Changes In miRNA Expression Research Articles

Early life and adult stress promote sex dependent changes in hypothalamic miRNAs and environmental enrichment prevents stress-induced miRNA and gene expression changes in rats

BackgroundThe hypothalamus plays a key role in the stress response. While early life stress (ELS) increases susceptibility to psychiatric disorders including major depressive disorder (MDD), acute stress during adulthood can also precipitate MDD after ELS.AimHere, we tested the expression of miRNAs following ELS and susceptibility to depression-like behavior and whether sex or acute stress exacerbates this response. We also tested whether environmental enrichment (Enr) promotes early life and adult behavioral stress resilience and its effect on hypothalamic miRNA and gene expression. Following rat maternal separation (MS) as an ELS model, Enr from weaning through adulthood, and restraint (RS) as acute adult stress, we tested both animal behavior and miRNA expression in the hypothalamus. Target genes and their enrichment and ontology were analyzed using bioinformatic tools. Target gene expression changes were tested using qPCR, and miRNA promoter methylation was studied using methylated-DNA immunoprecipitation qPCR.ResultsMS, Enr, RS, and sex altered hypothalamic miRNAs, including several previously reported in MS literature: miRs-29, − 124, − 132, − 144, − 504. Sex had a significant effect on the greatest number of miRNAs. Also, Enr reversed downregulation of miR-29b-1-5p and -301b-3p in MS. qPCR showed that MAPK6 and MMP19, targets of miR-301b-3p, were upregulated in MS and reversed by Enr. Additionally, miR-219a was hypermethylated in MS coinciding with decreased miR-219a expression.ConclusionsThis study found that sex plays a critical role in the hypothalamic miRNA response to both ELS and acute stress, with males expressing greater changes following postnatal stress. Moreover, enrichment significantly altered behavior as well as hypothalamic miRNA expression and their gene targets. Because of its role as the initiator of the autonomic stress response and connection to hedonic and motivational behavior, the hypothalamic miRNA landscape may significantly alter both the short and long-term behavioral response to stress.

Regulation of airway immunity by epithelial miRNAs.

The airway epithelium is essential to protect the host from inhaled pathogens and particles. It maintains immune homeostasis and mediates tissue repair after injury. Inflammatory diseases of the airways are associated with failure of epithelial functions, including loss of barrier integrity that results in increased tissue permeability and immune activation; excessive mucus secretion and impaired mucociliary clearance that leads to airflow obstruction and microbial overgrowth; and dysregulation of cellular signals that promotes inflammation and alters tissue structure and airway reactivity. MicroRNAs play crucial roles in mounting appropriate cellular responses to environmental stimuli and preventing disease, using a common machinery and mechanism to regulate gene expression in epithelial cells, immune cells of hematopoietic origin, and other cellular components of the airways. Respiratory diseases are accompanied by dramatic changes in epithelial miRNA expression that drive persistent immune dysregulation. In this review, we discuss responses of the epithelium that promote airway immunopathology, with a focus on miRNAs that contribute to the breakdown of essential epithelial functions. We emphasize the emerging role of miRNAs in regulation of epithelial responses in respiratory health and their value as diagnostic and therapeutic targets.

Integrative Analysis of DNA Methylation and microRNA Expression Reveals Mechanisms of Racial Heterogeneity in Hepatocellular Carcinoma.

Pathologic alterations in epigenetic regulation have long been considered a hallmark of many cancers, including hepatocellular carcinoma (HCC). In a healthy individual, the relationship between DNA methylation and microRNA (miRNA) expression maintains a fine balance; however, disruptions in this harmony can aid in the genesis of cancer or the propagation of existing cancers. The balance between DNA methylation and microRNA expression and its potential disturbance in HCC can vary by race. There is emerging evidence linking epigenetic events including DNA methylation and miRNA expression to cancer disparities. In this paper, we evaluate the epigenetic mechanisms of racial heterogenity in HCC through an integrated analysis of DNA methylation, miRNA, and combined regulation of gene expression. Specifically, we generated DNA methylation, mRNA-seq, and miRNA-seq data through the analysis of tumor and adjacent non-tumor liver tissues from African Americans (AA) and European Americans (EA) with HCC. Using mixed ANOVA, we identified cytosine-phosphate-guanine (CpG) sites, mRNAs, and miRNAs that are significantly altered in HCC vs. adjacent non-tumor tissue in a race-specific manner. We observed that the methylome was drastically changed in EA with a significantly larger number of differentially methylated and differentially expressed genes than in AA. On the other hand, the miRNA expression was altered to a larger extent in AA than in EA. Pathway analysis functionally linked epigenetic regulation in EA to processes involved in immune cell maturation, inflammation, and vascular remodeling. In contrast, cellular proliferation, metabolism, and growth pathways are found to predominate in AA as a result of this epigenetic analysis. Furthermore, through integrative analysis, we identified significantly differentially expressed genes in HCC with disparate epigenetic regulation, associated with changes in miRNA expression for AA and DNA methylation for EA.

Analysis of MicroRNA Expression Changes During the Course of Therapy In Rectal Cancer Patients.

MicroRNAs (miRNAs) regulate gene expression in a tissue-specific manner. However, little is known about the miRNA expression changes induced by the therapy in rectal cancer (RC) patients. We evaluated miRNA expression levels before and after therapy and identified specific miRNA signatures reflecting disease course and treatment responses of RC patients. First, miRNA expression levels were assessed by next-generation sequencing in two plasma samplings (at the time of diagnosis and a year after) from 20 RC patients. MiR-122-5p and miR-142-5p were classified for subsequent validation in plasma and plasma extracellular vesicles (EVs) on an independent group of RC patients (n=107). Due to the intrinsic high differences in miRNA expression levels between samplings, cancer-free individuals (n=51) were included in the validation phase to determine the baseline expression levels of the selected miRNAs. Expression levels of these miRNAs were significantly different between RC patients and controls (for all p <0.001). A year after diagnosis, miRNA expression profiles were significantly modified in patients responding to treatment and were no longer different from those measured in cancer-free individuals. On the other hand, patients not responding to therapy maintained low expression levels in their second sampling (miR-122-5p: plasma: p=0.05, EVs: p=0.007; miR-142-5p: plasma: p=0.008). Besides, overexpression of miR-122-5p and miR-142-5p in RC cell lines inhibited cell growth and survival. This study provides novel evidence that circulating miR-122-5p and miR-142-5p have a high potential for RC screening and early detection as well as for the assessment of patients’ outcomes and the effectiveness of treatment schedule.

Temporal brain microRNA expression changes in a mouse model of neonatal hypoxic-ischemic injury.

Neonatal hypoxic-ischemic brain injury (HIBI) results in significant morbidity and mortality despite current standard therapies. MicroRNAs (miRNAs) are a promising therapeutic target; however, there is a paucity of data on endogenous miRNA expression of the brain after HIBI during the primary therapeutic window (6-72 h after injury). Postnatal day 9 mouse pups underwent unilateral carotid ligation+hypoxia (HIBI), sham surgery+hypoxia, or sham surgery+normoxia (controls). miRNA sequencing was performed on the ipsilateral brain of each of the three groups plus the contralateral HIBI brain at 24 and 72 h after injury. Findings were validated in eight key miRNAs by quantitative polymerase chain reaction. Hypoxia resulted in significant differential expression of 38 miRNAs at both time points. Mir-2137, -335, -137, and -376c were significantly altered by neonatal HIBI at 24 and 72 h, with 3 of the 4 demonstrating multiphasic expression (different direction of differential expression at 24 versus 72 h). Our global assessment of subacute changes in brain miRNA expression after hypoxia or HIBI will advance research into targeted miRNA-based interventions. It will be important to consider the multiphasic miRNA expression patterns after HIBI to identify optimal timing for individual interventions. This study is the first to comprehensively define endogenous brain microRNA expression changes outside of the first hours after neonatal hypoxic-ischemic brain injury (HIBI). Mir-2137, -335, -137, and -376c were significantly altered by neonatal HIBI and therefore deserve further investigation as possible therapeutic targets. The expression profiles described will support the design of future studies attempting to develop miRNA-based interventions for infants with HIBI. At 24 h after injury, contralateral HIBI miRNA expression patterns were more similar to ipsilateral HIBI than to controls, suggesting that the contralateral brain is not an appropriate "internal control" for miRNA studies in this model.

Circulating miR-29c-3p is downregulated in patients with acromegaly

Background/aimmiRNAs control various biological functions, such as cell proliferation, differentiation, signaling pathways, apoptosis and metabolism. Recently, it has been shown that there is a relationship between changes in miRNA expression and the development of acromegaly. Studies are needed to identify new disease-specific miRNAs. The aim of the current study is to evaluate plasma miR-29c-3p, miR-31-5p and miR-18a-5p steady-state levels in acromegaly. Another aim is to investigate whether there is a difference in the levels of these miRNAs in patients with inadequate control and controlled acromegaly with somatostatin analog (SSA) therapy. These miRNAs targeting the IGF-1 gene were determined by in silico estimation.Materials and methodsThe study included 30 healthy controls (HC) and 20 patients with acromegaly. Anterior pituitary functions and disease activities of patients with acromegaly were evaluated at the time of study. The miR-29c-3p, miR-31-5p and miR-18a-5p levels were measured using quantitative real-time PCR (RT-qPCR).ResultsThe expression level of miR-29c-3p was significantly lower in patients with acromegaly compared to the HC group (p < 0.001). This downregulation was more pronounced in patients with inadequately controlled acromegaly than in patients with acromegaly controlled with somatostatin analogues (SSA) therapy (p = 0.016). Univariate logistic regression analysis results showed that down regulation of miR-29c-3p expression increases the risk of developing acromegaly [OR (95% Cl) = 1.605 (1.142-2.257), p = 0.006]. There was no significant difference between the groups in terms of miR-31-5p and miR-18a-5p expression levels (p = 0.375 and p = 0.649, respectively). ConclusionPlasma miR-29c-3p expression level is downregulated in patients with acromegaly, and this is more pronounced in patients with inadequate control.

Effects of single bouts of different endurance exercises with different intensities on microRNA biomarkers with and without blood flow restriction: a three-arm, randomized crossover trial

PurposePhysical activity is associated with altered levels of circulating microRNAs (ci-miRNAs). Changes in miRNA expression have great potential to modulate biological pathways of skeletal muscle hypertrophy and metabolism. This study was designed to determine whether the profile of ci-miRNAs is altered after different approaches of endurance exercise.MethodsEighteen healthy volunteers (aged 24 ± 3 years) participated this three-arm, randomized-balanced crossover study. Each arm was a single bout of treadmill-based acute endurance exercise at (1) 100% of the individual anaerobic threshold (IANS), (2) at 80% of the IANS and (3) at 80% of the IANS with blood flow restriction (BFR). Load-associated outcomes (fatigue, feeling, heart rate, and exhaustion) as well as acute effects (circulating miRNA patterns and lactate) were determined.ResultsAll training interventions increased the lactate concentration (LC) and heart rate (HR) (p < 0.001). The high-intensity intervention (HI) resulted in a higher LC than both lower intensity protocols (p < 0.001). The low-intensity blood flow restriction (LI-BFR) protocol led to a higher HR and higher LC than the low-intensity (LI) protocol without BFR (p = 0.037 and p = 0.003). The level of miR-142-5p and miR-197-3p were up-regulated in both interventions without BFR (p < 0.05). After LI exercise, the expression of miR-342-3p was up-regulated (p = 0.038). In LI-BFR, the level of miR-342-3p and miR-424-5p was confirmed to be up-regulated (p < 0.05). Three miRNAs and LC show a significant negative correlation (miR-99a-5p, p = 0.011, r = − 0.343/miR-199a-3p, p = 0.045, r = − 0.274/miR-125b-5p, p = 0.026, r = − 0.302). Two partial correlations (intervention partialized) showed a systematic impact of the type of exercise (LI-BFR vs. HI) (miR-99a-59: r = − 0.280/miR-199a-3p: r = − 0.293).ConclusionMiRNA expression patterns differ according to type of activity. We concluded that not only the intensity of the exercise (LC) is decisive for the release of circulating miRNAs—as essential is the type of training and the oxygen supply.

Analysis of lncRNA-miRNA-mRNA expression pattern in heart tissue after total body radiation in a mouse model

BackgroundRadiation therapy is integral to effective thoracic cancer treatments, but its application is limited by sensitivity of critical organs such as the heart. The impacts of acute radiation-induced damage and its chronic effects on normal heart cells are highly relevant in radiotherapy with increasing lifespans of patients. Biomarkers for normal tissue damage after radiation exposure, whether accidental or therapeutic, are being studied as indicators of both acute and delayed effects. Recent research has highlighted the potential importance of RNAs, including messenger RNAs (mRNAs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) as biomarkers to assess radiation damage. Understanding changes in mRNA and non-coding RNA expression will elucidate biological pathway changes after radiation.MethodsTo identify significant expression changes in mRNAs, lncRNAs, and miRNAs, we performed whole transcriptome microarray analysis of mouse heart tissue at 48 h after whole-body irradiation with 1, 2, 4, 8, and 12 Gray (Gy). We also validated changes in specific lncRNAs through RT-qPCR. Ingenuity Pathway Analysis (IPA) was used to identify pathways associated with gene expression changes.ResultsWe observed sustained increases in lncRNAs and mRNAs, across all doses of radiation. Alas2, Aplnr, and Cxc3r1 were the most significantly downregulated mRNAs across all doses. Among the significantly upregulated mRNAs were cell-cycle arrest biomarkers Gdf15, Cdkn1a, and Ckap2. Additionally, IPA identified significant changes in gene expression relevant to senescence, apoptosis, hemoglobin synthesis, inflammation, and metabolism. LncRNAs Abhd11os, Pvt1, Trp53cor1, and Dino showed increased expression with increasing doses of radiation. We did not observe any miRNAs with sustained up- or downregulation across all doses, but miR-149-3p, miR-6538, miR-8101, miR-7118-5p, miR-211-3p, and miR-3960 were significantly upregulated after 12 Gy.ConclusionsRadiation-induced RNA expression changes may be predictive of normal tissue toxicities and may indicate targetable pathways for radiation countermeasure development and improved radiotherapy treatment plans.

Dexmedetomidine Restores Autophagic Flux, Modulates Associated microRNAs and the Cholinergic Anti-inflammatory Pathway upon LPS-Treatment in Rats

Infections and perioperative stress can lead to neuroinflammation, which in turn is linked to cognitive impairments such as postoperative delirium or postoperative cognitive dysfunctions. The α2-adrenoceptor agonist dexmedetomidine (DEX) prevents cognitive impairments and has organo-protective and anti-inflammatory properties. Macroautophagy (autophagy) regulates many biological processes, but its role in DEX-mediated anti-inflammation and the underlying mechanism of DEX remains largely unclear. We were interested how a pretreatment with DEX protects against lipopolysaccharide (LPS)-induced inflammation in adult male Wistar rats. We used Western blot and activity assays to study how DEX modulated autophagy- and apoptosis-associated proteins as well as molecules of the cholinergic anti-inflammatory pathway, and qPCR to analyse the expression of autophagy and inflammation-associated microRNAs (miRNA) in the spleen, cortex and hippocampus at different time points (6 h, 24 h, 7 d). We showed that a DEX pretreatment prevents LPS-induced impairments in autophagic flux and attenuates the LPS-induced increase in the apoptosis-associated protein cleaved poly(ADP-ribose)-polymerase (PARP) in the spleen. Both, DEX and LPS altered miRNA expression and molecules of the cholinergic anti-inflammatory pathway in the spleen and brain. While only a certain set of miRNAs was up- and/or downregulated by LPS in each tissue, which was prevented or attenuated by a DEX pretreatment in the spleen and hippocampus, all miRNAs were up- and/or downregulated by DEX itself – independent of whether or not they were altered by LPS. Our results indicate that the organo-protective effect of DEX may be mediated by autophagy, possibly by acting on associated miRNAs, and the cholinergic anti-inflammatory pathway.Graphical abstractPreventive effects of DEX on LPS-induced inflammation. DEX restores the LPS-induced impairments in autophagic flux, attenuates PARP cleavage and alters molecules of the cholinergic system in the spleen. Furthermore, DEX alters and prevents LPS-induced miRNA expression changes in the spleen and brain along with LPS.

Multi-omics data integration reveals novel drug targets in hepatocellular carcinoma

BackgroundGenetic aberrations in hepatocellular carcinoma (HCC) are well known, but the functional consequences of such aberrations remain poorly understood.ResultsHere, we explored the effect of defined genetic changes on the transcriptome, proteome and phosphoproteome in twelve tumors from an mTOR-driven hepatocellular carcinoma mouse model. Using Network-based Integration of multi-omiCS data (NetICS), we detected 74 ‘mediators’ that relay via molecular interactions the effects of genetic and miRNA expression changes. The detected mediators account for the effects of oncogenic mTOR signaling on the transcriptome, proteome and phosphoproteome. We confirmed the dysregulation of the mediators YAP1, GRB2, SIRT1, HDAC4 and LIS1 in human HCC.ConclusionsThis study suggests that targeting pathways such as YAP1 or GRB2 signaling and pathways regulating global histone acetylation could be beneficial in treating HCC with hyperactive mTOR signaling.

Diet quality, gut microbiota, and microRNAs associated with mild cognitive impairment in middle-aged and elderly Chinese population

Diet, the gut microbiota, and microRNAs (miRNAs) have been recognized as factors possibly influencing the pathogenesis of Alzheimer's disease. However, the combined role of diet, the gut microbiota, and miRNAs in mild cognitive impairment (MCI) has not been extensively investigated. To examine the associations among diet quality, the gut microbiota, miRNAs, and risks of MCI in middle-aged and elderly Chinese population. Participants were drawn from the Effects and Mechanism Investigation of Cholesterol and Oxysterol on Alzheimer's disease (EMCOA) study. We included 2239 participants who completed comprehensive cognitive tests and dietary surveys at baseline and follow-up. Chinese Dietary Guidelines Index 2018 (CDGI-2018), Energy-Adjusted Dietary Inflammatory Index (E-DII) and Healthy Lifestyle Score (HLS) values were calculated based on data from a standard questionnaire. Changes of gut microbiota and serum miRNA expression were detected in 75 MCI and 52 healthy participants. The receiver operating characteristic curve was performed based on a logistic regression model distinguishing patients with MCI from healthy controls. Higher CDGI-2018 (RR, 0.75; 95% CI: 0.58-0.98) and HLS (RR, 0.54; 95% CI: 0.34-0.84) values and lower E-DII (RR, 1.46; 95% CI: 1.14-1.87) values were significantly associated with a lower risk of MCI. Lower microbial diversity and abundance of Faecalibacterium, unidentified Ruminococcaceae, and Alistipes were found in MCI participants compared with healthy controls. The abundance of ProteobacteriaandGammaproteobacteria was higher in MCI participants and was correlated with hsa-let-7g-5p, hsa-miR-107, and hsa-miR-186-3p. The predictive signatures of diet quality scores, differential gut microbiota, and serum miRNAs had good performance on distinguishing MCI participants from healthy individuals (AUC=0.91). A better diet quality is associated with a lower risk of MCI, which may be mediated by the microbiota composition and miRNA expression. These findings suggest that diet, the gut microbiota, and miRNAs may serve asa combinatorial biomarker to identify MCI patients and provide new insight for the diagnosis and prevention of MCI. This study was registered at the Chinese Clinical Trial Registry as ChiCTROOC-17011882.

Sensitivity of Plasma Micrornas as a Probable Non-Small Cell Lung Cancer Biomarker and Its Significance

Non-small cell lung cancer (NSCLC) is the chief origin of death due to cancer. The development of a less invasive diagnostic technique for NSCLC, especially at the beginning, can improve outcomes. By means of microarray platforms, we formerly diagnosed 12 microRNAs (miRNAs) abnormally expressed in primary cancer cells associated with early NSCLC. Objective: In this study, we will extend previous studies to determine if miRNAs may be beneficial as a NSCLC potential plasma biomarker. Methods: We primarily confirmed miRNA expression from PCR of 32 stage one NSCLC patients with lung tumor and plasma specimens were assessed, then assessed the investigative value of plasma miRNAs in 61 patients of NSCLC and 30 normal subjects. It was confirmed that the alteration of MiRNA expression influences the regeneration of neoplastic tumors. MiRNAs were steady and reliable in plasma measurements. The cohort consisted of 21 men and 11 women. Their age ranges from 47 to 80 years. AC tumors were classified in 17 and SCC in 15 patients. Smoking packets use in these patients were 39 ± 28. In addition, IV blood were taken from healthy subjects with matched data distribution to studied group as age, race, sex and smoking, and aided as a control to evaluate fluctuations in plasma in cancer patients. Results: Five out of 12 miRNAs showed a significant change of level in plasma and the corresponding tumor tissue (all r40850, all P&lt; 0.05). Four genes (miRNA-126, 21, 486-5 and 210p) set the best logistic regression pattern, with 87% sensitivity and 97% of specificity of at the time of NSCLC in differentiating from healthy patients. Moreover, the miRNA genes generated a sensitivity of 79.41% and 92.59% of specificity in patients diagnosed with SCC and AC. Moreover, genes have specificity of 96.67% in the analysis of lung adenocarcinoma and squamous cell carcinoma. (P less than 0.05). A change in square miRNA expression may offer potential NSCLC blood-derived biomarker. Conclusion: Finally, we show that miRNA expression identified from surgical tumor tissue can be easily and correctly determined in plasma. Though, prominently, a plasma miRNA recognition panel would be cast-off as a less invasive analytic method for NSCLC, including long-term adenocarcinoma. However, the presence of an independent potential biomarker requires further verification. Keywords: Lung cancer; Diagnosis; Plasma; Microrna; Qrt-PCR

1: MicroRNAs: Potential For Molecular Modulation Of Mechanically-induced Skin Growth During TE

Purpose:Tissue expansion (TE) is commonly utilized to promote skin growth prior to reconstructing a defect or deformity. In spite of its ubiquitous use, the role of molecular modulators during TE, such as microRNAs (miRNAs), has not previously been studied. MiRNAs are small endogenous molecules that regulate many biological processes, including cell proliferation, differentiation, and inflammatory response. Here, we investigate genome-wide changes in miRNA expression in skin during TE.Methods:Changes in miRNA expression were evaluated in a porcine TE model. Full-thickness skin biopsies were collected after 1 hour, 24 hours, 3 days, and 7 days of expansion, as well as from unexpanded skin (control). RNA extracted from biopsies was analyzed with next-generation sequencing (NGS). Differential expression analysis was performed using R software with the Bioconductor-DESeq2 package. Results were corrected for multiple testing using the Benjamini-Hochberg method. A combination of adjusted p-value < 0.05 and | log2 (fold change) | > 1 were used as the threshold to determine the significance of differentially expressed (DE) miRNAs. Potential target genes for DE miRNAs were identified by in silico analysis using three data bases: miRDB, miRTarBase, and DIANA Tools with Tarbase. Functional enrichment analysis of target genes was performed using the g:GOSt functional profiling tool, and results were visualized using R and Cytoscape software.Results:We identified 52 miRNAs that were differentially upregulated (n = 18) or downregulated (n = 34) during at least one of the tested timepoints during TE. At the four time points (1 hour, 24 hours, 3 days and 7 days), there were 15, 6, 22, and 20 DE miRNAs, respectively. Eight miRNAs (ssc-miR-193a-5p, ssc-miR-21, ssc-miR-9-1, ssc-miR-708-3p, ssc-miR-212, ssc-miR-196a, ssc-miR-15b, ssc-miR-184) were DE at more than one timepoint. The comparative analysis showed 9, 4, 16 and 15 unique miRNAs after 1 hour, 24, hours, 3 days and 7 days of TE, respectively. Gene Ontology and KEGG pathway analyses for predicted target genes demonstrated enrichment in cellular processes related to metabolism, transcription, translation, signal transduction, cell differentiation, migration, and angiogenesis.

Exposure to Fine Particulate Matter Air Pollution Alters mRNA and miRNA Expression in Bone Marrow-Derived Endothelial Progenitor Cells from Mice.

Exposure to fine particulate matter (PM2.5) air pollution is associated with quantitative deficits of circulating endothelial progenitor cells (EPCs) in humans. Related exposures of mice to concentrated ambient PM2.5 (CAP) likewise reduces levels of circulating EPCs and induces defects in their proliferation and angiogenic potential as well. These changes in EPC number or function are predictive of larger cardiovascular dysfunction. To identify global, PM2.5-dependent mRNA and miRNA expression changes that may contribute to these defects, we performed a transcriptomic analysis of cells isolated from exposed mice. Compared with control samples, we identified 122 upregulated genes and 44 downregulated genes in EPCs derived from CAP-exposed animals. Functions most impacted by these gene expression changes included regulation of cell movement, cell and tissue development, and cellular assembly and organization. With respect to miRNA changes, we found that 55 were upregulated while 53 were downregulated in EPCs from CAP-exposed mice. The top functions impacted by these miRNA changes included cell movement, cell death and survival, cellular development, and cell growth and proliferation. A subset of these mRNA and miRNA changes were confirmed by qRT-PCR, including some reciprocal relationships. These results suggest that PM2.5-induced changes in gene expression may contribute to EPC dysfunction and that such changes may contribute to the adverse cardiovascular outcomes of air pollution exposure.

MiR-1968-5p is involved in the pathogenesis of lupus nephritis of NZBWF1 mice by targeting csf1.

Lupus nephritis is one of the most common and severe systemic lupus erythematosus complications. However, the pathogenesis of lupus nephritis is still poorly understood. Increasing evidence has shown that microRNAs (miRNAs) are extensively involved in the pathophysiology of autoimmune diseases. NZBWF1 is the classical mouse model of lupus nephritis. The present study aimed to investigate the expression profiling of mRNA and miRNAs of NZBWF1 mice with lupus nephritis using microarray, and explored the potential molecular mechanism of miRNA. miRNA and mRNA microarrays were performed to identify miRNA and mRNA expression changes between pre-diseased (8-week-old) NZBWF1 mice and diseased NZBWF1 mice with lupus nephritis (28-week-old). Quantitative polymerase chain reaction (qPCR) validated these results. The target of miRNA was confirmed through a dual-luciferase reporter and stimulated mesangial cells experiment. The combined miRNA and mRNA analysis identified 43 differentially expressed miRNAs and 1796 differentially expressed mRNAs between pre-disease (8-week-old) (n = 4) and diseased (28-week-old) NZBWF1 mice. We found that miR-1968-5p was significantly decreased, and csf1 mRNA was significantly increased in lupus nephritis mouse and verified by RT-PCR. csf1 has been demonstrated to play important roles in SLE. Bioinformatics analysis predicted that the csf1 was a potential target gene of miR-1968-5p. A dual-luciferase reporter assay confirmed the target binding. In cell experiments, overexpression or knockdown of miR resulted in a decrease or increase of csf1 expression, respectively. These resultssuggest that miR-1968-5p may be involved in the pathogenesis of lupus nephritis of NZBWF1 mice by targeting csf1.

Abstract 2377: microRNA expression profile in urinary exosomes is dependent on non-invasive lymphoma induction in mice

Abstract Lymphoma accounts for approximately 4% of cancers in the United States, with an estimated 20,910 number of deaths per year. The standard method of diagnosis and staging of lymphoma involves surgical biopsy of the tumor - a procedure that has many negative associated risks. Exosomes are extracellular vesicles secreted in biological fluids that can serve as “liquid biomarkers”. Identification of unique cancer-related biomarkers in urinary exosomes may provide a novel non-invasive and cost-effective tool for lymphoma diagnosis. Analyses of biomarkers obtained from urinary exosomes have been used to evaluate urological cancers, however, these methods have not yet been translated to non-urological pathologies, such as lymphomas. The objective for this study was to determine the profile of microRNAs (miRNAs) expressed in urinary exosomes of mice challenged with lymphoma and compare it to miRNAs identified in urinary exosomes of control mice. Male and female C57BL/6 mice, (tumor (+), n=12), were injected with either 2.5x105 mouse EL-4 lymphoma cells or phosphate-buffered saline (tumor (-), n=12). Tumor growth was monitored for 20 days. Urine was collected for 48 hours starting on day 17 and serum, tumor tissues, and organs were collected on day 20. Extraction of urinary exosomes was followed by total RNA isolation and RT-qPCR for which a set of PCR arrays consisting of 709 mouse-specific miRNA primers was used. Fold changes in miRNA expression were quantified using the ΔΔCt method. Mice developed tumors by day 13 with initial tumor appearance around day seven. There were no statistically significant differences between final tumor mass, body weight, or food and water intake in tumor (+) versus tumor (-) mice. RT-qPCR arrays of miRNAs extracted from urinary exosomes revealed 464 miRNAs that were differentially expressed between tumor (+) and tumor (-). 215 miRNAs were up-regulated, while 249 miRNAs were down-regulated in tumor (+) mice. These results will be compared to miRNAs from serum and tumor tissues to identify tumor-specific miRNAs that can be used for potential application in the clinical setting. Citation Format: Brittany Wilson, Rebekah Betar, Alexander Martin, Zackaria Niazi, Michael Boyer, Lori Winter, Victor Babich, Francesca Di Sole, Elitsa Ananieva. microRNA expression profile in urinary exosomes is dependent on non-invasive lymphoma induction in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2377.

Combined drought and heat stresses trigger different sets of miRNAs in contrasting potato cultivars.

MicroRNAs are small, non-coding RNAs that are responsible for regulation of gene expression during plant growth and development. Although there are many studies on miRNAs in other plants, little work has been done to understand the role of miRNAs in abiotic stress tolerance in potatoes. This study investigates changes in miRNA profiles of two different potato cultivars (tolerant, Unica and susceptible, Russet Burbank) in response to heat, drought and their combination. Transcriptomic studies revealed that miRNA profiles depend on the susceptibility and tolerance of the cultivar and also the stress conditions. Large number of miRNAs were expressed in Unica, whereas Russet Burbank indicated lesser number of changes in miRNA expression. Physiological and transcriptional results clearly supported that Unica cultivar is tolerant to combined drought and heat stress compared to Russet Burbank. Moreover, psRNATarget analysis predicted that major miRNAs identified were targeting genes playing important roles in response to drought and heat stress and their important roles in genetic and post-transcriptional regulation, root development, auxin responses and embryogenesis were also observed. This study focused on eight miRNAs (Novel_8, Novel_9, Novel_105, miR156d-3p, miR160a-5p, miR162a-3p, miR172b-3p and miR398a-5p) and their putative targets where results indicate that they may play a vital role at different post-transcriptional levels against drought and heat stresses. We suggest that miRNA overexpression in plants can lead to increased tolerance against abiotic stresses; furthermore, there should be more emphasis on the studies to investigate the role of miRNAs in combined abiotic stress in plants.

LINGO-1 regulates Wnt5a signaling during neural stem and progenitor cell differentiation by modulating miR-15b-3p levels

BackgroundManipulation of neural stem and progenitor cells (NSPCs) is critical for the successful treatment of spinal cord injury (SCI) by NSPC transplantation, since their differentiation into neurons and oligodendrocytes can be inhibited by factors present in inflamed myelin. In this study, we examined the effects of LINGO-1 on spinal cord-derived NSPC (sp-NSPC) differentiation, the underlying mechanisms of action, and the functional recovery of mice after transplantation of manipulated cells.Methodssp-NSPCs were harvested from female adult C57/BL6 mice after SCI induced with an NYU impactor. These cells were infected with lentiviral vectors containing LINGO-1 shRNA sequence or a scrambled control and transplanted into SCI mice. Tuj-1- and GFAP-positive cells were assessed by immunofluorescence staining. Wnt5a, p-JNK, JNK, and β-catenin expression was determined by Western blot and RT-qPCR. miRNAs were sequenced to detect changes in miRNA expression. Motor function was evaluated 0–35 days post-surgery by means of the Basso Mouse Scale (BMS) and by the rotarod performance test.ResultsWe discovered that LINGO-1 shRNA increased neuronal differentiation of sp-NSPCs while decreasing astrocyte differentiation. These effects were accompanied by elevated Wnt5a protein expression, but unexpectedly, no changes in Wnt5a mRNA levels. miRNA-sequence analysis demonstrated that miR-15b-3p was a downstream mediator of LINGO-1 which suppressed Wnt5a expression. Transplantation of LINGO-1 shRNA-treated sp-NSPCs into SCI mice promoted neural differentiation, wound compaction, and motor function recovery.ConclusionsLINGO-1 shRNA promotes neural differentiation of sp-NSPCs and Wnt5a expression, probably by downregulating miR-15b-3p. Transplantation of LINGO-1 shRNA-treated NSPCs promotes recovery of motor function after SCI, highlighting its potential as a target for SCI treatment.

Translation of a circulating miRNA signature of melanoma into a solid tissue assay to improve diagnostic accuracy and precision.

Aim: Successful treatment of cutaneous melanoma depends on early and accurate diagnosis of clinically suspicious melanocytic skin lesions. Multiple international studies have described the challenge of providing accurate and reproducible histopathological assessments of melanocytic lesions, highlighting the need for new diagnostic tools including disease-specific biomarkers. Previously, a 38-miRNA signature (MEL38) was identified in melanoma patient plasma and validated as a novel biomarker. In this study, MEL38 expression in solid tissue biopsies representing the benign nevi to metastatic melanoma spectrum is examined. Patients& methods: Nanostring digital gene expression assessment of the MEL38 signature was performed on 308 formalin-fixed paraffin-embedded biopsies of nevi, melanoma insitu and invasive melanoma. Genomic data were interrogated using hierarchical clustering, univariateand multivariate statistical approaches. Classification scores computed from the MEL38 signature were analyzed for their association with demographic data and histopathology results, including MPATH-DX class, AJCC disease stage and tissue subtype. Results: The MEL38 score can stratify higher-risk melanomas (MPATH-Dx class V or more advanced) from lower-risk skin lesions (class I-IV) with an areaunder the curve of 0.97 (p<0.001). The genomic score ranges from 0 to 10 and is positively correlated with melanoma progression, with an intraclass correlation coefficient of 0.85 with stage 0-IV disease. Using an optimized classification threshold of ≥2.7 accurately identifies higher-risk melanomaswith 89% sensitivity and 94% specificity. Multivariate analysis showed the score to be a significant predictor of malignancy, independent of technical and clinical covariates. Application of the MEL38 signature to Spitz nevi reveals an intrasubtype profile, with elements in common to both nevi and melanoma. Conclusion: Melanoma-specific circulating miRNAs maintain their association with malignancy when measured in the hypothesized tissue of origin. The MEL38 signature is an accurate and reproducible metric of melanoma status, based on changes in miRNA expression that occur as the disease develops and spreads. Inclusion of the MEL38 score into routine practice would provide physicians with previously unavailable, personalized genomic information about their patient's skin lesions. Combining molecular biomarker data with conventional histopathology data may improve diagnostic accuracy, healthcare resource utilization and patient outcomes.

Identifying a novel biological mechanism for alcohol addiction associated with circRNA networks acting as potential miRNA sponges.

Our lab and others have shown that chronic alcohol use leads to gene and miRNA expression changes across the mesocorticolimbic (MCL) system. Circular RNAs (circRNAs) are noncoding RNAs that form closed‐loop structures and are reported to alter gene expression through miRNA sequestration, thus providing a potentially novel neurobiological mechanism for the development of alcohol dependence (AD). Genome‐wide expression of circRNA was assessed in the nucleus accumbens (NAc) from 32 AD‐matched cases/controls. Significant circRNAs (unadj. p ≤ 0.05) were identified via regression and clustered in circRNA networks via weighted gene co‐expression network analysis (WGCNA). CircRNA interactions with previously generated mRNA and miRNA were detected via correlation and bioinformatic analyses. Significant circRNAs (N = 542) clustered in nine significant AD modules (FWER p ≤ 0.05), within which we identified 137 circRNA hubs. We detected 23 significant circRNA–miRNA–mRNA interactions (FDR ≤ 0.10). Among these, circRNA‐406742 and miR‐1200 significantly interact with the highest number of mRNA, including genes associated with neuronal functioning and alcohol addiction (HRAS, PRKCB, HOMER1, and PCLO). Finally, we integrate genotypic information that revealed 96 significant circRNA expression quantitative trait loci (eQTLs) (unadj. p ≤ 0.002) that showed significant enrichment within recent alcohol use disorder (AUD) and smoking genome‐wide association study (GWAS). To our knowledge, this is the first study to examine the role of circRNA in the neuropathology of AD. We show that circRNAs impact mRNA expression by interacting with miRNA in the NAc of AD subjects. More importantly, we provide indirect evidence for the clinical importance of circRNA in the development of AUD by detecting a significant enrichment of our circRNA eQTLs among GWAS of substance abuse.