Transfer RNA-Derived Fragments and isomiRs Are Novel Components of Chronic TBI-Induced Neuropathology

Neuroinflammation is a secondary injury mechanism that evolves in the brain for months after traumatic brain injury (TBI). We hypothesized that an altered small non-coding RNA (sncRNA) signature plays a key role in modulating post-TBI secondary injury and neuroinflammation. At 3threemonths post-TBI, messenger RNA sequencing (seq) and small RNAseq were performed on samples from the ipsilateral thalamus and perilesional cortex of selected rats with a chronic inflammatory endophenotype, and sham-operated controls. The small RNAseq identified dysregulation of 2 and 19 miRNAs in the thalamus and cortex, respectively. The two candidates from the thalamus and the top ten from the cortex were selected for validation. In the thalamus, miR-146a-5p and miR-155-5p levels were upregulated, and in the cortex, miR-375-3p and miR-211-5p levels were upregulated. Analysis of isomiRs of differentially expressed miRNAs identified 3′nucleotide additions that were increased after TBI. Surprisingly, we found fragments originating from 16 and 13 tRNAs in the thalamus and cortex, respectively. We further analyzed two upregulated fragments, 3′tRF-IleAAT and 3′tRF-LysTTT. Increased expression of the full miR-146a profile, and 3′tRF-IleAAT and 3′tRF-LysTTT was associated with a worse behavioral outcome in animals with chronic neuroinflammation. Our results highlight the importance of understanding the regulatory roles of as-yet unknown sncRNAs for developing better strategies to treat TBI and neuroinflammation.

PATH-01. SMALL RNASEQ ANALYSIS OF MICRORNAS IN BRAIN METASTASIS

MicroRNAs (miRNAs) are a well-known subclass of short non-coding RNAs responsible for posttranscriptional gene silencing and have been described as dysregulated in many cancers. They have also been shown to be both specific diagnostic, prognostic, and predictive biomarkers as well as therapeutic targets. Therefore, specific miRNA expression patterns of BMs of various origins could serve as a promising diagnostic tool for determining both the original tumor and the prognosis in patients with BMs of unknown origin. For identifying significantly dysregulated miRNAs among BMs (n=90) with various origin and non-tumor brain tissues (n=12), small RNAseq analyses were used. cDNA libraries were prepared using QIAseq miRNA Library Kit and purified by Qiaseq beads. The final sequencing analyses were performed by Next 500/550 High Output v2 Kit-75 cycles using the NextSeq 500 instrument. For miRNA mapping and analysis, Miraligner and MirBase were used. Bioinformatic analysis of obtained sequencing data identified 472 significantly dysregulated miRNAs (logFc >2, adj.p-value< 0.05) between BM and non-tumor samples. The comparison of BMs origin from lung BMs (n = 26) with other BMs revealed 132 significantly dysregulated miRNAs, mainly miR-4662a-5p, miR-1179, miR-211-5p, miR-146a-5p, and miR-194-5p. The most significantly dysregulated miRNAs in breast BMs were miR-4728-3p, miR-211-5p, miR-184, miR-365b-5p, and miR-2115-3p. In BMs originating from melanoma, miR-200c-3p, miR-141-5p, miR-200b-5p, miR-514a-3p, and miR-200b-3p showed the most aberrant expression. We have demonstrated that miRNA profiling could be a potent tool for the partition of brain metastases based on their origin. We found that miRNA signatures corresponding to particular origins are rather distinct from the profiles of the rest of BMs. Our results suggest that after validation, miRNA profiling can be used to identify the origin of brain metastases and potentially for the refinement of the diagnosis. Supported by the Ministry of Health of the Czech Republic, grant nr. NV18-03-00398.

MicroRNA Expression Profiles in Autism Spectrum Disorder: Role for miR-181 in Immunomodulation

Background: MicroRNAs (miRNAs) are important regulators of molecular pathways in psychiatric disease. Here, we examine differential miRNAs expression in lymphoblastoid cell lines (LCLs) derived from 10 individuals with autism spectrum disorder (ASD) and compare them to seven typically developing unrelated age- and gender-matched controls and 10 typically developing siblings. Small RNAseq analysis identified miRNAs, and selected miRNAs were validated using quantitative real-time polymerase reaction (qRT-PCR). KEGG analysis identified target pathways, and selected predicted mRNAs were validated using qRT-PCR. Results: Small RNAseq analysis identified that multiple miRNAs differentiated ASD from unrelated controls and ASD from typically developing siblings, with only one, hsa-miR-451a_R-1, being in common. Verification with qRT-PCR showed that miR-320a differentiated ASD from both sibling and unrelated controls and that several members of the miR-181 family differentiated ASD from unrelated controls. Differential expression of AKT2, AKT3, TNF α and CamKinase II predicted by KEGG analysis was verified by qRT-PCR. Expression of CamKinase II βwas found to be correlated with the severity of stereotyped behavior of the ASD participants. Conclusions: This study provides insight into the mechanisms regulating molecular pathways in individuals with ASD and identifies differentiated regulated genes involved in both the central nervous system and the immune system.

BSCI-01. Small RNAseq analysis of microRNAs in brain metastasis

MicroRNAs (miRNAs) are a well-known subclass of short non-coding RNAs responsible for posttranscriptional gene silencing and have been described as dysregulated in many cancers. They have also been shown to be both specific diagnostic, prognostic, and predictive biomarkers as well as therapeutic targets. Therefore, specific miRNA expression patterns of BMs of various origins could serve as a promising diagnostic tool for determining both the original tumor and the prognosis in patients with BMs of unknown origin. For identifying significantly dysregulated miRNAs among BMs (n = 90) with various origin and non-tumor brain tissues (n = 12), small RNAseq analyses were used. cDNA libraries were prepared using QIAseq miRNA Library Kit and purified by Qiaseq beads. The final sequencing analyses were performed by Next 500/550 High Output v2 Kit-75 cycles using the NextSeq 500 instrument. For miRNA mapping and analysis, Miraligner and MirBase were used. Bioinformatic analysis of obtained sequencing data identified 472 significantly dysregulated miRNAs (logFc>2, adj.p-value<0.05) between BM and non-tumor samples. The comparison of BMs origin from lung BMs (n = 26) with other BMs revealed 132 significantly dysregulated miRNAs, mainly miR-4662a-5p, miR-1179, miR-211-5p, miR-146a-5p, and miR-194-5p. The most significantly dysregulated miRNAs in breast BMs were miR-4728-3p, miR-211-5p, miR-184, miR-365b-5p, and miR-2115-3p. In BMs originating from melanoma, miR-200c-3p, miR-141-5p, miR-200b-5p, miR-514a-3p, and miR-200b-3p showed the most aberrant expression. We have demonstrated that miRNA profiling could be a potent tool for the partition of brain metastases based on their origin. We found that miRNA signatures corresponding to particular origins are rather distinct from the profiles of the rest of BMs. Our results suggest that after validation, miRNA profiling can be used to identify the origin of brain metastases and potentially for the refinement of the diagnosis. Supported by the Ministry of Health of the Czech Republic, grant nr. NV18-03-00398.

P–290 Time-course analysis of endometrial miR/isomiR expression dynamics during hCG-primed menstrual-cycle phase transitions

Study question What is the qualitative and quantitative profile of microRNAs (miR) and their sequence variants - isomiRs, and how it changes during the menstrual-cycle phase transitions? Summary answer Time-course analysis of endometrial miR/isomiR profiles has shown that menstrual-phase transitions cause widespread and complex changes in miR gene expression and processing. What is known already Embryo implantation depends on the receptivity of the endometrium during the window of implantation, when ovarian hormones and genetic factors coordinate the development of the uterine lining and prepare it for embryo implantation. The most important factors for successful implantation studied so far are the embryo itself, the histological dating of the endometrium and its molecular genetic characteristics, including miRs. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes have the potential to produce not only miR but also variants (isomiRs) thereof, which can differ in sequence and length and can be functionally significant. Study design, size, duration miR/isomiR landscape was assessed by small RNA sequencing of endometrial biopsy samples at 4 time points of endometrial cycle covering the proliferative and secretory phases. Healthy, fertile, female volunteers took part in the study lasting one and a half years. For accurate phase dating, human chorionic gonadotropin (hCG) was administrated, and ultrasonic, histological and hormonal assessments were done at each time point. Statistically significant data of miR/isomiR identification and expression dynamics was considered for analysis. Participants/materials, setting, methods Participant choice criteria - at least one child born, problem-free pregnancies, no diseases or allergies; hCG application time determined according follicle and endometrium ultrasound scanning, and ovarian hormone levels; endometrial biopsies taken at hGC (before hormone application), hGC+2, hGC+7, hGC+9 time points; small RNAseq completed by Karolinska Institute, Sweden; miR/isomiR identified using local Galaxy instance with an in-built workflow and tools developed by our laboratory; differential expression and target prediction evaluated with DESeq2 and miRDB,resp. Main results and the role of chance Within the cohort of patients, across the four study time points, the small RNAseq data revealed numbers of miRs and isomiRs to be changed. The largest statistically significant changes in their expression were found at LH + 9. The miR families that showed the largest number of members with altered expression were miR125a, miR30d, miR449c, miR92a/b and miR99a. The expression levels tended to decrease in the miR125a and miR92a families and to increase in the miR10a and miR449c families during the three studied time points of the cycle compared to the proliferative phase. Among those affected, the number of isomiRs, including templated and non-templated isomiRs, was much higher than that of miRs. For example, the ratio of the significantly altered miRs/templated isomiRs/non-templated isomiRs was 6/16/11 at LH + 9. Templated isomiRs of hsa-miR–148a–3p, hsa-miR–30d–5p and hsa-miR–449c–5p were among the most upregulated, while several templated and non-templated isomiRs of hsa-miR–125–5p were the most downregulated at LH + 9. Of particular interest are those isomiRs, in which the seed site is shifted compared to the reference miRs and results in altered target transcripts. Target prediction of the most affected isomiR of hsa-miR–449c–5p identified new targets of target scores much higher than of the reference miR. Limitations, reasons for caution IsomiRs are a source of novel biomarkers for clinical diagnosis. An important next step is the validation of the in-silico predicted miRs/isomiRs and their target transcripts by RT-qPCR in larger number of individuals. Expression profiles should be associated with the dominant cell type in the endometrial biopsy preparation. Wider implications of the findings: MiR/isomiR signatures, together with those of their target mRNAs, can be applied to distinguish the endometrial phases, especially the implantation window, as well as for diagnosing endometrial dysfunction. It is worth investigating the possibility of miRs/isomiRs being used as biomarkers not only in endometrial biopsy but also in liquid biopsy. Trial registration number The Bulgarian National Science Fund КП–06 Н31/2

Reciprocal Changes in miRNA Expression with Pigmentation and Decreased Proliferation Induced in Mouse B16F1 Melanoma Cells by l-Tyrosine and 5-Bromo-2′-Deoxyuridine

Background: Many microRNAs have been identified as critical mediators in the progression of melanoma through its regulation of genes involved in different cellular processes such as melanogenesis, cell cycle control, and senescence. However, microRNAs’ concurrent participation in syngeneic mouse B16F1 melanoma cells simultaneously induced decreased proliferation and differential pigmentation by exposure to 5-Brd-2′-dU (5’Bromo-2-deoxyuridine) and L-Tyr (L-Tyrosine) respectively, is poorly understood. Aim: To evaluate changes in the expression of microRNAs and identify which miRNAs in-network may contribute to the functional bases of phenotypes of differential pigmentation and reduction of proliferation in B16F1 melanoma cells exposed to 5-Brd-2′-dU and L-Tyr. Methods: Small RNAseq evaluation of the expression profiles of miRNAs in B16F1 melanoma cells exposed to 5-Brd-2′-dU (2.5 μg/mL) and L-Tyr (5 mM), as well as the expression by qRT-PCR of some molecular targets related to melanogenesis, cell cycle, and senescence. By bioinformatic analysis, we constructed network models of regulation and co-expression of microRNAs. Results: We confirmed that stimulation or repression of melanogenesis with L-Tyr or 5-Brd-2′-dU, respectively, generated changes in melanin concentration, reduction in proliferation, and changes in expression of microRNAs 470-3p, 470-5p, 30d-5p, 129-5p, 148b-3p, 27b-3p, and 211-5p, which presented patterns of coordinated and reciprocal co-expression, related to changes in melanogenesis through their putative targets Mitf, Tyr and Tyrp1, and control of cell cycle and senescence: Cyclin D1, Cdk2, Cdk4, p21, and p27. Conclusions: These findings provide insights into the molecular biology of melanoma of the way miRNAs are coordinated and reciprocal expression that may operate in a network as molecular bases for understanding changes in pigmentation and decreased proliferation induced in B16F1 melanoma cells exposed to L-Tyr and 5-Brd-2′-dU.

Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle

The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles. A subset of high (H) and low (L) responders was selected depending on their response to superstimulation and EV-miRNA profiles were analysed at both time-points in each heifer. Approximately 200 known miRNAs were detected in each sample with 144 commonly detected in all samples. A total of 12 and 14 miRNAs were dysregulated in UH vs. UL and in SH vs. SL heifers, respectively. Interestingly, miR-206 and miR-6517 exhibited the same differential expression pattern in H compared to L heifers both before and after superstimulation. Pathway analysis indicated that circadian rhythm and signaling pathways were among the top pathways enriched with genes targeted by dysregulated miRNAs in H vs. L responding heifers. In conclusion, heifers with divergent ovarian responses exhibited differential expression of plasma EV-miRNAs which may be used as a potential biomarker to predict superstimulation response.

STAT3 Mediated miR-30a-5p Inhibition Enhances Proliferation and Inhibits Apoptosis in Colorectal Cancer Cells

Signal transducer and activator of transcription 3 (STAT3), a transcriptional factor involved in tumorigenesis and cancer stemness formation, contributes to drug resistance in cancer therapies. STAT3 not only mediates gene transcription but also participates in microRNA suppression. This study identified a STAT3-downstream micro RNA (miRNA) involved in drug resistance against regorafenib in colorectal cancer stem-like tumorspheres. Small RNAseq was used to investigate differential microRNAs in colorectal cancer cell-derived tumorspheres and in a STAT3-knockdown strain. The miRNA-mediated genes were identified by comparing RNAseq data with gene targets predicted using TargetScan. Assays for detecting cell viability and apoptosis were used to validate findings. The formation of colorectal cancer stem-like tumorspheres was inhibited by BBI608, a STAT3 inhibitor, but not by regorafenib. Additional investigations for microRNA expression demonstrated an increase in 10 miRNAs and a decrease in 13 miRNAs in HT29-derived tumorspheres. A comparison of small RNAseq results between tumorspheres and HT29shSTAT3 cells revealed the presence of four STAT3-mediated miRNAs in HT29-derived tumorspheres: hsa-miR-215-5p, hsa-miR-4521, and hsa-miR-215-3p were upregulated, whereas miR-30a-5p was downregulated. Furthermore, hsa-miR-4521 was associated with poor overall survival probability, and miR-30a-5p was associated with better overall survival probability in patients with rectum cancer. Comparisons of RNAseq findings between HCT116- and HT29-derived tumorspheres revealed that HSPA5 were mediated by the STAT3-miR-30a-5p axis, which is overexpressed in colorectal tumorspheres associating to anti-apoptosis. In addition, the transfection of miR-30a-5p and inhibition of HSPA5 by HA15 significantly reduced cell viability and increased apoptosis in HT29 cells. In conclusion, a STAT3-miR-30a-5p-HSPA5 axis was observed against regorafenib-mediated apoptosis in colorectal cancer tumorspheres. The expression of miR-30a-5p was repressed by STAT3; in addition, HSPA5 was identified as the target gene of miR-30a-5p and contributed to both tumorsphere formation and anti-apoptosis.

Extracellular vesicles and their miRNA cargo in retinal health and degeneration: mediators of homeostasis, and vehicles for targeted gene therapy

1.1.AbstractPurposePhotoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases, however the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs including exosomes encapsulate and transfer nucleic acids, including microRNA (miRNA), to recipient cells which in disease may result in dysfunctional immune responses and a loss of homeostatic regulation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina.MethodsIsolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and in situ hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion.ResultsResults demonstrated an inverse correlation between s-mEV secretion and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage.ConclusionsTaken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.

GENE-60. THE EPITRANSCRIPTOMIC CODE IN LGG: METABOLICALLY REPROGRAMMED IDH-MUTANT GLIOMAS ALTER tRNA MODIFICATION LANDSCAPE

BACKGROUND Diffuse lower grade gliomas (LGGs) are generally slow growing primary central nervous system tumors that occur in early adult life. The prevalence of isocitrate dehydrogenase (IDH) mutations is high in LGG, and induces excess production of the oncometabolite 2-hydroxyglutarate (2-HG). These gain-of-function mutations play a key role in promoting metabolic reprogramming of the cancer cell that affects activity of α-KG dependent demethylases. Inhibition of DNA demethylase activity leads to glioma with a CpG island methylator phenotype (G-CIMP). Whether the activity of RNA demethylases and methylation status of tRNAs in LGG are modulated by changes in IDH-status is unknown. AIM: To investigate whether IDH mutations play a role in reprogramming of tRNA modifications in adult glioma. MATERIALS AND METHODS We combined small RNAseq and LC-MS/MS analysis to identify distinct tRNA processing patterns and methylation signatures in LGG tissues. To address important experimental bottlenecks that limit RNAseq-based detection of tRNA and possibly other modified small noncoding RNAs, we employed a tailored small RNAseq method with validation of specific methylation sites by mass-spectrometry. RESULTS Our customized small RNAseq approach yielded >100 fold increase in sequencing reads per tRNA type, thereby dramatically improving tRNA detection when compared to currently used small RNAseq approaches. Moreover, LC-MS/MS analysis revealed a higher abundance of modified nucleosides in tRNA from IDH-mutant LGG compared to IDH-wildtype LGG. Analysis of tRNA from IDH-mutant and IDH-wildtype LGG using the combination of our tailored small RNAseq and LC-MS/MS methodology demonstrated strong differential tRNA expression, tRFs processing and tRNA methylation. CONCLUSION We described an approach that makes use of tailored small RNA sequencing combined with mass-spectrometry that enables insights into cancer driven alterations in tRNA methylation patterns and differential tRNA processing signatures. Our data implies that tumor metabolic reprogramming deregulates tRNA methylation, contributing to an altered epitranscriptomic code in IDH-mutant LGG.