Global miRNA Expression Research Articles

Streptococcus gordonii Supragingival Bacterium Oral Infection-Induced Periodontitis and Robust miRNA Expression Kinetics.

Streptococcus gordonii (S. gordonii, Sg) is one of the early colonizing, supragingival commensal bacterium normally associated with oral health in human dental plaque. MicroRNAs (miRNAs) play an important role in the inflammation-mediated pathways and are involved in periodontal disease (PD) pathogenesis. PD is a polymicrobial dysbiotic immune-inflammatory disease initiated by microbes in the gingival sulcus/pockets. The objective of this study is to determine the global miRNA expression kinetics in S. gordonii DL1-infected C57BL/6J mice. All mice were randomly divided into four groups (n = 10 mice/group; 5 males and 5 females). Bacterial infection was performed in mice at 8 weeks and 16 weeks, mice were euthanized, and tissues harvested for analysis. We analyzed differentially expressed (DE) miRNAs in the mandibles of S. gordonii-infected mice. Gingival colonization/infection by S. gordonii and alveolar bone resorption (ABR) was confirmed. All the S. gordonii-infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, and a significant increase in mandible and maxilla ABR (p < 0.0001). miRNA profiling revealed 191 upregulated miRNAs (miR-375, miR-34b-5p) and 22 downregulated miRNAs (miR-133, miR-1224) in the mandibles of S. gordonii-infected mice at the 8-week mark. Conversely, at 16 weeks post-infection, 10 miRNAs (miR-1902, miR-203) were upregulated and 32 miRNAs (miR-1937c, miR-720) were downregulated. Two miRNAs, miR-210 and miR-423-5p, were commonly upregulated, and miR-2135 and miR-145 were commonly downregulated in both 8- and 16-week-infected mice mandibles. Furthermore, we employed five machine learning (ML) algorithms to assess how the number of miRNA copies correlates with S. gordonii infections in mice. In the ML analyses, miR-22 and miR-30c (8-week), miR-720 and miR-339-5p (16-week), and miR-720, miR-22, and miR-339-5p (combined 8- and 16-week) emerged as the most influential miRNAs.

Exosomal non-coding RNA exhibit sex-specific profiles in salt sensitive hypertension

Exosomes are secreted by various living cells and can be found in all body fluids, especially in cerebrospinal fluid (CSF) and plasma, thereby providing a source of biomarkers for cardiovascular diseases that can be used for patients’ diagnostic. Using high-throughput sequencing technologies, exosomes have been found to contain different RNA populations particularly non-coding RNA. Recently, many non-coding RNA have been recognized as key regulators in the progression of hypertension. In this study we hypothesized that female and male C57BI/6J mice exhibit different microRNA (miR) profiles after deoxycorticosterone acetate (DOCA)+salt hypertension treatment. CSF (10 μl) and plasma (100 μl) were collected from 3-month-old mice and again after 3 weeks of DOCA-salt treatment before exosomes isolation and processing for high throughput small RNA sequencing (Creative Biolabs). Analysis was performed using R language and RStudio ( https://rstudio.com/ ) as an integrated development environment. Global miRNA analysis of CSF exosomes shows different expression miRNA profiles in DOCA+salt male mice compared with their own baseline values. In addition, we observed a remarkable difference in global miRNA expression profile between DOCA+salt male mice and DOCA+salt females (p-value &lt; 2.2e-16), while no significant difference was detected between DOCA+salt females and their baseline values (p-value = 0.6679). Moreover, we observed that there are three clusters of around 200 miR that have different expression levels in DOCA+salt males. Our analysis identified around 74 upregulated miRNA in DOCA+salt males compared to DOCA+salt females. The maximum level of expression belonged to the muscle-specific miRNA (myomiR) miR-1a-3p, miR-206-3p and miR-26a-5p which were highly accumulated in CSF derived exosomes. These top three upregulated miRNA were associated with 282 validated target genes among which, two pivotal genes, ACE2 and ADAM17, were identified in the PPI network, miRTargetLink, and by KEGG analysis. miR-26a-5p was mostly associated with ACE2 and ADAM17 and the mRNA expression of ADAM17 was associated with reduced miR-206-3p. In conclusion, our bioinformatic analysis identified three miRNA and associated them with two key molecules involved in salt sensitive hypertension. Further analysis is likely to highlight more diagnostic biomarkers and potential candidates for the development of new therapeutic approaches. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Defining the contribution of microRNA-specific Argonautes with slicer capability in animals.

microRNAs regulate gene expression through interaction with an Argonaute protein. While some members of this protein family retain an enzymatic activity capable of cleaving RNA molecules complementary to Argonaute-bound small RNAs, the role of the slicer residues in the canonical microRNA pathway is still unclear in animals. To address this, we created Caenorhabditis elegans strains with mutated slicer residues in the endogenous ALG-1 and ALG-2, the only two slicing Argonautes essential for the miRNA pathway in this animal model. We observe that the mutation in ALG-1 and ALG-2 catalytic residues affects overall animal fitness and causes phenotypes reminiscent of miRNA defects only when grown and maintained at restrictive temperature. Furthermore, the analysis of global miRNA expression shows that the slicer residues of ALG-1 and ALG-2 contribute differentially to regulate the level of specific subsets of miRNAs in young adults. We also demonstrate that altering the catalytic tetrad of those miRNA-specific Argonautes does not result in any defect in the production of canonical miRNAs. Together, these data support that the slicer residues of miRNA-specific Argonautes contribute to maintaining levels of a set of miRNAs for optimal viability and fitness in animals particularly exposed to specific growing conditions.

MicroRNA-mRNA Networks Linked to Inflammation and Immune System Regulation in Inflammatory Bowel Disease.

By performing a comprehensive meta-analysis, we integrated miRNA expression data from nine studies in IBD. We obtained detailed information on significantly deregulated miRNAs (fold change, FC ≥ 2 and p < 0.05), sample type and number, and platform applied for analysis in the training and validation sets. Further bioinformatic analyses were performed to identify miRNA target genes, by using the microRNA Data Integration Portal tool. We also sought to identify statistically enriched pathways of genes regulated by miRNAs using ToppGene Suite. Additional analyses were performed to filter for genes expressed in intestinal tissue using the European Bioinformatics Institute (EBI) database. Our findings showed the upregulation of 15 miRNAs in CD and 33 in UC. Conversely, six miRNAs were downregulated in CD, while seven were downregulated in UC. These results indicate a greater deregulation of miRNAs in UC compared to CD. Of note, miRNA target genes were enriched for immune system regulation pathways. Among significantly deregulated miRNAs with a higher number of miRNA-target gene interactions, we identified miR-199a-5p and miR-362-3p altered in CD, while among UC case patients, miRNA-target gene interactions were higher for miR-155-5p. The identified miRNAs play roles in regulating genes associated with immune system regulation and inflammation in IBD. Such miRNAs and their target genes have the potential to serve as clinically relevant biomarkers. These findings hold promise for enhancing the accuracy of diagnoses and facilitating the development of personalized treatment strategies for individuals with various forms of IBD.

Mesenchymal stem cell-regulated miRNA-mRNA landscape in acute-on-chronic liver failure

BackgroundAcute-on-chronic liver failure (ACLF) is a major challenge in the field of hepatology. While mesenchymal stem cell (MSC) therapy can improve the prognosis of patients with ACLF, the molecular mechanisms through which MSCs attenuate ACLF remain poorly understood. We performed global miRNA and mRNA expression profiling via next-generation sequencing of liver tissues from MSC-treated ACLF mice to identify important signaling pathways and major factors implicated in ACLF alleviation by MSCs. MethodsCarbon tetrachloride-induced ACLF mice were treated with saline or mouse bone marrow-derived MSCs. Mouse livers were subjected to miRNA and mRNA sequencing. Related signal transduction pathways were obtained through Gene Set Enrichment Analysis. Functional enrichment, protein-protein interaction, and immune infiltration analyses were performed for the differentially expressed miRNA target genes (DETs). Hub miRNA and mRNA associated with liver injury were analyzed using LASSO regression. The expression levels of hub genes were subjected to Pearson's correlation analysis and verified using RT-qPCR. The biological functions of hub genes were verified in vitro. ResultsThe tricarboxylic acid cycle and peroxisome proliferator-activated receptor pathways were activated in the MSC-treated groups. The proportions of liver-infiltrating NK resting cells, M2 macrophages, follicular helper T cells, and other immune cells were altered after MSC treatment. The expression levels of six miRNAs and 10 transcripts correlated with the degree of liver injury. miR-27a-5p was downregulated in the mouse liver after MSC treatment, while its target gene E2f2 was upregulated. miR-27a-5p inhibited E2F2 expression, suppressed G1/S phase transition and proliferation of hepatocytes, in addition to promoting their apoptosis. ConclusionsThis is the first comprehensive analysis of miRNA and mRNA expression in the liver tissue of ACLF mice after MSC treatment. The results revealed global changes in hepatic pathways and immune subpopulations. The miR-27a-5p/E2F2 axis emerged as a central regulator of the MSC-induced attenuation of ACLF. The current findings improve our understanding of the molecular mechanisms through which MSCs alleviate ACLF.

Screening of microRNAs and target genes involved in Sclerotinia sclerotiorum (Lib.) infection in Brassica napus L.

BackgroundRapeseed (Brassica napus L.) is the third largest source of vegetable oil in the world, and Sclerotinia sclerotiorum (Lib.) is a major soil-borne fungal plant pathogen that infects more than 400 plant species, including B. napus. Sclerotinia stem rot caused an annual loss of 10 − 20% in rapeseed yield. Exploring the molecular mechanisms in response to S. sclerotiorum infection in B. napus is beneficial for breeding and cultivation of resistant varieties. To gain a better understanding of the mechanisms regarding B. napus tolerance to Sclerotinia stem rot, we employed a miRNAome sequencing approach and comprehensively investigated global miRNA expression profile among five relatively resistant lines and five susceptible lines of oilseed at 0, 24, and 48 h post-inoculation.ResultsIn this study, a total of 40 known and 1105 novel miRNAs were differentially expressed after S. sclerotiorum infection, including miR156, miR6028, miR394, miR390, miR395, miR166, miR171, miR167, miR164, and miR172. Furthermore, 8,523 genes were predicted as targets for these differentially expressed miRNAs. These target genes were mainly associated with disease resistance (R) genes, signal transduction, transcription factors, and hormones. Constitutively expressing miR156b (OX156b) plants strengthened Arabidopsis resistance against S. sclerotiorum accompanied by smaller necrotic lesions, whereas blocking miR156 expression in Arabidopsis (MIM156) led to greater susceptibility to S. sclerotiorum disease, associated with extensive cell death of necrotic lesions.ConclusionsThis study reveals the distinct difference in miRNA profiling between the relatively resistant lines and susceptible lines of B. napus in response to S. sclerotiorum. The identified differentially expressed miRNAs related to sclerotinia stem rot resistance are involved in regulating resistance to S. sclerotiorum in rapeseed by targeting genes related to R genes, signal transduction, transcription factors, and hormones. miR156 positively modulates the resistance to S. sclerotiorum infection by restricting colonization of S. sclerotiorum mycelia. This study provides a broad view of miRNA expression changes after S. sclerotiorum infection in oilseed and is the first to elucidate the function and mechanism underlying the miR156 response to S. sclerotiorum infection in oilseed rape.

Exposure to sublethal concentrations of thiacloprid insecticide modulated the expression of microRNAs in honeybees (Apis mellifera L.)

This study aimed to investigate the sublethal effects of thiacloprid on microRNA (miRNA) expression in honeybees (Apis mellifera L.) and the role of a specific miRNA, ame-miR-283–5p, in thiacloprid resistance. The high-throughput small RNA-sequencing was used to analyze global miRNA expression profile changes in honeybees orally exposed to sublethal concentrations of thiacloprid (20 mg/L and 4 mg/L) for 72 h. Thiacloprid at 20 mg/L had no observed adverse effects. In addition, bees were fed with miRNA mimics or inhibitors to increase or decrease ame-miR-283–5p expression, and its effects on P450 gene expression (CYP9Q2 and CYP9Q3) were examined. Thiacloprid susceptibility was also detected. The results showed that treatment with thiacloprid at 20 mg/L and 4 mg/L induced 11 and five differentially expressed miRNAs (DEMs), respectively. Bioinformatic analysis suggested that the DEMs are mainly involved in the regulation of growth and development, metabolism, nerve conduction, and behavior. ame-miR-283–5p was downregulated by both concentrations, which was validated using quantitative real-time reverse transcription PCR analysis. Enhancing ame-miR-283–5p expression significantly inhibited CYP9Q2 mRNA and protein expression, and increased thiacloprid toxicity, while reducing ame-miR-283–5p expression significantly promoted CYP9Q2 expression, and decreased thiacloprid susceptibility. Our study revealed a novel role of miRNAs in insecticide resistance in honeybees.

Oral Spirochete Treponema denticola Intraoral Infection Reveals Unique miR-133a, miR-486, miR-126-3p, miR-126-5p miRNA Expression Kinetics during Periodontitis.

miRNAs are major regulators of eukaryotic gene expression and host immunity, and play an important role in the inflammation-mediated pathways in periodontal disease (PD) pathogenesis. Expanding our previous observation with the global miRNA profiling using partial human mouth microbes, and lack of in vivo studies involving oral spirochete Treponema denticola-induced miRNAs, this study was designed to delineate the global miRNA expression kinetics during progression of periodontitis in mice infected with T. denticola by using NanoString nCounter® miRNA panels. All of the T. denticola-infected male and female mice at 8 and 16 weeks demonstrated bacterial colonization (100%) on the gingival surface, and an increase in alveolar bone resorption (p < 0.0001). A total of 70 miRNAs with at least 1.0-fold differential expression/regulation (DE) (26 upregulated and 44 downregulated) were identified. nCounter miRNA expression profiling identified 13 upregulated miRNAs (e.g., miR-133a, miR-378) and 25 downregulated miRNAs (e.g., miR-375, miR-34b-5p) in T. denticola-infected mouse mandibles during 8 weeks of infection, whereas 13 upregulated miRNAs (e.g., miR-486, miR-126-5p) and 19 downregulated miRNAs (miR-2135, miR-142-3p) were observed during 16 weeks of infection. One miRNA (miR-126-5p) showed significant difference between 8 and 16 weeks of infection. Interestingly, miR-126-5p has been presented as a potential biomarker in patients with periodontitis and coronary artery disease. Among the upregulated miRNAs, miR-486, miR-126-3p, miR-126-5p, miR-378a-3p, miR-22-3p, miR-151a-3p, miR-423-5p, and miR-221 were reported in human gingival plaques and saliva samples from periodontitis and with diabetes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed various functional pathways of DE miRNAs, such as bacterial invasion of epithelial cells, Ras signaling, Fc gamma R-mediated phagocytosis, osteoclast differentiation, adherens signaling, and ubiquitin mediated proteolysis. This is the first study of DE miRNAs in mouse mandibles at different time-points of T. denticola infection; the combination of three specific miRNAs, miR-486, miR-126-3p, and miR-126-5p, may serve as an invasive biomarker of T. denticola in PD. These miRNAs may have a significant role in PD pathogenesis, and this research establishes a link between miRNA, periodontitis, and systemic diseases.

MicroRNA Biogenesis during Cellular Senesence Induced by Chronic Stress of the Endoplasmic Reticulum

MicroRNAs are small non-coding regulatory RNAs about 22 nt long, post-transcriptional and transcriptional regulators of gene expression that stabilize the cellular phenotype and play an important role in differentiation, development, and apoptosis. MicroRNA biogenesis includes several precisely controlled post-transcriptional stages of processing and transport, including cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER with the formation of a mature duplex included in the RISC complex. The role of miRNA and its biogenesis are not well understood in such an important process as cellular stress. Cellular stress is a non-specific cellular response to non-physiological stimuli that can switch a cell to death or cellular senescence. The global decrease in microRNA levels is a key feature of cancer cells and an important reason for the formation of a malignant phenotype. In this work, using flow cytometry and high-throughput analysis of gene expression, we showed that chronic endoplasmic reticulum (ER) stress, one of the types of cellular stress associated with impaired protein folding in the ER, leads to the formation of a cellular aging phenotype in fibroblast-like FRSN cells. Despite the fact that acute ER stress can reduce miRNA biogenesis, chronic stress does not lead to a significant drop in global miRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Under chronic ER stress, we found an increase in cell population heterogeneity in terms of lysosomal beta-galactosidase activity, which does not exclude induced or initial cell heterogeneity and in terms of expression of microRNA biogenesis pathway components.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: We previously performed global miRNA expression profiling of cHL vs. non-Hodgkin lymphoma (NHL) cell lines and sorted germinal centre B-cells (GCB). In the group of 79 deregulated miRNAs (p < 0.05) we became particularly interested in the upregulated miR-23a-3p, a potential oncomiR, predicted to regulate the expression of TNFAIP3 and the downregulated miR-148a-3p, a potential tumor suppressor miRNA, predicted to regulate the expression of IL15. Therefore, we aimed to decipher the role of miR-23a-3p and miR-148a-3p in cHL pathogenesis. Methods: Expression analyses were performed with TaqMan Advanced miRNA Assays versus two miRNA controls. Laser capture microdissection was used for primary HRS collection. 1,000 (miRNA expression) and 2 × 200 (DNA methylation) HRS cells were collected per case. Bisulfite pyrosequencing assay was designed using the PyroMark Software and sequencing performed using the PyroMark Q24 sequencer. MiRNA-mRNAs interactions were validated with Dual-Glo Luciferase Assay using respective miRNA mimics. For overexpression pre-miRNAs were cloned into the pCDH-CMV-MCS-EF1α-GreenPuro vector and transductions were performed in triplicate. Cell proliferation was measured using CCK-8 in four replications in three independent reactions by the GloMax®96 Microplate reader. Results: We confirmed the overexpression of miR-23a-3p (p < 0.001) and downregulation of miR-148a-3p (p < 0.014) in microdissected HRS cells (n = 10) vs. GCB cells (n = 10). Moreover, we observed inverse correlation of miR-148a-3p expression with DNA methylation level of an adjacent CpG island (r = −0.72, p < 0.01; cHL n = 7, NHL n = 10). Elevated DNA methylation of this CpG island was detected also in 2/6 cHL primary cases. Next, we validated interactions between miR-23a-3p and miR-148a-3p and their target genes. A reduction by 34% (p < 0.001) and 40% (p < 0.01) of the luciferase signal was observed in consequence of the interaction of miR-23a-3p mimic—TNFAIP3 3’UTR and miR-148a-3p mimic—IL15 3’UTR, respectively. Moreover, the abundance of TNFAIP3 protein was reduced after overexpression of the miR-23a (coexpressed with miR-27a) in L428 (p = 0.0018) and GCB6-16 (p = 0.0083) cell lines, compared to empty vector transductions. Whereas overexpression of miR-148a in three cHL cell lines caused a 32% decrease (p < 0.05) in proliferation in the KM-H2 cell line compared to empty vector transductions. The research was funded by: This research was funded by the National Science Center, Poland grants: 2014/14/M/NZ2/00529 to M.G. and the 2019/32/T/NZ5/00441 Etiuda scholarship to JP. SH is supported by the German Research Foundation (DFG, HA6145/3-1). MG, ADz-K and NR received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952304. Keywords: Hodgkin lymphoma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

Genome-wide profiling of miRNA-gene regulatory networks in mouse postnatal heart development-implications for cardiac regeneration.

After birth, mammalian cardiomyocytes substantially lose proliferative capacity with a concomitant switch from glycolytic to oxidative mitochondrial energy metabolism. Micro-RNAs (miRNAs) regulate gene expression and thus control various cellular processes. Their roles in the postnatal loss of cardiac regeneration are however still largely unclear. Here, we aimed to identify miRNA-gene regulatory networks in the neonatal heart to uncover role of miRNAs in regulation of cell cycle and metabolism. We performed global miRNA expression profiling using total RNA extracted from mouse ventricular tissue samples collected on postnatal day 1 (P01), P04, P09, and P23. We used the miRWalk database to predict the potential target genes of differentially expressed miRNAs and our previously published mRNA transcriptomics data to identify verified target genes that showed a concomitant differential expression in the neonatal heart. We then analyzed the biological functions of the identified miRNA-gene regulatory networks using enriched Gene Ontology (GO) and KEGG pathway analyses. Altogether 46 miRNAs were differentially expressed in the distinct stages of neonatal heart development. For twenty miRNAs, up- or downregulation took place within the first 9 postnatal days thus correlating temporally with the loss of cardiac regeneration. Importantly, for several miRNAs, including miR-150-5p, miR-484, and miR-210-3p there are no previous reports about their role in cardiac development or disease. The miRNA-gene regulatory networks of upregulated miRNAs negatively regulated biological processes and KEGG pathways related to cell proliferation, while downregulated miRNAs positively regulated biological processes and KEGG pathways associated with activation of mitochondrial metabolism and developmental hypertrophic growth. This study reports miRNAs and miRNA-gene regulatory networks with no previously described role in cardiac development or disease. These findings may help in elucidating regulatory mechanism of cardiac regeneration and in the development of regenerative therapies.

Abstract 5843: miR-224 activates cancer-associated fibroblasts to promote cancer cell invasion in lung tumor microenvironment

Abstract Cancer-associated fibroblasts (CAFs) promote EMT (epithelial-mesenchymal transition), invasion, and metastasis of cancer cells in the tumor microenvironment. To identify microRNAs (miRNAs) associated with the reprogramming of normal lung fibroblasts (LFs) into CAFs, global miRNA expression was profiled in LFs and CAFs through Nanostring nCounter. miR-224 was one of the increased miRNAs in CAFs, and its expression was higher in tumors than in normal tissues in dbDEMC and TCGA_lung adenocarcinoma data. miR-224 levels were then checked by qRT-PCR in paraffine-embedded tumor samples from patients with lung adenocarcinoma. Kaplan-Meier survival analysis showed that high miR-224 expression was related to poor survival. These data show that lung cancer progression is promoted by miR-224. To discover the roles of increased miR-224 in CAFs, miR-224 was overexpressed by lentiviral infection in LFs. LF_miR-224 overexpression enhanced the expression of CAF activation markers including Fap, S100a4, and Vim. In spheroid invasion assay using co-culture of LFs and cancer cells, as an experimental model of the tumor microenvironment, LF_miR-224 promoted invasion of lung cancer cells compared with control LFs. In transwell migration assay, LF_miR-224 stimulated migration of lung cancer cells more than control LFs. Collectively, these results indicate that CAFs reprogrammed by miR-224 promote the migration and invasion of lung cancer cells. Citation Format: Seonyeong Oh, Inyoung Cheon, Young-Ho Ahn. miR-224 activates cancer-associated fibroblasts to promote cancer cell invasion in lung tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5843.

Computational approaches for discovering significant microRNAs, microRNA-mRNA regulatory pathways, and therapeutic protein targets in endometrial cancer.

Endometrial cancer (EC) is a urogenital cancer affecting millions of post-menopausal women, globally. This study aims to identify key miRNAs, target genes, and drug targets associated with EC metastasis. The global miRNA and mRNA expression datasets of endometrial tissue biopsies (24 tumors +3 healthy tissues for mRNA and 18 tumor +4 healthy tissues for miRNAs), were extensively analyzed by mapping of DEGs, DEMi, biological pathway enrichment, miRNA-mRNA networking, drug target identification, and survival curve output for differentially expressed genes. Our results reveal the dysregulated expression of 26 miRNAs and their 66 target genes involved in focal adhesions, p53 signaling pathway, ECM-receptor interaction, Hedgehog signaling pathway, fat digestion and absorption, glioma as well as retinol metabolism involved in cell growth, migration, and proliferation of endometrial cancer cells. The subsequent miRNA-mRNA network and expression status analysis have narrowed down to 2 hub miRNAs (hsa-mir-200a, hsa-mir-429) and 6 hub genes (PTCH1, FOSB, PDGFRA, CCND2, ABL1, ALDH1A1). Further investigations with different systems biology methods have prioritized ALDH1A1, ABL1 and CCND2 as potential genes involved in endometrial cancer metastasis owing to their high mutation load and expression status. Interestingly, overexpression of PTCH1, ABL1 and FOSB genes are reported to be associated with a low survival rate among cancer patients. The upregulated hsa-mir-200a-b is associated with the decreased expression of the PTCH1, CCND2, PDGFRA, FOSB and ABL1 genes in endometrial cancer tissue while hsa-mir-429 is correlated with the decreased expression of the ALDH1A1 gene, besides some antibodies, PROTACs and inhibitory molecules. In conclusion, this study identified key miRNAs (hsa-mir-200a, hsa-mir-429) and target genes ALDH1A1, ABL1 and CCND2 as potential biomarkers for metastatic endometrial cancers from large-scale gene expression data using systems biology approaches.

Macrophage-derived exosomal miR-195a-5p impairs tubular epithelial cells mitochondria in acute kidney injury mice.

Macrophages (Mφ) infiltration is a common characteristic of acute kidney injury (AKI). Exosomes-mediated cell communication between tubular epithelial cells (TECs) and Mφ has been suggested to be involved in AKI. Exosomes-derived from injured TECs could regulate Mφ polarization during AKI. However, little is known regarding how activated Mφ regulates kidney injury. To explore the role of activated Mφ in the AKI process, we revealed that Mφ-derived exosomes from AKI mice (ExosAKI ) caused mitochondria damage and induced TECs injury. Then, we detected the global miRNA expression profiles of MφNC and MφAKI and found that among the upregulated miRNAs, miR-195a-5p, which regulates mitochondria metabolism in cancer, was significantly increased in MφAKI . Due to the enrichment of miR-195a-5p in ExosAKI , the miR-195a-5p level in the kidney was elevated in AKI mice. More interestingly, based on the high expression of pri-miR-195a-5p in kidney-infiltrated Mφ, and the reduction of miR-195a-5p in kidney after depletion of Mφ in AKI mice, we confirmed that miR-195a-5p may be produced in infiltrated Mφ, and shuttled into TECs via ExosMφ . Furthermore, in vitro inhibition of miR-195a-5p alleviated the effect of ExosAKI induced mitochondrial dysfunction and cell injury. Consistently, antagonizing miR-195a-5p with a miR-195a-5p antagomir attenuated cisplatin-induced kidney injury and mitochondrial dysfunction in mice. These findings revealed that the Mφ exosomal miR-195a-5p derived from AKI mice played a critical pathologic role in AKI progression, representing a new therapeutic target for AKI.

Global miRNA expression reveals novel nuclear and mitochondrial interactions in Type 1 diabetes mellitus.

Considering the potential role of miRNAs as biomarkers and their interaction with both nuclear and mitochondrial genes, we investigated the miRNA expression profile in type 1 diabetes (T1DM) patients, including the pathways in which they are involved considering both nuclear and mitochondrial functions. We analyzed samples of T1DM patients and control individuals (normal glucose tolerance) by high throughput miRNA sequencing (miRNome). Next, five miRNAs - hsa-miR-26b-5p, hsa-let-7i-5p, hsa-miR-143-3p, hsa-miR-501-3p and hsa-miR-100-5p - were validated by RT-qPCR. The identification of target genes was extracted from miRTarBase and mitoXplorer database. We also performed receiver operating characteristic (ROC) curves and miRNAs that had an AUC > 0.85 were considered potential biomarkers. Overall, 41 miRNAs were differentially expressed in T1DM patients compared to control. Hsa-miR-21-5p had the highest number of predicted target genes and was associated with several pathways, including insulin signaling and apoptosis. 34.1% (14/41) of the differentially expressed miRNAs also targeted mitochondrial genes, and 80.5% (33/41) of them targeted nuclear genes involved in the mitochondrial metabolism. All five validated miRNAs were upregulated in T1DM. Among them, hsa-miR-26b-5p showed AUC>0.85, being suggested as potential biomarker to T1DM. Our results demonstrated 41 DE miRNAs that had a great accuracy in discriminating T1DM and control group. Furthermore, we demonstrate the influence of these miRNAs on numerous metabolic pathways, including mitochondrial metabolism. Hsa-miR-26b-5p and hsa-miR-21-5p were highlighted in our results, possibly acting on nuclear and mitochondrial dysfunction and, subsequently, T1DM dysregulation.

Differential expression of miRNAs from extracellular vesicles in chronic graft-versus-host disease: A preliminary study.

Chronic graft-versus-host disease (cGvHD) is a complex disorder that typically manifests after allogeneic hematopoietic stem cell transplantation (HSCT). It is a major cause of non-relapse mortality, which makes finding biomarkers associated with its occurrence a priority. Recent studies increasingly indicate that microRNAs (miRNAs, short regulatory RNA molecules) can be used as biomarkers of various disorders. They can circulate in patients' bodies encapsulated within extracellular vesicles (EVs). To identify miRNAs associated with the occurrence of cGvHD in EVs isolated from the plasma of patients after allogeneic HSCT. We performed global miRNA expression profiling in a pilot cohort of 3 cGvHD cases and 4 non-cGvHD patients without disease symptoms 90 days after the transplantation (control group). The 2 groups were naturally clustered according to their miRNA profiles using unsupervised hierarchical clustering analysis. We identified 3 miRNAs that were differentially expressed in the cGvHD patients compared to the non-cGvHD patients. The levels of hsa-miR-630 and hsa-miR-374b-5p were lower in the cGvHD patients: 4.1-fold (p = 0.002) and 2.7-fold (p = 0.044), respectively. In contrast, the levels of hsa-miR-29c-3p were 5.8-fold higher (p = 0.004). Our results suggest that miRNA profiles from plasma EVs may act as markers of cGvHD onset.

The combination of SMRT sequencing and Illumina sequencing highlights organ-specific and age-specific expression patterns of miRNAs in Sika Deer.

Due to the lack of high-quality Sika Deer (Cervus nippon) transcriptome and sRNAome across multiple organs or development stages, it is impossible to comprehensively analyze the mRNA and miRNA regulatory networks related to growth, development and immunity response. In this study, we used single molecule-real time sequencing (SMRT-seq) and Illumina sequencing methods to generate transcriptome and sRNAome from ten tissues and four age groups of Sika Deer to help us understand molecular characteristics and global miRNA expression profiles. The results showed that a total of 240,846 consensus transcripts were generated with an average length of 2,784 bp. 4,329 Transcription factors (TFs), 109,000 Simple Sequence Repeats (SSRs) and 18,987 Long non-coding RNAs (LncRNAs) were identified. Meanwhile, 306 known miRNAs and 143 novel miRNAs were obtained. A large number of miRNAs showed organ-specific and age-specific differential expression patterns. In particular, we found that the organ-specific miRNAs were enriched in the brain, some of which shared only between the brain and adrenal. These miRNAs were involved in maintaining specific functions within the brain and adrenal. By constructing miRNA96mRNA interaction networks associated with Sika Deer immunity, we found that miRNAs (miR-148a, miR-26a, miR-214, let-7b, etc.) and mRNAs (CD6, TRIM38, C3, CD163, etc.) might play an important role in the immune response of Sika Deer spleen. Together, our study generated an improved transcript annotation for Sika Deer by SMRT-seq and revealed the role of miRNA in regulating the growth, development and immunity response of Sika Deer.

BIOM-53. SPECIFIC MICRORNA EXPRESSION PATTERN PREDICTS RECURRENCE IN ATYPICAL MENINGIOMA

Abstract Meningiomas are the most common primary tumors of the central nervous system (CNS). Atypical meningioma (AM) recurs in 40% of patients despite total resection and radiotherapy (RT). No consensus on optimal adjuvant management was found, and it is difficult to identify patients insensitive to RT in real-life clinical practice. A promising group of biomarkers represent microRNAs (miRNAs), short non-coding RNAs that regulate most biological processes, including cell proliferation, differentiation, and apoptosis. The study aims to identify tissue miRNAs capable of predicting patients with AM who could benefit from the indicated adjuvant RT. The study includes 80 patients with AM in the exploratory phase and 400 patients with meningioma in the validation phase. Total RNA enriched with miRNAs was isolated from FFPE tissue using the mirVana miRNA Isolation Kit (TF Scientific). Subsequently, RNA quantity and quality controls were measured using NanoDrop 2000 (TF Scientific) and Qubit 2.0 (TF Scientific) instruments. A global miRNA expression profile was generated using the TaqMan Array Human MicroRNA Cards (TF Scientific), which allow the detection of up to 754 miRNAs simultaneously. Obtained data were processed and integrated through bioinformatics algorithms with clinicopathological data of patients with AM.The study identified significantly dysregulated miRNAs among AM patients with and without recurrence (p &amp;lt; 0.05). Results also suggest dysregulated miRNA expression profile in AM patients with indicated RT who did/did not develop a recurrence (p &amp;lt; 0.05). Lists of individual miRNAs and detailed graphical analyses will be included in the conference presentation.The results will help predict the prognosis of surgically intervened patients more accurately and can help determine which patients will benefit from the adjuvant RT. This research is supported by the AZV grant from the Ministry of Health of the Czech Republic (reg. No. NV19-03-00559) and Conceptual development of research organization (FNBr, 65269705). All rights reserved.

Ikaros Regulates microRNA Networks in Acute Lymphoblastic Leukemia.

The hematopoietic transcription factor Ikaros (IKZF1) regulates normal B cell development and functions as a tumor suppressor in precursor B cell acute lymphoblastic leukemia (B-ALL). MicroRNAs (miRNAs) are small regulatory RNAs that through post-transcriptional gene regulation play critical roles in intracellular processes including cell growth in cancer. However, the role of Ikaros in the regulation of miRNA expression in developing B cells is unknown. In this study, we examined the Ikaros-regulated miRNA targets using human IKZF1-mutated Ph+ B-ALL cell lines. Inducible expression of wild-type Ikaros (the Ik1 isoform) caused B-ALL growth arrest and exit from the cell cycle. Global miRNA expression analysis revealed a total of 31 miRNAs regulated by IK1, and ChIP-seq analysis showed that Ikaros bound to several Ik1-responsive miRNA genes. Examination of the prognostic significance of miRNA expression in B-ALL indicate that the IK1-regulated miRNAs hsa-miR-26b, hsa-miR-130b and hsa-miR-4649 are significantly associated with outcome in B-ALL. Our findings establish a potential regulatory circuit between the tumor-suppressor Ikaros and the oncogenic miRNA networks in IKZF1-mutated B-ALL. These results indicate that Ikaros regulates the expression of a subset of miRNAs, of which several may contribute to B-ALL growth.

Global microRNA and protein expression in human term placenta.

Description of the global expression of microRNAs (miRNAs) and proteins in healthy human term placentas may increase our knowledge of molecular biological pathways that are important for normal fetal growth and development in term pregnancy. The aim of this study was to explore the global expression of miRNAs and proteins, and to point out functions of importance in healthy term placentas. Placental samples (n = 19) were identified in a local biobank. All samples were from uncomplicated term pregnancies with vaginal births and healthy, normal weight newborns. Next-generation sequencing and nano-scale liquid chromatographic tandem mass spectrometry were used to analyse miRNA and protein expression, respectively. A total of 895 mature miRNAs and 6,523 proteins were detected in the placentas, of which 123 miRNAs and 346 proteins were highly abundant. The miRNAs were in high degree mapped to chromosomes 19, 14, and X. Analysis of the highly abundant miRNAs and proteins showed several significantly predicted functions in common, including immune and inflammatory response, lipid metabolism and development of the nervous system. The predicted function inflammatory response may reflect normal vaginal delivery, while lipid metabolism and neurodevelopment may be important processes for the term fetus. The data presented in this study, with complete miRNA and protein findings, will enhance the knowledge base for future research in the field of placental function and pathology.