Predicted mRNA Targets Research Articles

Integrative mRNA/miRNA expression analysis in healing human gingiva.

MicroRNAs (miRNAs) are implicated in the epigenetic regulation of complex biological processes. Their possible role in human oral wound healing, a process that differs from cutaneous wound healing by being faster and typically scar-free, has been unexplored. This report presents the miRNA expression profile of experimental human oral wounds and an integrative analysis of mRNA/miRNA expression. Nine healthy volunteers provided standardized normal and 5-day healing palatal biopsies, used for next generation miRNA and mRNA sequencing analysis, correlation and network analysis, real-time PCR (qPCR) and immunohistochemistry. On average, 169 significantly regulated precursor miRNAs were detected, including 21 novel miRNAs, selectively confirmed by PCR. Hsa-miR-223-3p and hsa-miR-124-3p were, respectively, the most up- and downregulated miRNAs in healing gingiva. Hsa-miR-124-3p had the most predicted mRNA target interactions, with angiogenesis-related genes the most enriched. Correlation analysis showed the highest correlation between hsa-miR-181a-3p and SERPINB1; hsa-miR-223-5p and SLC2A3; hsa-miR-1301 and MS4A7. In addition, SERPINB1 mRNA had the most associations with differentially regulated miRNAs. IL33 was the only cytokine significantly correlated with miRNAs (ρ>0.95). qPCR and immunohistochemistry verified the significant upregulation of SERPINB1 and IL33 in healing gingiva. This study is the first to report on the miRNome of healing human gingiva and to provide an integrative analysis of miRNA/mRNA expression during human oral wound healing; the results offer novel insights into the participating molecular mechanisms and raise the possibility of SERPINB1 and IL-33 as potential wound healing therapeutic targets.

Exploration of microRNA profiles in human colostrum.

BackgroundColostrum is well known to have excellent nutritional value for newborns. The aim of this study was to investigate the dynamic expression pattern of microRNA in human colostrum and mature milk. Furthermore, we identified the specific microRNA in human colostrum and analyzed the regulatory function of human colostrum.MethodsWe collected breast milk samples from 18 lactating volunteers. The expression of microRNA in breast milk was detected by microarray analysis. The expression differences were characterized by log2FC (|log2fold change| >1.58) and associated P values (P<0.05). Furthermore, the prediction of microRNA targets, bioinformatics analysis and network generation were carried out using network database.ResultsOur results showed that during the human lactation process, the composition of microRNAs in human milk changes dynamically. Compared to the microRNA expression profile in human mature milk, the expression levels of 49 microRNAs were significantly different and 67 microRNAs were specifically expressed in human colostrum. Based on the results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the predicted target mRNAs of the identified colostrum-specific microRNAs were involved in the regulation of distinct biological processes, such as signal transduction, positive regulation of GTPase activity, and protein phosphorylation. Moreover, the predicted mRNA targets were from large spectrums of signaling pathways, such as the MAPK, Ras, Hippo, Wnt, and mTOR signaling pathways, as well as the longevity regulating pathway.ConclusionsOur study illuminates the landscape of microRNA expressions in human colostrum and mature milk, and emphasizes the value of microRNAs as nutritional additives in milk-related commercial products.

Abstract 1400: An additive effect of ebv-miR-BART5-5p and hsa-miR18a-5p in the downregulation of CDH1 transcript in Epstein-Barr virus associated gastric cancer

Abstract Background. Nine percent of Gastric Cancer (GC) is associated with Epstein-Barr virus (EBV-associated GC), with significant variations in the global prevalence rates. Emerging evidence shows that, in addition to cellular miRNAs, viral miRNAs can also bind human mRNAs. EBV miRNAs are examples of this binding and are associated with oncogenic capacities of EBV. ebv-miR-BART5-5p shares the exact seed sequence with hsa-miR-18a-5p. However, common mRNA targets have not been fully described. The aim of this study is to determine predicted targets shared by both miRNAs in EBV-associated GC. Methodology. By in house method we identified predicted mRNA targets for ebv-miR-BART5-5p and hsa-miR-18a-5p. These targets were queried in published results from the supplementary files of the STAD TCGA study and miRwalk 2.0 software. Validation was performed in 7 EBV-associated GC (cases) and 6 EBV-negative GC (controls) samples by miQ-PCR (ebv-miR-BART5-5p and hsa-miR-18a-5p) and qRT-PCR (predicted targets). Results. We identified 25 downregulated and 11 upregulated mRNAs in EBV-associated GC cases compared with EBV-negative tumors in validated databases. Interestingly CDH1, which has been associated with diffuse and hereditary GC, was among the predicted targets by miRwalk 2.0. In clinical samples of EBV-associated GC, ebv-miR-BART5-5p and hsa-miR-18a-5p were overexpressed compared with control samples (p=0.0253 and p=0.0205, respectively). Furthermore, CDH1 was downregulated in EBV-associated tumors (p=0.0290). Conclusion: We identified a common human mRNA target, the CDH1 transcript for ebv-miR-BART5-5p and hsa-miR-18a-5p in EBV-associated GC. An additive effect can be suggested between both miRNAs, which may explain the unique downregulation of CDH1 in EBV-associated GC. This work warrants future validation in vitro, as well as in a larger cohort of EBV-associated GC. Grant support: CONICYT-FONDAP-15130011, Fondecyt Regular-1191928 and Fondecyt PostDoc-3201028 Citation Format: Keila Torres, Natalia Landeros, Ignacio Wichmann, Allan Peñaloza-Otárola, Iva Polakovicova, Vinicius Maracaja-Coutinho, Maria Jose Maturana, Franz Villaroel-Espindola, Alejandro Corvalan. An additive effect of ebv-miR-BART5-5p and hsa-miR18a-5p in the downregulation of CDH1 transcript in Epstein-Barr virus associated gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1400.

MicroRNA and mRNA profiling in the idiopathic inflammatory myopathies

BackgroundThe idiopathic inflammatory myopathies (IIMs) are heterogeneous autoimmune conditions of skeletal muscle inflammation and weakness. MicroRNAs (miRNAs) are short, non-coding RNA which regulate gene expression of target mRNAs. The aim of this study was to profile miRNA and mRNA in IIM and identify miRNA-mRNA relationships which may be relevant to disease.MethodsmRNA and miRNA in whole blood samples from 7 polymyositis (PM), 7 dermatomyositis (DM), 5 inclusion body myositis and 5 non-myositis controls was profiled using next generation RNA sequencing. Gene ontology and pathway analyses were performed using GOseq and Ingenuity Pathway Analysis. Dysregulation of miRNAs and opposite dysregulation of predicted target mRNAs in IIM subgroups was validated using RTqPCR and investigated by transfecting human skeletal muscle cells with miRNA mimic.ResultsAnalysis of differentially expressed genes showed that interferon signalling, and anti-viral response pathways were upregulated in PM and DM compared to controls. An anti-Jo1 autoantibody positive subset of PM and DM (n = 5) had more significant upregulation and predicted activation of interferon signalling and highlighted T-helper (Th1 and Th2) cell pathways. In miRNA profiling miR-96-5p was significantly upregulated in PM, DM and the anti-Jo1 positive subset. RTqPCR replicated miR-96-5p upregulation and predicted mRNA target (ADK, CD28 and SLC4A10) downregulation. Transfection of a human skeletal muscle cell line with miR-96-5p mimic resulted in significant downregulation of ADK.ConclusionMiRNA and mRNA profiling identified dysregulation of interferon signalling, anti-viral response and T-helper cell pathways, and indicates a possible role for miR-96-5p regulation of ADK in pathogenesis of IIM.

Comparison of miRNA Cargo within Wharton's Jelly mesenchymal stem cell-derived exosomes manufactured in FBS and hPLT expansion media

Background & Aim Wharton's Jelly Mesenchymal Stem Cell (WJMSCs)-derived exosomes have been studied extensively in animal models of degenerative and chronic diseases. Thus, the development of a large-scale manufacturing procedure to harvest clinical-grade exosomes is required for the translation of exosome to the clinic. We have developed an exosome production technique to manufacture large quantities of exosomes using the quantum bioreactor (Terumo BCT) with both fetal bovine serum (FBS) and human platelet lysate (hPLT) supplemented expansion media. AWe aimed to compare the miRNA cargo of exosomes isolated from FBS-expanded and PLT-expanded WJMSC to determine if growth media is a significant contributor to exosome product preparation. Methods, Results & Conclusion WJMSCs were isolated from Wharton's Jelly and expanded to passage 1. After harvest, 30 × 106 cells were loaded into the quantum bioreactor and expanded for 1 week to achieve maximum confluence. Expansion media was washed and replaced with serum-free aMEM media. Conditioned media was collected every 24 hours for 120 hours and exosomes were precipitated by sequential centrifugation and ultracentrifugation. NanoSight analysis of the final products revealed a production of 15.0 × 1012± 0.32 × 1012 total particles with an average mode size of 111±4.78 nm. Surface protein analysis revealed positive expression of exosome markers CD9, CD63, and CD81. Exosome products were subjected to miRNA isolation, sequencing, and miRNA identification for bioinformatics analysis with the Qiagen IPA software. miRNA analysis was completed using 3 WJMSC donors grown in 20% FBS or 5%PLT supplemented media. Sequencing results identified 157 miRNAs in the PLT group and 154 miRNAs in the FBS group (with a 100-copy minimum expression cut off). FBS and PLT comparison revealed 142 common miRNAs with 15 unique to PLT and 12 unique to FBS. Further RNA target analysis revealed 68 miRNAs that target 1024 mRNA in the PLT group and 64 miRNA that target 1048 mRNA in the FBS group. The two groups share 998 common mRNA targets with 27 unique to PLT and 51 unique to FBS. Canonical pathway analysis of the unique targets reveals the top pathway hits, serotonin receptor and JAK/Stat signaling for PLT and IL-7 and RANK signaling for FBS. We found both expansion medias to be effective for large scale expansion and downstream exosome production. miRNA sequencing revealed small variations in the total number of predicted mRNA targets, suggesting a conserved therapeutic effect between the two groups. Wharton's Jelly Mesenchymal Stem Cell (WJMSCs)-derived exosomes have been studied extensively in animal models of degenerative and chronic diseases. Thus, the development of a large-scale manufacturing procedure to harvest clinical-grade exosomes is required for the translation of exosome to the clinic. We have developed an exosome production technique to manufacture large quantities of exosomes using the quantum bioreactor (Terumo BCT) with both fetal bovine serum (FBS) and human platelet lysate (hPLT) supplemented expansion media. AWe aimed to compare the miRNA cargo of exosomes isolated from FBS-expanded and PLT-expanded WJMSC to determine if growth media is a significant contributor to exosome product preparation. WJMSCs were isolated from Wharton's Jelly and expanded to passage 1. After harvest, 30 × 106 cells were loaded into the quantum bioreactor and expanded for 1 week to achieve maximum confluence. Expansion media was washed and replaced with serum-free aMEM media. Conditioned media was collected every 24 hours for 120 hours and exosomes were precipitated by sequential centrifugation and ultracentrifugation. NanoSight analysis of the final products revealed a production of 15.0 × 1012± 0.32 × 1012 total particles with an average mode size of 111±4.78 nm. Surface protein analysis revealed positive expression of exosome markers CD9, CD63, and CD81. Exosome products were subjected to miRNA isolation, sequencing, and miRNA identification for bioinformatics analysis with the Qiagen IPA software. miRNA analysis was completed using 3 WJMSC donors grown in 20% FBS or 5%PLT supplemented media.

Deregulation of miR-27a may contribute to canine fibroblast activation after coculture with a mast cell tumour cell line.

The tumour microenvironment comprises a diverse range of cells, including fibroblasts, immune cells and endothelial cells, along with extracellular matrix. In particular, fibroblasts are of significant interest as these cells are reprogrammed during tumorigenesis to become cancer‐associated fibroblasts (CAFs), which in turn support cancer cell growth. MicroRNAs (miRNAs) have been shown to be involved in this intercellular crosstalk in humans. To assess whether miRNAs are also involved in the activation of fibroblasts in dogs, we cocultured primary canine skin fibroblasts with the canine mast cell tumour cell line C2 directly or with C2‐derived exosomes, and measured differential abundance of selected miRNAs. Expression of the CAF markers alpha‐smooth muscle actin (ACTA2) and stanniocalcin 1 confirmed the activation of our fibroblasts after coculture. We show that fibroblasts displayed significant downregulation of miR‐27a and let‐7 family members. These changes correlated with significant upregulation of predicted target mRNAs. Furthermore, RNA interference knockdown of miR‐27a revealed that cyclin G1 (CCNG1) exhibited negative correlation at the mRNA and protein level, suggesting that CCNG1 is a target of miR‐27a in canine fibroblasts and involved in their activation. Importantly, miR‐27a knockdown itself resulted in fibroblast activation, as demonstrated by the formation of ACTA2 filaments. In addition, interleukin‐6 (IL‐6) was strongly induced in our fibroblasts when cocultured, indicating potential reciprocal signalling. Taken together, our findings are consistent with canine fibroblasts being reprogrammed into CAFs to further support cancer development and that downregulation of miR‐27a may play an important role in the tumour–microenvironment crosstalk.

MiRNA-mRNA Integrated Analysis Reveals Roles for miRNAs in a Typical Halophyte, Reaumuria soongorica, during Seed Germination under Salt Stress.

MicroRNAs (miRNAs) are endogenous small RNAs that play a crucial role in plant growth, development, and environmental stress responses. Reaumuria soongorica is a typical halophyte that is widely distributed in saline–alkali desert regions. Under salt stress, R. soongorica can complete germination, a critical biological process in the life cycle of seed plants. To identify miRNAs and predict target mRNAs involved in seed germination during salt stress, nine small-RNA libraries were constructed and analyzed from R. soongorica seeds treated with various concentrations of NaCl. We also obtained transcriptome data under the same treatment conditions. Further analysis identified 88 conserved miRNAs representing 25 defined families and discovered 13 novel miRNAs from nine libraries. A co-expression analysis was performed on the same samples to identify putative miRNA–mRNA interactions that were responsive to salt stress. A comparative analysis of expression during germination under 273 (threshold) and 43 mM (optimal) NaCl treatments identified 13 differentially expressed miRNAs and 23 corresponding target mRNAs, while a comparison between 43 mM NaCl and non-salt-stress conditions uncovered one differentially expressed miRNA and one corresponding target mRNA. These results provide basic data for further study of molecular mechanisms involved in the germination of salt-stressed R. soongorica seeds, and also provide a reference for the improvement of salt tolerance during plant germination.

Eldecalcitol (ED-71)-induced exosomal miR-6887-5p suppresses squamous cell carcinoma cell growth by targeting heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1).

Heparin-binding protein 17/fibroblast growth factor-binding protein-1 (HBp17/FGFBP-1) was purified from A431 cell-conditioned media based on its capacity to bind to fibroblast growth factor 1 and 2 (FGF-1 and FGF-2). HBp17/FGFBP-1 has been observed to induce the tumorigenic potential of epithelial cells and is highly expressed in oral cancer cell lines and tissues. HBp17/FGFBP-1 is also recognized as a pro-angiogenic molecule as a consequence of its interaction with FGF-2. We have previously reported that Eldecalcitol (ED-71), an analog of 1α,25(OH)2D3, downregulated the expression of HBp17/FGFBP-1 and inhibited the proliferation of squamous cell carcinoma (SCC) cells in vitro and in vivo through NF-κb inhibition. To explore the possibility of microRNA (miRNA) control of HBp17/FGFBP-1, we analyzed exosomal miRNAs from medium conditioned by A431 cells treated with ED-71. Microarray analysis revealed that 12 exosomal miRNAs were upregulated in ED-71-treated A431 cells. Among them, miR-6887-5p was identified to have a predicted mRNA target matching the 3' untranslated region (3'-UTR) of HBp17/FGFBP-1. The 3'-UTR of HBp17/FGFBP-1 was confirmed to be a direct target of miR-6887-5p in SCC/OSCC cells, as assessed with a luciferase reporter assay. Functional assessment revealed that overexpression of miR-6887-5p in SCC/OSCC cells inhibited cell proliferation and colony formation in vitro, and inhibited tumor growth in vivo compared with control. In conclusion, our present study supports a novel anti-cancer mechanism involving the regulation of HBp17/FGFBP-1 function by exosomal miR-6887-5p in SCC/OSCC cells, which has potential utility as a miRNA-based cancer therapy.

Regulatory Noncoding Small RNAs Are Diverse and Abundant in an Extremophilic Microbial Community.

Regulatory small RNAs (sRNAs) play large-scale and essential roles in many cellular processes across all domains of life. Microbial sRNAs have been extensively studied in model organisms, but very little is known about the dynamics of sRNA synthesis and their roles in the natural environment. In this study, we discovered hundreds of intergenic (itsRNAs) and antisense (asRNAs) sRNAs expressed in an extremophilic microbial community inhabiting halite nodules (salt rocks) in the Atacama Desert. For this, we built SnapT, a new sRNA annotation pipeline that can be applied to any microbial community. We found asRNAs with expression levels negatively correlated with that of their overlapping putative target and itsRNAs that were conserved and significantly differentially expressed between 2 sampling time points. We demonstrated that we could perform target prediction and correlate expression levels between sRNAs and predicted target mRNAs at the community level. Functions of putative mRNA targets reflected the environmental challenges members of the halite communities were subjected to, including osmotic adjustments to a major rain event and competition for nutrients.IMPORTANCE Microorganisms in the natural world are found in communities, communicating and interacting with each other; therefore, it is essential that microbial regulatory mechanisms, such as gene regulation affected by small RNAs (sRNAs), be investigated at the community level. This work demonstrates that metatranscriptomic field experiments can link environmental variation with changes in RNA pools and have the potential to provide new insights into environmental sensing and responses in natural microbial communities through noncoding RNA-mediated gene regulation.

A survey of non-coding RNAs in the social and predatory myxobacterium Myxococcus xanthus DK1622.

Prokaryotic ncRNAs are important regulators of gene expression, and can be involved in complex signalling networks. The myxobacteria are model organisms for studies into multicellular development and microbial predation, being particularly renowned for their large genomes and exceptionally sophisticated signalling networks. However, apart from two specific examples, little is known about their regulatory ncRNAs. Here, we integrate bioinformatic predictions and transcriptome sequence data to provide a comprehensive survey of the ncRNAs made by the exemplar myxobacterium M. xanthus DK1622. M. xanthus RNA-seq data from four experimental conditions was interrogated to identify transcripts mapping outside coding sequences and to known ncRNAs. The resulting 37 ncRNAs were clustered on the genome and most (30/37) were conserved across the myxobacteria. A majority of ncRNAs (22/37) were intergenic, while 13 were at least partially antisense to protein-coding genes. Predicted promoter and terminator sequences explained the start/stop sites of 18 ncRNAs. mRNA targets for the ncRNAs were predicted, including plausible candidates for a known regulatory ncRNA. 22 ncRNAs were differentially expressed by nutrient availability and expression of 25 predicted targets was found to correlate strongly with that of their regulatory ncRNAs. Sharing of predicted mRNA targets by multiple ncRNAs suggests that some ncRNAs might regulate each other within signalling networks. This genomic survey of M. xanthus ncRNA biology provides a starting point for further studies of myxobacterial ncRNAs, which are likely to have important functions in these industrially important and sophisticated organisms.

Integrated microRNA/mRNA expression profiling of the skin of psoriasis patients

BackgroundPsoriasis is a chronic inflammatory disease characterized by demarcated, raised, and scaling skin lesions. It often serves as a model for immune-mediated disorders. Gene expression profiling of affected skin has allowed insights into psoriasis pathogenesis. However, the mechanisms leading to specific mRNA expression alterations in psoriasis are barely understood. ObjectivesTo perform integrated microRNA-mRNA expression studies of non-lesional, peri-lesional, and lesional skin from psoriasis patients. MethodsCutaneous microRNA and mRNA expression profiles of 14 patients using Nanostring nCounter-technology and RNA sequencing as well as in vitro keratinocyte stimulation and qPCR studies. ResultsOnly 3.5 % of microRNAs manifested a robust gradual expression trend from non-lesional to paired lesional skin, with 61 % being upregulated and 39 % being downregulated. Relevance of these microRNA regulations was supported by their inverse association with 57 % of the mRNA species found to be regulated during psoriatic lesion development. Many of the involved mRNAs were downregulated and functionally related to keratinocyte metabolism, barrier function, and neuronal signaling, and were already regulated in peri-lesional skin. An integrated correlation analysis revealed a robust interaction for 134 microRNAs/mRNAs pairs. In vitro keratinocyte studies of selected microRNAs/mRNAs revealed regulations of all analyzed microRNAs in a psoriasis-like manner by IL-17A/TNF-α (e.g. hsa-miR-23a-3p), IFN-γ (e.g. hsa-miR-106a-5p/miR-17-5p), or IL-24 (e.g. hsa-miR-203a-3p). Moreover, most of their predicted target mRNAs (e.g. ID4, EPHB2) were respectively altered by the same cytokines. ConclusionOur study suggests that, during development of psoriatic lesions, defined aspects of psoriasis pathogenesis are regulated by the action of microRNAs.

973P - Advanced clear-cell renal cell carcinoma (accRCC): Association of microRNAs (miRNAs) with molecular subtypes, mRNA targets and outcome

BackgroundBased on mRNA expression, accRCC can be divided in four molecular subtypes (ccrcc1-4), associated with prognosis, angiogenic signature, immune phenotype and therapy response. miRNAs are master regulators of mRNA function. We assessed the correlation of miRNA expression with (1) ccrccc1-4 subtypes, (2) mRNA targets and (3) outcome. MethodsWe performed miRNome analysis of 128 accRCC. (1) Unsupervised hierarchical clustering was done using 50 miRNAs with the most variable expression across all samples. Results were validated on TCGA data (n=255). (2) For miRNAs with subtype-specific expression, we performed pathway analysis of predicted mRNA targets (IPA, KEGG) and assessed target-downregulation in TCGA. (3) We used Cox regression to correlate miRNA expression with overall survival (OS) since diagnosis. Hazard ratios (HR) were correlated with subtype-specific expression (Pearson). Results(1) Samples separated in two miRNA clusters, that partially overlapped with molecular subtypes. Cluster 1 consisted of 69% favourable subtypes (ccrcc2 or 3), cluster 2 of 77% unfavourable (ccrcc1 or 4) (p<2.2e-16). (2) Pathway analysis of predicted mRNA targets and targets suppressed in TCGA, suggested that favourable subtypes exhibit more angiogenic signaling, whereas unfavourable subtypes activate pathways involved in tumor invasiveness. (3) Several miRNAs were significantly associated with OS. There was a robust correlation in the entire dataset between HR for OS and differential expression between favourable/unfavourable subtypes (r=0.322; p<0.0001).Table973PTablemiRNASubtype-specific expression log2FoldChange <0: higher in ccrcc1 or -4 (unfavourable) log2FoldChange >0: higher in ccrcc2 or -3 (favourable)*p<0.05 ** false discovery rate <0.05Overall survival (hazard ratio)182-5p-1,43**1,25**3529-3p-1,42**1,37**335-3p-1,33**1,25**34c-5p-1,22**1,25**193b-3p-1,17**1,31**370-3p-1,07**1,16**199b-5p-1,05**1,26**21-3p-0,89**1,47**574-5p-0,89**1,40**4792-0,88**0,88*let-7i-5p-0,81**1,66**652-3p-0,75**1,39**1301-3p-0,70**1,39**222-3p-0,66**1,31*320a-0,63**1,28**425-5p-0,62**1,66**149-5p-0,61**1,32*146b-5p-0,54*1,49**193b-5p-0,491,21**185-5p-0,49**1,42**21-5p-0,42*1,68**7641-0,330,80**320b-0,281,25**1260b-0,150,79**44540,150,80**6880-5p0,250,64**27b-3p0,36**0,71*1260a0,380,73**79770,390,73**23b-3p0,48**0,81**23920,550,80**194-5p0,55*0,74**30c-5p0,70**0,78*190a-5p0,86**0,83**204-5p1,23**0,71**135a-5p1,37**0,86** ConclusionsaccRCC molecular subtypes exhibit different miRNA expression patterns. Differentially expressed miRNAs are implicated in pathways driving tumor biology and strongly correlated with OS. These findings underscore the robustness of the molecular subtypes and are, to the best of our knowledge, the first to show an association of global miRNome expression with outcome. Legal entity responsible for the studyThe authors. FundingFonds Wetenschappelijk Onderzoek Vlaanderen. DisclosureM. Albersen: Research grant / Funding (self): Ipsen. B. Beuselinck: Advisory / Consultancy, Research grant / Funding (self): Bristol Meyers Squibb; Speaker Bureau / Expert testimony: Ipsen; Speaker Bureau / Expert testimony: Merck; Research grant / Funding (self): Pfizer. All other authors have declared no conflicts of interest.

Milk-derived miRNA profiles elucidate molecular pathways that underlie breast dysfunction in women with common genetic variants in SLC30A2

Studies in humans and pre-clinical animal models show milk-derived miRNAs reflect mammary gland function during lactation. The zinc transporter SLC30A2/ZnT2 plays a critical role in mammary gland function; ZnT2-null mice have profound defects in mammary epithelial cell (MEC) polarity and secretion, resulting in sub-optimal lactation. Non-synonymous genetic variation in SLC30A2 is common in humans, and several common ZnT2 variants are associated with changes in milk components that suggest breast dysfunction in women. To identify novel mechanisms through which dysfunction might occur, milk-derived miRNA profiles were characterized in women harboring three common genetic variants in SLC30A2 (D103E, T288S, and Exon 7). Expression of ten miRNAs differed between genotypes, and contributed to distinct spatial separation. Studies in breast milk and cultured MECs confirmed expression of ZnT2 variants alters abundance of protein levels of several predicted mRNA targets critical for breast function (PRLR, VAMP7, and SOX4). Moreover, bioinformatic analysis identified two novel gene networks that may underlie normal MEC function. Thus, we propose that genetic variation in genes critical for normal breast function such as SLC30A2 has important implications for lactation performance in women, and that milk-derived miRNAs can be used to identify novel mechanisms and for diagnostic potential.

IDENTIFICATION OF NOVEL MICRORNAS ASSOCIATED ATHEROSCLEROTIC LESION FORMATION IN THE AORTA OF HYPERCHOLESTEROLEMIC APOE-/- MICE

Objective: Evidence is accumulating that microRNAs (miRNAs), members of the non-coding RNA family, may be effective biomarkers and therapeutic targets in atherosclerosis. The aim of this study was to identify novel miRNA expression patterns underlying vascular inflammation and oxidative stress associated with atheroma formation in human and experimental atherosclerosis. Design and method: Human non-atherosclerotic (superior thyroid artery) and atherosclerotic (carotid artery) arterial specimens, ApoE-/- mice, and in vitro polarized pro-, and anti-inflammatory mouse macrophages (Mac) were used. Male ApoE-/- mice were maintained on normal (ND) or high-fat cholesterol rich (HD) diet for 6 months to accelerate de development of advanced atherosclerotic lesions along the aorta. Atherosclerotic lesions were analyzed by Oil Red O staining and immunohistochemistry (markers of immune cell infiltration, CD45, CD68). Human and mouse miRCURY LNA™ miRNome real-time PCR panels were employed to determine the expression pattern of 752 different miRNAs. Microarray-based (Agilent SurePrint G3 Human/Mouse gene Expression v2 8x60K) genome-wide gene expression profiling was done to determine the ratio between a specific miRNA and the expression level of its predicted mRNA target. miRNA target prediction tools were used (miRanda, MiRBase, miRWalk, Target Scan, etc). Results: miRNA expression profiling revealed that 272 miRNAs are significantly up-regulated and 224 down-regulated in the atherosclerotic aorta of ApoE-/- (HD) mice as compared to values obtained for ApoE-/- (ND) animals. A cluster of 49 miRNAs with upregulated levels in atherosclerotic aorta of mice were detected in cultured pro-inflammatory mouse Mac as compared to resting Mac. In cultured anti-inflammatory Mac, the augmented expression of 63 miRNA was detected. Similarly regulated (up/down) homologous miRNAs were determined in both human and mouse atherosclerotic tissues. Candidate miRNAs linked to vascular inflammation and oxidative stress in atherosclerosis were predicted by in silico analysis. Conclusions: We found new miRNA panels reporting information about key pathological processes linked to atheroma formation such as inflammation and oxidative stress in both human and experimental atherosclerosis. The data of this study point to the novel atherosclerotic tissue-expressed miRNAs as potential biomarkers and/or therapeutic targets in atherosclerosis-related cardiovascular disorders.

Hepatocyte-Derived Exosomes Promote Liver Immune Tolerance: Possible Implications for Idiosyncratic Drug-Induced Liver Injury.

Most idiosyncratic drug-induced liver injury appears to result from an adaptive immune attack on the liver. Recent evidence suggests that the T-cell response may be facilitated by the loss of immune tolerance. In this study, we explored the hypothesis that constitutively released hepatocyte-derived exosomes (HDE) are important for maintaining normal liver immune tolerance. Exosomes were isolated from the conditioned medium of primary human hepatocytes via polymer precipitation. Mock controls were prepared by processing fresh medium that was not hepatocyte exposed with precipitation reagent. THP-1 monocytes were then treated with HDE or an equivalent volume of mock control for 24 h, followed by a 6-h stimulation with LPS. HDE exposure resulted in a significant decrease in the LPS-induced media levels of interleukin-1β and interleukin-8. Gene expression profiling performed in THP-1 cells just prior to LPS-induced stimulation identified a significant decrease among genes associated with innate immune response. MicroRNA (miRNA) profiling was performed on the HDE to identify exosome contents that may drive immune suppression. Many of the predicted mRNA target genes for the most abundant microRNAs in HDE were among the differentially expressed genes in THP-1 cells. Taken together, our data suggest that HDE play a role in maintaining normal liver immune tolerance. Future experiments will explore the possibility that drugs causing idiosyncratic liver injury promote the loss of homeostatic HDE signaling.

Integrated Genomic and Functional microRNA Analysis Identifies miR-30-5p as a Tumor Suppressor and Potential Therapeutic Nanomedicine in Head and Neck Cancer.

To identify deregulated and inhibitory miRNAs and generate novel mimics for replacement nanomedicine for head and neck squamous cell carcinomas (HNSCC). We integrated miRNA and mRNA expression, copy number variation, and DNA methylation results from The Cancer Genome Atlas (TCGA), with a functional genome-wide screen. We reveal that the miR-30 family is commonly repressed, and all 5 members sharing these seed sequence similarly inhibit HNSCC proliferation in vitro. We uncover a previously unrecognized inverse relationship with overexpression of a network of important predicted target mRNAs deregulated in HNSCC, that includes key molecules involved in proliferation (EGFR, MET, IGF1R, IRS1, E2F7), differentiation (WNT7B, FZD2), adhesion, and invasion (ITGA6, SERPINE1). Reexpression of the most differentially repressed family member, miR-30a-5p, suppressed this mRNA program, selected signaling proteins and pathways, and inhibited cell proliferation, migration, and invasion in vitro. Furthermore, a novel miR-30a-5p mimic formulated into a targeted nanomedicine significantly inhibited HNSCC xenograft tumor growth and target growth receptors EGFR and MET in vivo. Significantly decreased miR-30a/e family expression was related to DNA promoter hypermethylation and/or copy loss in TCGA data, and clinically with decreased disease-specific survival in a validation dataset. Strikingly, decreased miR-30e-5p distinguished oropharyngeal HNSCC with poor prognosis in TCGA (P = 0.002) and validation (P = 0.007) datasets, identifying a novel candidate biomarker and target for this HNSCC subset. We identify the miR-30 family as an important regulator of signal networks and tumor suppressor in a subset of HNSCC patients, which may benefit from miRNA replacement nanomedicine therapy.

Genome-wide profiling of long noncoding RNA expression patterns and CeRNA analysis in mouse cortical neurons infected with different strains of borna disease virus

Borna disease virus 1 (BoDV-1) is neurotropic prototype of Bornaviruses causing neurological diseases and maintaining persistent infection in brain cells of mammalian species. Long non-coding RNA (lncRNA) is transcript of more than 200 nucleotides without protein-coding function regulating various biological processes as proliferation, apoptosis, cell migration and viral infection. However, regulatory of lncRNAs in BoDV-1 infection remains unknown. To identify differential expression profiles and predict functions of lncRNA in BoDV-1 infection, microarray data showed that 3528 lncRNAs and 2661 lncRNAs were differentially expressed in Strain V and Hu-H1 BoDV-infected groups compared with control groups, respectively. Gene Ontology (GO) and pathway analysis suggested that differential lncRNAs may be involved in regulation of metabolic, biological regulation, cellular process, endocytosis, viral infections and cell adhesion processes, cancer in both BoDV-infected strains. ENSMUST00000128469 was found down-regulated in both BoDV-infected groups compared with control groups consistent with microarray (p < 0.05). ceRNA analysis indicated possible interaction networks as ENSMUST00000128469/miR-22-5p, miR-206-3p, miR-302b-5p, miR-302c-3p, miR-1a-3p/Igf1. Igf1 was found up-regulated in both BoDV-infected groups compared with control groups (p < 0.05). Possible functions of predicted target mRNAs and miRNAs of ENSMUST00000128469 were involved in cell proliferation, transcriptional misregulation and proteoglycan pathways enriched in cancer. lncRNA may be involved in regulation of Hu-H1 inhibited cell proliferation and promoted apoptosis through NF-kB, JNK/MAPK signaling, BCL2 and CDK6/E2F1 pathways different from Strain V. Possible interaction networks as ENSMUST00000128469/miR-22-5p, miR-206-3p, miR-302b-5p, miR-302c-3p, miR-1a-3p/Igf1 may involve in regulation of cell proliferation, apoptosis, and cancer.

The role of microRNAs in chronic pseudomonas lung infection in Cystic fibrosis.

Cystic Fibrosis (CF) is the most common life limiting genetic disorder, characterized by chronic respiratory failure secondary to inflammation and chronic bacterial lung infection. Pseudomonas aeruginosa lung infection is associated with more severe lung disease and rapid progression of respiratory failure when compared to Staphylococcus aureus infection. We hypothesized that a specific signature of epigenetic factors targeting specific gene transcripts contributes to the increased morbidity seen in CF patients with chronic Pseudomonas infection. We collected exhaled breath condensate (EBC) from 27 subjects and evaluated miRNA signatures in these samples using commercial PCR array. We identified predicted mRNA targets and associated signaling pathways using Ingenuity Pathway Analysis. We found 11 differentially expressed miRNAs in EBC of patients infected with Pseudomonas aeruginosa compared to EBC from CF patients who were not chronically infected with Pseudomonas aeruginosa (p < 0.05). Six of these miRNAs (hsa-miRNA-1247, hsa-miRNA-1276, hsa-miRNA-449c, hsa-miRNA-3170, hsa-miRNA-432-5p and hsa-miR-548) were significantly different in the CF Pseudomonas positive group when compared to both the CF Pseudomonas negative group and healthy control group. Ingenuity pathway analysis (IPA) revealed organismal injury and abnormalities, reproductive system disease and cancer as the top diseases and bio functions associated with these miRNAs. IPA also detected RELA, JUN, TNF, IL-10, CTNNB1, IL-13, SERPINB8, CALM1, STARD3NL, SFI1, CD55, RPS6KA4, TTC36 and HIST1H3D as the top target genes for these miRNAs. Our study identified 6 miRNAs as epigenetic factors specifically associated with chronic Pseudomonas infection in patients with CF.

Edwardsiella tarda-induced miR-7a functions as a suppressor in PI3K/AKT/GSK3β signaling pathway by targeting insulin receptor substrate-2 (IRS2a and IRS2b) in Paralichthys olivaceus

To study the effect of Edwardsiella tarda infection on miRNAs expression profile in Japanese flounder, fish were injected intraperitoneally with E. tarda. The miRNAs involved in regulating immune responses were analyzed by high-throughput sequencing. A total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed (DE miRNAs) after E. tarda infection, indicating that they were immune-related miRNAs. To further examine the relationship between the miRNAs and their predicted target mRNAs, a total of 22 predicted target mRNAs, mainly related to endocytic signaling pathway, NF-κB signaling pathway, and p53 signaling pathway, were detected with miRNA mimics in HEK-293T cells by dual-luciferase reporter experiments. Finally, we confirmed that insulin receptor substrate-2 (IRS2a and IRS2b) were regulated by miR-7a. And the target sites of the 3’ untranslated region (UTR) of IRS2a and IRS2b were verified by dual-luciferase reporter experiments. Furthermore, we found that the E. tarda and LPS significantly increased host miR-7a expression. In vivo and in vitro studies revealed that IRS2-mediated PI3K/AKT/GSK3β signaling pathway was suppressed. Taken together, these results implied that miR-7a might be a key regulator of PI3K/AKT/GSK3β signaling pathway via suppressing the IRS2a and IRS2b genes.

Exposure to enriched environment rescues anxiety-like behavior and miRNA deregulated expression induced by perinatal malnutrition while altering oligodendrocyte morphology

Maternal malnutrition is one of the major early-life adversities affecting the development of newborn's brain and is associated with an increased risk to acquire cognitive and emotional deficiencies later in life. Studies in rodents have demonstrated that exposure to an enriched environment (EE) can reverse the negative consequences of early adversities. However, rescue of emotional disorders caused by perinatal malnutrition and the mechanisms involved has not been determined. We hypothesized that exposure to an EE may attenuate the anxiety-like disorders observed in mice subjected to perinatal protein malnutrition and that this could be mediated by epigenetic mechanisms. Male CF-1 mice were subject to perinatal protein malnutrition until weaning and then exposed to an EE for 5 weeks after which small RNA-seq was performed. In parallel, dark–light box and elevated plus maze tests were conducted to evaluate anxiety traits. We found that exposure to an EE reverses the anxiety-like behavior in malnourished mice. This reversal is paralleled by the expression of three miRNAs that become dysregulated by perinatal malnutrition (miR-187-3p, miR-369-3p and miR-132-3p). The predicted mRNA targets of these miRNAs are mostly related to axon guidance pathway. Accordingly, we also found that perinatal malnutrition leads to reduction in the cingulum size and altered oligodendrocyte morphology. These results suggest that EE-rescue of anxiety disorders derived from perinatal malnutrition is mediated by the modulation of miRNAs associated with the regulation of genes involved in axonal guidance.