Expression profiling of key pathways in rat liver after a one-year feeding trial with transgenic maize MON810.

In a recent one-year feeding study, we observed no adverse effects on tissue level in organs of rats fed with the genetically-modified maize MON810. Here, we assessed RNA expression levels of 86 key genes of the apoptosis-, NF-кB-, DNA-damage response (DDR)-, and unfolded-protein response (UPR) pathways by RT-qPCR in the rat liver. Male and female rats were fed either with 33% MON810 (GMO), isogenic- (ISO), or conventional maize (CONV) and RNAs were quantified from eight rats from each of the six feeding groups. Only Birc2 transcript showed a significant (p ≤ 0.05) consistent difference of ≥1.5-fold between the GMO and ISO groups in both sexes. Unsupervised cluster analysis showed a strong separation of male and female rats, but no clustering of the feeding groups. Individual analysis of the pathways did not show any clustering of the male or female feeding groups either, though transcript levels of UPR pathway-associated genes caused some clustering of the male GMO and CONV feeding group samples. These differences were not seen between the GMO and ISO control or within the female cohort. Our data therefore does not support an adverse effect on rat liver RNA expression through the long-term feeding of MON810 compared to isogenic control maize.

MicroRNA-15 family targets the hedgehog signaling pathway during postnatal development of porcine intestine.

The hedgehog signaling pathway is a crucial regulator of the postnatal intestinal development. Regulation of hedgehog expression itself is poorly understood. MicroRNAs were demonstrated to control differentiation and proliferation in postnatal intestinal development. This study identifies members of the miR-15 family to regulate the expression of key hedgehog factors employing in silico and in vitro experiments. Physiological relevance is demonstrated by incorporation of in vivo expression data from the ileum and colon from 7 to 56 days old piglets. Results presented in this study improve the understanding of the complex regulation of hedgehog signaling during intestinal development and disease.

Transcriptomic Analysis of Intestinal Tissues from Two 90-Day Feeding Studies in Rats Using Genetically Modified MON810 Maize Varieties.

Background: Global as well as specific expression profiles of selected rat tissues were characterized to assess the safety of genetically modified (GM) maize MON810 containing the insecticidal protein Cry1Ab. Gene expression was evaluated by use of Next Generation Sequencing (NGS) as well as RT-qPCR within rat intestinal tissues based on mandatory 90-day rodent feeding studies. In parallel to two 90-day feeding studies, the transcriptional response of rat tissues was assessed as another endpoint to enhance the mechanistic interpretation of GM feeding studies and/or to facilitate the generation of a targeted hypothesis. Rats received diets containing 33% GM maize (MON810) or near-isogenic control maize. As a site of massive exposure to ingested feed the transcriptomic response of ileal and colonic tissue was profiled via RT-qPCR arrays targeting apoptosis, DNA-damage/repair, unfolded protein response (UPR). For global RNA profiling of rat ileal tissue, we applied NGS. Results: No biological response to the GM-diet was observed in male and in female rat tissues. Transcriptome wide analysis of gene expression by RNA-seq confirmed these findings. Nevertheless, gene ontology (GO) analysis clearly associated a set of distinctly regulated transcripts with circadian rhythms. We confirmed differential expression of circadian clock genes using RT-qPCR and immunoassays for selected factors, thereby indicating physiological effects caused by the time point of sampling. Conclusion: Prediction of potential unintended effects of GM-food/feed by transcriptome based profiling of intestinal tissue presents a novel approach to complement classical toxicological testing procedures. Including the detection of alterations in signaling pathways in toxicity testing procedures may enhance the confidence in outcomes of toxicological trials. In this study, no significant GM-related changes in intestinal expression profiles were found in rats fed GM-maize MON810. Relevant alterations of selected cellular pathways (apoptosis, DNA damage and repair, UPR) pointing toward intestinal toxicity of the diets were not observed. Transcriptomic profiles did not reveal perturbations of pathways associated with toxicity, underlining the study results revealed by classical OECD endpoints.

One-year oral toxicity study on a genetically modified maize MON810 variety in Wistar Han RCC rats (EU 7th Framework Programme project GRACE).

The GRACE (GMO Risk Assessment and Communication of Evidence; www.grace-fp7.eu ) project was funded by the European Commission within the 7th Framework Programme. A key objective of GRACE was to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of a 1-year feeding trial with a GM maize MON810 variety, its near-isogenic non-GM comparator and an additional conventional maize variety are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 452. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after a chronic exposure.

Structural analysis of microRNA-target interaction by sequential seed mutagenesis and stem-loop 3' RACE.

BACKGROUND: As a consequence of recent RNAseq efforts, miRNAomes of diverse tissues and species are available. However, most interactions between microRNAs and regulated mRNAs are still to be deciphered. While in silico analysis of microRNAs results in prediction of hundreds of potential targets, bona-fide interactions have to be verified e.g. by luciferase reporter assays using fused target sites as well as controls incorporating mutated seed sequences. The aim of this study was the development of a straightforward approach for sequential mutation of multiple target sites within a given 3' UTR. METHODOLOGY/PRINCIPAL FINDINGS: The established protocol is based on Seed Mutagenesis Assembly PCR (SMAP) allowing for rapid identification of microRNA target sites. Based on the presented approach, we were able to determine the transcription factor NKX3.1 as a genuine target of miR-155. The sequential mutagenesis of multiple microRNA target sites was examined by miR-29a mediated CASP7 regulation, which revealed one of two predicted target sites as the predominant site of interaction. Since 3' UTR sequences of non-model organisms are either lacking in databases or computationally predicted, we developed a Stem-Loop 3' UTR RACE PCR (SLURP) for efficient generation of required 3' UTR sequence data. The stem-loop primer allows for first strand cDNA synthesis by nested PCR amplification of the 3' UTR. Besides other applications, the SLURP method was used to gain data on porcine CASP7 3'UTR evaluating evolutionary conservation of the studied interaction. CONCLUSIONS/SIGNIFICANCE: Sequential seed mutation of microRNA targets based on the SMAP approach allows for rapid structural analysis of several target sites within a given 3' UTR. The combination of both methods (SMAP and SLURP) enables targeted analysis of microRNA binding sites in hitherto unknown mRNA 3' UTRs within a few days.

Quantification and accurate normalisation of small RNAs through new custom RT-qPCR arrays demonstrates Salmonella-induced microRNAs in human monocytes.

BACKGROUND: Small interfering and non-coding RNAs regulate gene expression across all kingdoms of life. MicroRNAs constitute an important group of metazoan small RNAs regulating development but also disease. Accordingly, in functional genomics microRNA expression analysis sheds more and more light on the dynamic regulation of gene expression in various cellular processes. RESULTS: We have developed custom RT-qPCR arrays allowing for accurate quantification of 31 small RNAs in triplicate using a 96 well format. In parallel, we provide accurate normalisation of microRNA expression data based on the quantification of 5 reference snRNAs. We have successfully employed such arrays to study microRNA regulation during human monocyte differentiation as well as Salmonella infection. Besides well-known protagonists such as miR-146 or miR-155, we identified the up-regulation of miR-21, miR-222, miR-23b, miR-24, miR-27a as well as miR-29 upon monocyte differentiation or infection, respectively. CONCLUSIONS: The provided protocol for RT-qPCR arrays enables straight-forward microRNA expression analysis. It is fully automatable, compliant with the MIQE guidelines and can be completed in only 1 day. The application of these arrays revealed microRNAs that may mediate monocyte host defence mechanisms by regulating the TGF-ß signalling upon Salmonella infection. The introduced arrays are furthermore suited for customised quantification of any class of small non-coding RNAs as exemplified by snRNAs and thus provide a versatile tool for ubiquitous applications.