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.

Molecular diagnosis of polycystic ovary syndrome in obese and non-obese women by targeted plasma miRNA profiling

Objective Polycystic ovary syndrome (PCOS) is diagnosed based on the clinical signs, but its presentation is heterogeneous and potentially confounded by concurrent conditions, such as obesity and insulin resistance. miRNA have recently emerged as putative pathophysiological and diagnostic factors in PCOS. However, no reliable miRNA-based method for molecular diagnosis of PCOS has been reported. The aim of this study was to develop a tool for accurate diagnosis of PCOS by targeted miRNA profiling of plasma samples, defined on the basis of unbiased biomarker-finding analyses and biostatistical tools. Methods A case–control PCOS cohort was cross-sectionally studied, including 170 women classified into four groups: non-PCOS/lean, non-PCOS/obese, PCOS/lean, and PCOS/obese women. High-throughput miRNA analyses were performed in plasma, using NanoString technology and a 800 human miRNA panel, followed by targeted quantitative real-timePCR validation. Statistics were applied to define optimal normalization methods, identify deregulated biomarker miRNAs, and build classification algorithms, considering PCOS and obesity as major categories. Results The geometric mean of circulating hsa-miR-103a-3p, hsa-miR-125a-5p, and hsa-miR-1976, selected among 125 unchanged miRNAs, was defined as optimal reference for internal normalization (named mR3-method). Ten miRNAs were identified and validated after mR3-normalization as differentially expressed across the groups. Multinomial least absolute shrinkage and selection operator regression and decision-tree models were built to reliably discriminate PCOS vs non-PCOS, either in obese or non-obese women, using subsets of these miRNAs as performers. Conclusions We define herein a robust method for molecular classification of PCOS based on unbiased identification of miRNA biomarkers and decision-tree protocols. This method allows not only reliable diagnosis of non-obese women with PCOS but also discrimination between PCOS and obesity. Capsule We define a novel protocol, based on plasma miRNA profiling, for molecular diagnosis of PCOS. This tool not only allows proper discrimination of the condition in non-obese women but also permits distinction between PCOS and obesity, which often display overlapping clinical presentations.

Characterization and miRNA Profiling of Extracellular Vesicles from Human Osteoarthritic Subchondral Bone Multipotential Stromal Cells (MSCs)

Osteoarthritis (OA) is a heterogeneous disease in which the cross-talk between the cells from different tissues within the joint is affected as the disease progresses. Extracellular vesicles (EVs) are known to have a crucial role in cell-cell communication by means of cargo transfer. Subchondral bone (SB) resident cells and its microenvironment are increasingly recognised to have a major role in OA pathogenesis. The aim of this study was to investigate the EV production from OA SB mesenchymal stromal cells (MSCs) and their possible influence on OA chondrocytes. Small EVs were isolated from OA-MSCs, characterized and cocultured with chondrocytes for viability and gene expression analysis, and compared to small EVs from MSCs of healthy donors (H-EVs). OA-EVs enhanced viability of chondrocytes and the expression of chondrogenesis-related genes, although the effect was marginally lower compared to that of the H-EVs. miRNA profiling followed by unsupervised hierarchical clustering analysis revealed distinct microRNA sets in OA-EVs as compared to their parental MSCs or H-EVs. Pathway analysis of OA-EV miRNAs showed the enrichment of miRNAs implicated in chondrogenesis, stem cells, or other pathways related to cartilage and OA. In conclusion, OA SB MSCs were capable of producing EVs that could support chondrocyte viability and chondrogenic gene expression and contained microRNAs implicated in chondrogenesis support. These EVs could therefore mediate the cross-talk between the SB and cartilage in OA potentially modulating chondrocyte viability and endogenous cartilage regeneration.

Targeting M2 Macrophages Alleviates Airway Inflammation and Remodeling in Asthmatic Mice via miR-378a-3p/GRB2 Pathway

Background: Asthma is a complex respiratory disease characterized by airway inflammation and remodeling. MicroRNAs (miRNAs) mediate various cellular processes including macrophage polarization and play an important role in the pathogenesis of asthma. In present study, we aimed to screen miRNA profiling involved in macrophage polarization and investigate its possible functions and mechanisms.Methods: An OVA-sensitized mouse model was established and 2-chloroadenosine (2-CA) was used to interfere with macrophages. The airway inflammation and remodeling were assessed. The identification and function of M2 alveolar macrophages were assessed by flow cytometry, RT-qPCR, arginase activity and co-culture experiment. Microarray screening was used to select miRNAs which were related to macrophage polarization and RNA interference (RNAi) technique was performed to confirm the function of the selected miRNA and its target gene.Results: Alveolar macrophages of asthmatic mice showed significant M2 polarization. 2-CA alleviated airway inflammation and remodeling as well as M2 polarization. In vitro, IL-4-induced M2 macrophages promoted the proliferation of α-SMA-positive cells. And miRNA profiling showed a remarkable increased expression of miR-378a-3p in IL-4 induced M2 macrophages. Dual luciferase reporter assay confirmed growth factor receptor binding protein 2 (GRB2) was a target gene of miR-378a-3p. A miR-378a-3p inhibitor and knockdown of GRB2 repolarized alveolar macrophages from M1 to M2 phenotype.Conclusion: Our findings suggest that miR-378a-3p/GRB2 pathway regulates the polarization of alveolar macrophages which acts as a potential therapeutic target for airway inflammation and remodeling in asthma.

A New Approach for Prostate Cancer Diagnosis by miRNA Profiling of Prostate-Derived Plasma Small Extracellular Vesicles

Vesicular miRNA has emerged as a promising marker for various types of cancer, including prostate cancer (PC). In the advanced stage of PC, the cancer-cell-derived small extracellular vesicles (SEVs) may constitute a significant portion of circulating vesicles and may mediate a detectable change in the plasma vesicular miRNA profile. However, SEVs secreted by small tumor in the prostate gland constitute a tiny fraction of circulating vesicles and cause undetectable miRNA pattern changes. Thus, the isolation and miRNA profiling of a specific prostate-derived fraction of SEVs can improve the diagnostic potency of the methods based on vesicular miRNA analysis. Prostate-specific membrane antigen (PSMA) was selected as a marker of prostate-derived SEVs. Super-paramagnetic beads (SPMBs) were functionalized by PSMA-binding DNA aptamer (PSMA–Apt) via a click reaction. The efficacy of SPMB–PSMA–Apt complex formation and PSMA(+)SEVs capture were assayed by flow cytometry. miRNA was isolated from the total population of SEVs and PSMA(+)SEVs of PC patients (n = 55) and healthy donors (n = 30). Four PC-related miRNAs (miR-145, miR-451a, miR-143, and miR-221) were assayed by RT-PCR. The click chemistry allowed fixing DNA aptamers onto the surface of SPMB with an efficacy of up to 89.9%. The developed method more effectively isolates PSMA(+)SEVs than relevant antibody-based technology. The analysis of PC-related miRNA in the fraction of PSMA(+)SEVs was more sensitive and revealed distinct diagnostic potency (AUC: miR-145, 0.76; miR-221, 0.7; miR-451a, 0.65; and miR-141, 0.64) than analysis of the total SEV population. Thus, isolation of prostate-specific SEVs followed by analysis of vesicular miRNA might be a promising PC diagnosis method.

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.