp=2.2×10−7 , although the effect of miR-449c-5p did not reach statistical significance using p< 0.05 threshold. miR-769-3p does not induce neurite outgrowth, and, however, is the only exception among the identified differentiation-inducing seed families.miRNA seed sequences are central in determining their target genes -41. We tThe above observations lead us to identify potential miRNA targets based on seed sequence matches. We first identified 48 genes that have been previously demonstrated to regulate neuroblastoma differentiation . using ICDK4 and STAT3. Figure CDK4 and STAT3 expression induces neurite outgrowth, and that their combined repression has an enhanced effect on neurite outgrowth relative to individual repression. These results indicate that CDK4 and STAT3 mediate, at least partially, the differentiation-inducing function of miR-506-3p/miR-124-3p, and suggest that the effect of miR-506-3p/miR-124-3p on cell differentiation are most likely mediated by concordantly down-regulating multiple target genes.We further investigated the targets of miR-506-3p/miR-124-3p family. Table In this study, we developed a HCS approach to facilitate the discovery of novel differentiation-inducing agents for neuroblastoma. Several HCS approaches based on quantification of neurite outgrowth have been described , 42-44. Our study provides the first comprehensive and direct functional analysis of miRNA species in inducing neuroblastoma cell differentiation. Our screen not only recapitulated several previous findings, but also identified miRNAs that have not been known to regulate neuroblastoma differentiation. For example, we are the first to identify the function of miR-506-3p in promoting neuroblastoma cell differentiation. Our further investigations show that endogenous miR-506-3p expression was dramatically increased in differentiated neuroblastoma cells. In addition, published data have indicated the tissue-specific expression of miR-506 in adrenal gland, the primary tissue of origin for neuroblastoma . This evin vivo studies, and to compare their clinical relevance in neuroblastoma tumorigenesis in clinical studies.In our HCS, several miRNAs that were previously reported to induce neuroblastoma cell differentiation were not identified as potent inducers of cell differentiation , 30. OneAnother intriguing finding in our study is the overrepresentation of a set of miRNA seed families in our identified differentiation-inducing miRNAs. This observation is beyond providing convincing evidence supporting the reliability of our HCS approach. It leads us to an interesting speculation: the conservation of seed sequence among different miRNAs located at different genomic regions may be an important evolution trait that is selected by nature to guard the normal cell differentiation process during development; if one miRNA fails its expression in cells, expression of another seed-family member will perform similar function to prevent differentiation error. If this is true, we expect that miRNA seed sequence families are more likely to be involved in cell differentiation than miRNAs with unique sequences. There are certainly other possible explanations. Whether miRNA seed families are also overrepresented in other differentiation/development processes and what are the pathophysiological significances of this overrepresentation are certainly interesting questions to pursue in the future.in vitro and in vivo, in order to identify the optimal design that has the most potent effect on neuroblastoma differentiation and has minimal non-specific cytotoxicity on normal cells and tissues.Previous studies have demonstrated the promise of synthetic miRNA mimics as therapeutic agents in cancer treatment , 22. Our-RA, raising the possibility of applying differentiation-inducing miRNA mimics to remission induction therapy. Further investigations in in vivo models are certainly warranted to examine the therapeutic efficacy of miR-506-3p mimics.Currently, differentiation agents are limited to be used for post-remission maintenance therapy in high-risk neuroblastoma . One of In this study, we preliminarily analyzed the predicted targetomes of the identified top 14 differentiation-inducing miRNAs using informatics tools, and took a small step toward validating the predicted targets of miR-506-3p/miR-124-3p family. Our investigation suggests that the differentiation-inducing function of a miRNA is likely mediated by concordantly down-regulating multiple targets. Our current analysis is limited to genes that are known to regulate neuroblastoma differentiation. It is highly likely that there are undiscovered targets of these miRNAs that play important roles in mediating their differentiation-inducing functions. Indeed, our expression array analyses indicate that miR-506-3p/miR-124-3p overexpression down-regulates many more of their predicted targets than we investigated in this study (data not shown). Whether these targets play roles in mediating their differentiation-inducing function warrants further investigation.In summary, we have established a HCS platform to screen for novel differentiation-inducing substances in neuroblastoma cells. We have identified novel miRNAs and miRNA seed families that induce neuroblastoma cell differentiation. Our study not only provides the first comprehensive understanding of the role of miRNAs in neuroblastoma differentiation, but also provides novel leads for developing miRNA-based differentiation agents for neuroblastoma treatment. We expect that further investigation based on our findings will not only promote better understanding of the mechanisms of neuroblastoma tumorigenesis, but may also provide new therapeutic strategies for treating neuroblastoma.ATRA and cis-RA were from Sigma . Dharmacon miRNA mimic library and individual miRNA mimics were from Thermo Fisher Scientific . miRNA precursors were purchased from Ambion . Rabbit anti-GAP43, anti-NSE, and anti-β-TUBULIN III were from Abcam . Rabbit anti-CALNEXIN, anti-GAPDH and goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) were from Santa Cruz . Rabbit anti-PARP (cleaved), anti-CASPASE-3, anti-STAT3, and anti-CDK4, were from Cell Signaling . Rabbit anti-Ki67 was from Millipore .BE(2)-C cells were purchased from the ATCC. Other cell lines were obtained from the cell line repository at the Greehey Children's Cancer Research Institute. Cells were grown in DMEM/F12 supplemented with 10% fetal bovine serum.Cells were plated and treated in 96-well plates. For detecting neurite outgrowth, cells were placed into ZOOM IncuCyte Imaging System (Essen BioScience) and cell images were taken under 20X microscopic magnification. For detecting neurite outgrowth in a time-dependent manner, cell images were taken every 6 h. The neurite lengths associated with each treatment were calculated using the neurite definition defined for each specific cell line using the NeuroTrack system (Essen BioScience).The relative neurite length associated with cells in each well on the screen plates was determined as above. In order to allow for plate-to-plate comparison, neurite length associated with each well in each plate was first internally normalized to the mean of the corresponding plate, and multiple screen plates were then aggregated together to generate the neurite length distribution. The data was then further analyzed to determine the distribution of the unaffected cells as described below, and to identify differentiation-inducing miRNA mimics as illustrated in p<0.05 considered as rejecting the null hypothesis that the neurite length distribution fits Gamma distribution.In order to examine whether the neurite length distribution of the untreated cells fit Gamma distribution, Gaussian kernel smoothing was first performed to generate the empirical density curve based on the neurite length distribution histogram of the untreated cells, and Gamma model parameters  were then estimated by minimizing the area in between empirical and Gamma distribution curves. Statistical significance of the fitness to Gamma distribution was examined by Kolmogorov-Smirnov goodness-of-fit test, with Cell lysates were prepared using RIPA buffer. Protein concentration was determined using the Pierce BCA assay (Thermo Fisher Scientific). For electrophoresis, equal amounts of cell lysate were resolved by SDS-PAGE and transferred to Immun-Blot PVDF membranes (Bio-Rad Laboratories). Membranes were blocked and probed with primary antibodies to specific proteins. Bound antibodies were detected with secondary antibodies conjugated with horseradish peroxidase (HRP) visualized by enhanced chemiluminescent (ECL) substrate (Pierce).Cells were plated in 96-well plates and were treated with specified conditions. Cells were placed into the IncuCyte imaging system and cell confluence was monitored every 6 h for 120 h. Cell growth curves were derived by comparing the cell confluences at different time points.Cells were plated in 96-well format and treated as specified. After culturing for 120 h, cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega).Cells were transfected as specified and cultured overnight. 500 cells were re-plated in 10 cm dishes. After 14 days, colonies were visualized by staining with 1% crystal violet. Colony numbers and sizes were analyzed using Image J .8 permutations were run to determine the probability that at least × non-unique seed sequences appear in a randomly selected set of 14 miRNAs (p(X)).We use random permutation to examine whether miRNA seed families are significantly enriched in the identified set of differentiation-inducing miRNAs. The miRNA mimic library includes mimics for 1231 human miRNAs listed in miRBase 16.0. Among these miRNAs, 900 miRNAs have unique seed sequences; 331 miRNAs share seed sequences with at least one other miRNA, constituting 125 seed sequence families. To calculate the probability of miRNAs from the same seed families randomly appears in a set of 14 miRNAs, 14 miRNAs were randomly drawn from the 1231 miRNAs, and the number of non-unique seed sequences within the 14 miRNAs is counted (X). 10nd - 8th or 1st - 7th nucleotides, 5'-3') as a potential target site of this miRNA. To identify targets that potentially mediate the differentiation-inducing function of our identified miRNAs, we first identified the genes that have been known to relate to neuroblastoma differentiation using IPA. From this gene list, we identified the predicted targets for each miRNA and generated the predicted miRNA:target interaction network mediating the differentiation-inducing function of the identified miRNAs.miRNA target sites in 3'UTRs were predicted based on seed sequence complementarity and were identified using the IPA Pathway analysis function, which identifies any 7-nucleotide region (3'-5') in a given 3'UTR completely complementary to the seed sequence of a miRNA  replaced by CACGGUU. The 3'UTRs were cloned downstream of the firefly luciferase coding sequences into the pmirGLO dual-luciferase reporter (Promega), a vector containing both firefly and Renilla luciferase cDNAs under the control of separate promoter/terminator systems. The firefly luciferase was used as the primary reporter for miRNA regulation of the 3'UTR. The Renilla luciferase is an internal control for normalization. BE(2)-C cells were co-transfected with luciferase reporters (0.8 ng/ul) and miRNA mimics or control oligonucleotide (oligo) (25 nM). Luciferase activities were measured after 72 h using the Dual-Glo Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity to evaluate the effect of the miRNAs.The segments of the wildtype 3'UTRs for p-value for neurite length associated with each miRNA mimic was evaluated by assuming the distribution of neurite lengths follows a Gamma distribution, and FDR was determined by Benjamini-Hochberg correction method for multiple tests [t-test by comparing the treatment group with control, with p< 0.05 considered statistically significant.For HCS, le tests . We cons


We found a good correlation between off-rates and cellular responses on cloned CD8 T cells from cancer patients[Adoptive transfer of high avidity tumor-specific CD8 T cells is a promising treatment for late stage cancers. Because most tumor antigens are self-antigens, tumor-specific T cells typically express low affinity T cell antigen receptors (TCR) due to thymic negative selection, which accounts mostly for their poor tumoricidal activity. To identify rare, most tumor-reactive T cells we developed a robust and accurate screening assay. The functional avidity of T cell responses is related to pMHC-TCR binding parameters, with an assumed optimal range of pMHC-TCR affinity for the most potent interaction. We recently described the development of NTAmers, reversiIn conclusion, NTAmers and pMHC dimers enable precise measurement of pMHC-TCR binding parameters under physiological conditions and subsequent isolation of live functionally active tumor-specific CD8 T cells. Translation of these techniques to the single cell level for high-throughput screening and isolation of rare and highly functional tumor-specific CD8 T cells directly from cancer patients is in progress.


Silencing of ODC also led to significantly reduced concentrations of polyamines , tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion  proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.Transgenic plants of  These plants were identical in growth habit and morphology to those of non-transgenic parental line SC 58. Seeds of all lines were surface-sterilized and germinated in vitro on Murashige and Skoog (MS) agar plates containing 3% sucrose and 75 μg mL−1 kanamycin sulphate according to −1 of compost mix) as recommended by the manufacturer .Homozygous TN. tabacum plants (~10–12 leaf stage) either remained non-wounded (C) or were mechanically injured in one of three ways as follows. (1) Designated ‘W’ for wounded; a fabric pattern wheel was drawn across the lamina twice on each side of the mid-vein of the two uppermost (>50%) expanded leaves to simulate insect attack  Desortho-phthaldialdehyde/ethanethiol/fluorenylmethoxycarbonyl derivatives as described by Concentrations of amino acids, amines, alkaloids and phenolamides were determined using portions of homogeneous powdered tissue that had previously been freeze-dried for a minimum of 48h. One hundred milligrams of freeze-dried ground leaf or root powder was pre-weighed and aliquoted into 1.5mL Eppendorf tubes containing a sterile stainless steel ball to aid extraction for subsequent metabolite analyses. Alkaloids and phenolamides were extracted from leaf and root samples using an optimized 40% methanol extraction method described by 2 using a hot phenol method adapted from Nicotiana species (TM SYBR no-ROX (Bioline) following the manufacturer’s recommendations. Previously published gene-specific primers (Supplementary Table S1 at JXB online). Results were obtained from analysis of three independent samples per treatment, each containing three technical replicates. Data were analysed using the 2–ΔΔCT method  and R-Studio .All statistical tests were performed using R 3.1.2  and 25-fold lower in roots  and a ~2-fold increase in nicotine content of roots (rising to ~2.4mg g−1 dwt); a ~2-fold increase in nornicotine concentrations (rising to ~20 μg−1 dwt in leaves and ~80 μg−1 dwt in roots); and a ~10-fold increase in anatabine concentrations  in baseline levels of ornithine in leaf and root tissues of non-wounded odc-RNAi transgenics compared with VC plants  by leaf wounding alone and by apex removal (5- to 6-fold) relative to similarly treated VC plants  and root (30–40%) tissues compared with VC plants. Wounding did not significantly alter arginine concentrations in leaves of VC plants but there was a significant, albeit <2-fold, increase in arginine concentration of roots in response to the combined leaf wounding and apex removal treatment  and also alkaloid production and mobilization  (SAMDC) or were reduced (SAMS) in roots of wounded vs. non-wounded VC control plants  . In contl plants .1odc-RNAi plants  levels of ADC transcript in roots of wounded odc-RNAi plants compared with VC counterparts, increasing in magnitude with plants damaged by leaf wounding < apex removal < combined leaf wounding and apex removal treatments  than in comparable VC plants. We observed even greater relative differences in roots of wounded odc-RNAi plants, where QPT transcript levels were 5- to 6-fold lower than in similarly wounded VC plants. Even in plants that experienced the most severe combined leaf wounding and apex removal treatment, QPT transcript levels of odc-RNAi plants were not significantly elevated above levels observed in non-wounded VC plants  and/or apex removal (6- to 7-fold) in VC plants, compared with non-wounded counterparts. A marked reduction (~50%) in relative baseline SPDS transcript levels was observed in non-wounded odc-RNAi plants compared with VC plants. This difference was further exacerbated in wounded odc-RNAi plants, with SPDS transcript levels being 75–80% lower than that of similarly wounded VC plants. Silencing of ODC also resulted in ~1.5- to 2-fold reductions in relative SAMS and SAMDC transcript levels in both non-wounded and wounded odc-RNAi-plants, compared with appropriate VC plants  leaves, as well as in the stem and roots. Tyramine concentrations were also lower in leaves and roots of wounded odc-RNAi lines than in VC plants but, interestingly, the converse was true in stem tissues where levels were ~2-fold higher in wounded odc-RNAi lines compared with similarly wounded VC plants  controlling the synthesis of defensive alkaloids in N. tabacum, particularly in response to wounding. One or more mechanism(s) linking ornithine metabolism directly with pyridine alkaloid biosynthetic capacity in Nicotiana may also help explain previous observations whereby over-expression of a yeast ODC gene led to both elevated putrescine levels and increased concentrations of nicotine in hairy roots of N. rustica  in cells over-expressing the mouse ODC gene compared with vector controls (ODC was down-regulated in tobacco using an RNAi approach, diminished transcript levels of the polyamine synthesis genes SAMDC, SAMS, and SPDS in roots of odc-RNAi plants was observed compared with vector-only controls. Such reductions in transcript abundance in odc-RNAi transgenics may be directly linked with reduced putrescine supply and likely underpin the lower spermidine and spermine levels that we observed in these plants compared with their VC counterparts. On the other hand, an increase in the arginine concentration of these plants, together with elevated ADC transcript, is in line with previous observations suggesting compensatory increases in ADC activity in plants with lowered ODC activity (With regards to effects of ng yeast  and the ramonium . ExperimODC-silenced plants, and unlike our earlier observations with plants of the T1 generation (2 lines that were homozygous for the introduced odc-RNAi construct, compared with VC controls. Anomalies such as sporadic periodic production of chlorotic, bleached and brittle leaves may be linked to changes in photosynthetic machinery or nutritional deficiencies associated with reduced putrescine supply (odc-RNAi transgenics compared with vector-only controls, such as shorter internodes, reduced root biomass, slower rates of release of dormant axillary buds following decapitation, and also markedly reduced vigour and delayed onset of flowering in odc-RNAi plants when grown in soil, are broadly in line with morphological alterations that have been reported previously in polyamine mutants of tobacco (Nicotiana plants treated with the ODC biochemical inhibitor difluoromethylornithine (DFMO; per se (ODC gene less likely to be down-regulated by the Nicotiana odc-RNAi construct used here, for example the ODC gene from yeast that was previously expressed in N. rustica (odc-RNAi transgenic lines of N. tabacum.In addition to changes in the metabolic profile of  tobacco ; in NicoJXB online.Supplementary data are available at Table S1. Sequences of gene-specific primers used for qRT-PCR


People with Anorexia Nervosa exhibit difficulties flexibly adjusting behaviour in response to environmental changes. This has previously been attributed to problematic behavioural shifting, characterised by a decrease in fronto-striatal activity. Additionally, alterations of instrumental learning, which relies on fronto-striatal networks, may contribute to the observation of inflexible behaviour. The authors sought to investigate the neural correlates of cognitive flexibility and learning in Anorexia Nervosa.Thirty-two adult females with Anorexia Nervosa and thirty-two age-matched female control participants completed the Wisconsin Card Sorting Task whilst undergoing functional magnetic resonance imaging. Event-related analysis permitted the comparison of cognitive shift trials against those requiring maintenance of rule-sets and allowed assessment of trials representing learning.Although both groups performed similarly, we found significant interactions in the left middle frontal gyrus, precuneus and superior parietal lobule whereby blood-oxygenated-level dependent response was higher in Anorexia Nervosa patients during shifting but lower when maintaining rule-sets, as compared to healthy controls. During learning, posterior cingulate cortex activity in healthy controls decreased whilst increasing in the Anorexia Nervosa group, whereas the right precuneus exhibited the opposite pattern. Furthermore, learning was associated with lower blood-oxygenated-level dependent response in the caudate body, as compared to healthy controls.People with Anorexia Nervosa display widespread changes in executive function. Whilst cognitive flexibility appears to be associated with aberrant functioning of the fronto-parietal control network that mediates between internally and externally directed cognition, fronto-striatal alterations, particularly within the caudate body, were associated with instrumental learning. Together, this shows how perseverative tendencies could be a substrate of multiple high-order processes that may contribute to the maintenance of Anorexia Nervosa. We chose to use the WCST because it permits investigation of separable forms of set-shifting inefficiencies that have previously been linked with distinct neural patterns. For instance, failure to respond appropriately to negative feedback has been associated with decreased regional cerebral bloodflow in the middle frontal gyrus whereas the inability to keep in mind previously unsuccessful strategies was related to decreased regional cerebral bloodflow in the inferior parietal lobule . Such chThe WCST also allows measurement of cortical function during instrumental learning and reinforcement, arguably vital processes for efficient flexible behaviour. Indeed, abnormal activity within the dorsal striatum, which plays a major role in forming stimulus-response and action-outcome associations , has beeWe hypothesised that patients with anorexia would 1) perform poorly on the WCST, particularly with regards to perseverative errors, 2) exhibit abnormal fronto-striatal activity during learning and reinforcement and 3) utilise the fronto-parietal control network to a greater degree than healthy controls (HC) during cognitive set-shifting.http://www.b-eat.co.uk/) . All AN participants had been diagnosed by eating disorder clinicians as fulfilling DSM-IV criteria and included 24 restrictive and 8 binge/purge subtypes. Fifteen were taking ≥1 psychoactive medications during the study  were recruited from the South London and Maudsley (SLaM) eating disorder service and from a B-EAT community sample  screeneThis study was carried out in accordance with the Declaration of Helsinki under approval of the National Research Ethics Committee, London (Ref 11/LO/0952). Written informed consent was obtained following complete description of the study.Participants performed a modified version of the WCST during the scan. At the start of each trial, a single ‘stimulus’ card was presented in the centre of a grey screen, surrounded by four ‘reference’ cards . ‘RefereParticipants had 3000ms from the start of each trial to match the ‘stimulus’ card to one of the four ‘references’ using a 4-way joystick, according to either shape, colour or quantity. The chosen ‘reference’ was then immediately outlined for 2000ms; in green if correct, in red if incorrect, and the ‘stimulus’ card was replaced with corresponding written feedback. This was followed by a rest period lasting until the trial had elapsed 6000ms, at which point a new trial started . ParticiSiS-P) and recurrent (Rec-P) [Eff-Sh), both traditionally classified as non-perseverative errors [F-Corr and S-Corr respectively) were also considered separately for fMRI analysis as they represent learning and reinforcement processes. A high-level baseline of consecutive correct trials was used in order to subtract behavioural flexibility processes from events of interest. Error set, omission, anticipatory and ambiguous trials not conforming to selected event criteria were coded as nuisance covariates.Reaction time and responses were recorded during scanning and classified as discrete events according to previous literature . Notably (Rec-P) , and adoe errors . The forfMRI data were acquired on a 1.5-Tesla GE Signa HDx system running 14m5 software  at the Centre for Neuroimaging Sciences of the Institute of Psychiatry, King’s College London. A body coil was used for RF transmission and an 8-channel head coil for RF reception. T2*-weighted gradient echo echo-planar images (GE-EPI) depicting blood-oxygen-level-dependent (BOLD) contrast were acquired in the axial plane, parallel to the anterior commissure—posterior commissure (AC-PC) line, with the following parameters: repetition time (TR) = 2000ms, echo time (TE) = 40ms, flip angle = 70°, slice thickness = 5mm, slice gap = 0.5mm, field of view = 24x24cm, matrix size = 64x64. Whole-brain coverage was acquired with 25 slices and 300 T2*-weighted whole-brain volumes were acquired for each subject.To facilitate co-registration of fMRI data in standard space, a whole-brain high-resolution GE-EPI volume consisting of 43 slices parallel to the AC-PC line was acquired for each participant . Data quality was assured using an automated quality control procedure .t-tests were used to assess between-group differences unless Shapiro-Wilk test indicated non-normality of data, in which case non-parametric Mann-Whitney U comparisons were conducted. Performance measures included total number of correct trials, completed sets , stuck-in-set perseverations, recurrent perseverations and loss-of-set errors. Reaction times for these events were also analysed, as well as for first correct, second correct and efficient shift trials.Demographic, clinical and performance data were analysed using Statistical Package for the Social Sciences (SPSS 20.0) . Paramethttp://brainmap.co.uk).fMRI data were analysed with XBAM v4.1, developed at King’s College London’s Institute of Psychiatry, which utilises a non-parametric permutation-based approach to minimize assumptions and reduce the effect of outliers  were then detected by time-series analysis using a linear model in which each component of the experimental design was convolved separately with a pair of Poisson kernels (λ = 4 and 8 seconds) to allow variability in the haemodynamic delay. Events were modelled as continuous periods between two sequential participant responses, where the type of event was defined by the combination of these two responses  and F-Corr vs. Eff-Sh (cognitive flexibility) by group interactions were performed using split-plot analyses of variance (ANOVA) in order to ascertain whether any brain regions exhibit group dependency in differential event-related activation patterns.Between and within-group comparisons were performed by fitting the data at each intracerebral voxel at which all subjects have non-zero data using a linear model of the type:The AN group had a significantly lower BMI and cognitive flexibility scale score and higher EDE-Q global, HADS-depression and HADS-anxiety scores compared to HC . There wWCST performance and reaction times are illustrated in F-Corr and Eff-Sh trials are adaptive events that are necessary for the completion of the task. However, these events differ on the basis of the response action carried out; Eff-Sh trials are those in which the participant shifts to a different untested rule following negative feedback whilst F-Corr trials are those which follow a correct rule application and followed by continued application of that rule. The former requires a shift whilst the latter requires maintenance therefore we reasoned that comparing these events would allow visualisation of neural regions involved in cognitive flexibility.Both F-Corr and Eff-Sh trials were compared for each group separately. HC exhibited significantly greater activation in the left precuneus, right anterior cingulate, right middle frontal gyrus, left inferior frontal gyrus and right cerebellum during Eff-Sh and in the right putamen, right postcentral gyrus and left paracentral lobule during F-Corr. AN exhibited greater activation in the middle frontal gyri bilaterally, left precuneus, right superior frontal gyrus, left medial frontal gyrus, right inferior parietal lobule and left cerebellum during Eff-Sh and in the left middle frontal gyrus and left dorsal posterior cingulate cortex during F-Corr  interaction revealed that the AN group exhibited greater activation during Eff-Sh trials but lesser activation during F-Corr trials in the left middle frontal gyrus, left inferior precuneus and left superior parietal lobule, as compared to the HC group  by event (HC group .SiS-P (50%) and Rec-P (37.5%) errors over the course of the task with these participants also showing low event frequency  and second correct (S-Corr) trials both represent events in which the participant has applied the correct sorting rule. Although a requisite of F-Corr is maintenance and application of the rule in the next trial, the period modelled may be associated with uncertainty; first after because the correct rule is found through trial and error and second because the participant may not be sure of the rule even a correct response. As such, the event following repeated application (S-Corr) represents verification of what participants believe to be the correct rule. We reasoned that comparing these events would allow assessment of neural mechanisms involved in learning.First correct  interaction revealed that the AN group had increasing activation in the right posterior cingulate cortex from F-Corr to S-Corr whereas the HC group exhibited a decreasing trend. Oppositely, AN show decreasing activation in the right precuneus from F-Corr to S-Corr whilst HC exhibited an increasing trend  by event (ng trend .The current study used the Wisconsin Card Sorting Test (WCST) in conjunction with whole-brain event-related fMRI to elucidate the neural underpinnings of instrumental learning and cognitive flexibility in AN. With regards to behavioural measures, whilst AN attained lower scores on the cognitive flexibility scale and a measure of general WCST performance (completed sets), we did not detect differences with regards to perseverations. Previous neuropsychological studies using the WCST consistently identify heightened perseverative tendency in AN ,6. HowevS-Corr), although no difference was found after first correct trials (F-Corr). This was somewhat surprising, given that previous fMRI investigations have found hyperactivity during various executive processes, including reward and cognitive flexibility [Despite this, lower activation was found in the left caudate body in AN as compared to HC after consolidating a new rule , which rk Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with strong evidence for viral carcinogenesis. The association of MCC with the Merkel cell polyomavirus (MCPyV) may explain the explicit immunogenicity of MCC. Indeed, MCPyV-encoded proteins are likely targets for cytotoxic immune responses to MCC as they are both foreign to the host and necessary to maintain the oncogenic phenotype. However, to date only a single MCPyV-derived CD8 T-cell epitope have been described, thus impeding specific monitoring of T-cell responses to MCC.To overcome this limitation, we scanned the MCPyV oncoproteins large T and small T antigen and the virus-capsid protein VP1 for potential T-cell epitopes, and tested for major histocompatibility complex (MHC) class I affinity. We confirmed the relevance of these epitopes using a high-throughput platform for T-cell enrichment and combinatorial encoding of MHC class I multimers.ex vivo further substantiated the relevance of the identified epitopes.In peripheral blood from 38 MCC patients and 30 healthy donors we identified 53 MCPyV-directed CD8+ T-cell responses against 35 different peptide sequences. Strikingly, T-cell responses against oncoproteins were exclusively present in MCC patients, but not in healthy donors. We further demonstrate both the processing and presentation of the oncoprotein-derived epitopes, as well as the lytic activity of oncoprotein-specific T cells towards MHC-matched MCC cells. Demonstrating the presence of oncoprotein-specific T cells among tumour infiltrating lymphocytes These T-cell epitopes represent ideal targets for antigen specific immune therapy of MCC, and enables tracking and characterisation of MCPyV specific immune responses.


MicroRNAs (miRNAs) are short, 22-25 nucleotide long transcripts that may suppress entire signaling pathways by interacting with the 3’-untranslated region (3’-UTR) of coding mRNA targets, interrupting translation and inducing degradation of these targets. The long 3’-UTRs of brain transcripts compared to other tissues predict important roles for brain miRNAs. Supporting this notion, we found that brain miRNAs co-evolved with their target transcripts, that non-coding pseudogenes with miRNA recognition elements compete with brain coding mRNAs on their miRNA interactions, and that Single Nucleotide Polymorphisms (SNPs) on such pseudogenes are enriched in mental diseases including autism and schizophrenia, but not Alzheimer’s disease (AD). Focusing on evolutionarily conserved and primate-specific miRNA controllers of cholinergic signaling (‘CholinomiRs’), we find modified CholinomiR levels in the brain and/or nucleated blood cells of patients with AD and Parkinson’s disease, with treatment-related differences in their levels and prominent impact on the cognitive and anti-inflammatory consequences of cholinergic signals. Examples include the acetylcholinesterase (AChE)-targeted evolutionarily conserved miR-132, whose levels decline drastically in the AD brain. Furthermore, we found that interruption of AChE mRNA’s interaction with the primate-specific CholinomiR-608 in carriers of a SNP in the AChE’s miR-608 binding site induces domino-like effects that reduce the levels of many other miR-608 targets. Young, healthy carriers of this SNP express 40% higher brain AChE activity than others, potentially affecting the responsiveness to AD’s anti-AChE therapeutics, and show elevated trait anxiety, inflammation and hypertension. Non-coding regions affecting miRNA-target interactions in neurodegenerative brains thus merit special attention.


Epithelial ovarian cancer (EOC) is typically diagnosed at advanced stages, and is associated with a high relapse rate. Patients in remission are ideal candidates for immunotherapy aimed at cure or prolonging disease-free periods. However, immunosuppressive pathways in the tumor microenvironment are obstacles to durable anti-tumor immunity. In a metastatic syngeneic mouse model of EOC, immunosuppressive macrophages and myeloid-derived suppressor cells (MDSCs) accumulate in the local tumor environment. In addition, resident peritoneal macrophages from non-tumor-bearing mice were highly immunosuppressive, abrogating stimulated T cell proliferation in a cell contact-dependent manner. Immunization with microparticles containing TLR9 and NOD-2 ligands (MIS416) significantly prolonged survival in tumor-bearing mice. The strategy of MIS416 immunization followed by anti-CD11b administration further delayed tumor progression, thereby establishing the proof of principle that myeloid depletion can enhance vaccine efficacy. In patients with advanced EOC, ascites analysis showed substantial heterogeneity in the relative proportions of myeloid subsets and their immunosuppressive properties. Together, these findings point to immunosuppressive myeloid cells in the EOC microenvironment as targets to enhance vaccination. Further studies of myeloid cell accumulation and functional phenotypes in the EOC microenvironment may identify patients who are likely to benefit from vaccination combined with approaches that deplete tumor-associated myeloid cells. Results are expressed as net counts per minute (cpm) [average cpm from mixed cultures of T cells with PECs in presence of CD3/CD28 - ].To evaluate the immunosuppressive properties of peritoneal myeloid cells, PECs were FACS-sorted to isolate macrophages (CD11bescribed . T cellsTime to euthanasia was plotted using Kaplan-Meier curves and analyzed using the log-rank method. Comparisons between two groups were assessed by the Mann-Whitney test, and the Kruskal-Wallis test was used for multiple group comparisons. Statistical analysis was performed using Graph Pad Prism 6 software.


In the present study, the antitumor effect of the combination of a PS-targeting antibody with antibodies that inhibit the downstream immune checkpoints PD-1 or CTLA-4 antibody in the K1735 mouse melanoma model was examined. Tumor-bearing mice were treated with each antibody alone or the combination at 5 to 10 mg/kg, twice a week. Combination therapy potently suppressed tumor growth and improved overall survival compared to single agent treatment. Flow cytometry revealed that combination therapy induced the highest ratio of tumor-infiltrating immune effector to suppressor cells. Importantly, combination treatment also significantly decreased the levels of myeloid-derived suppressor cells (MDSC) in the spleen. In addition, inhibition of PS and PD-1 or CTLA-4 resulted in significantly more IL-2 and IFNg-secreting splenic CD4+ and CD8+ T cells than any single agent treatment. Finally, combined immune checkpoint blockade did not induce any observable toxicity following multiple treatment doses. In summary, our findings demonstrate that the combination of antibody-mediated PS blockade with an inhibition of established immune checkpoints  represents a promising strategy for cancer immunotherapy.Phosphatidylserine (PS) is an upstream immune checkpoint that drives global immunosuppression. Previous work has shown that PS targeting agents can override PS-driven immunosuppression and re-program the tumor microenvironment from immunosuppressive to immunosupportive, break tumor immune tolerance, and elicit potent