miR Expression Patterns Research Articles

Epigenetic Mechanisms of Aluminum-Induced Neurotoxicity and Alzheimer's Disease: A Focus on Non-Coding RNAs.

Aluminum (Al) is known to induce neurotoxic effects, potentially contributing to Alzheimer's disease (AD) pathogenesis. Recent studies suggest that epigenetic modification may contribute to Al neurotoxicity, although the mechanisms are still debatable. Therefore, the objective of the present study was to summarize existing data on the involvement of epigenetic mechanisms in Al-induced neurotoxicity, especially AD-type pathology. Existing data demonstrate that Al exposure induces disruption in DNA methylation, histone modifications, and non-coding RNA expression in brains. Alterations in DNA methylation following Al exposure were shown to be mediated by changes in expression and activity of DNA methyltransferases (DNMTs) and ten-eleven translocation proteins (TETs). Al exposure was shown to reduce histone acetylation by up-regulating expression of histone deacetylases (HDACs) and impair histone methylation, ultimately contributing to down-regulation of brain-derived neurotrophic factor (BDNF) expression and activation of nuclear factor κB (NF-κB) signaling. Neurotoxic effects of Al exposure were also associated with aberrant expression of non-coding RNAs, especially microRNAs (miR). Al-induced patterns of miR expression were involved in development of AD-type pathology by increasing amyloid β (Aβ) production through up-regulation of Aβ precursor protein (APP) and β secretase (BACE1) expression (down-regulation of miR-29a/b, miR-101, miR-124, and Let-7c expression), increasing in neuroinflammation through NF-κB signaling (up-regulation of miR-9, miR-125b, miR-128, and 146a), as well as modulating other signaling pathways. Furthermore, reduced global DNA methylation, altered histone modification, and aberrant miRNA expression were associated with cognitive decline in Al-exposed subjects. However, further studies are required to evaluate the contribution of epigenetic mechanisms to Al-induced neurotoxicity and/or AD development.

Abstract 17798: Plasma Cardiac miRNA Expression During ST Elevation Myocardial Infarction

Introduction: MicroRNAs (miRs), short sequences of RNA that silence gene expression, have been proposed as biomarkers potentially capable of detecting myocardial infarction earlier and more accurately than troponin. Few studies have assessed expression patterns of cardiac-relevant miRs at the time of ST elevation myocardial infarction (STEMI). Hypothesis: Plasma levels of miRs known to regulate myocardial processes are altered in the plasma of patients early after STEMI onset. Aim: To establish a signature of miRs differentially expressed in plasma of patients with STEMI at the time of presentation. Methods: 10cc blood was obtained at the time of coronary angiography for patients presenting with STEMI(n=9), and plasma was recovered after centrifugation. Patients without coronary heart disease who had undergone coronary angiography served as referent normals(n=5). Following RNA extraction, RT-qPCR was performed to quantify levels of 84 cardiac miRs (Qiagen, Germany) with fold change calculated by the 2 - ΔΔCt method. Results: The mean age was similar between STEMI and referent normal control patients (59±9 vs 60±8), and the mean troponin was numerically higher in the STEMI group (15.4±21 vs 0.00±0; p=0.13). As shown in the table, the expression levels of 25 miRs were significantly increased, and 2 were decreased as compared with control patients (p<0.10). Seven miRs were not detected, and the remaining 47 detectable miRs were not different between the groups(p≥0.10). Cardiac-specific miR-499a-5p and miR-208b-3p demonstrated the greatest fold-change relative to referent normal patients. Conclusions: Cardiac-specific miRs, most notably miR-499a-5p and 208b-3p, as well as many other miRs are differentially expressed early after STEMI onset. Further work is needed to determine whether miRs can detect STEMI earlier or more accurately than troponin or provide incremental diagnostic value for patients presenting with acute coronary syndromes.

The deleted in oral cancer (DOC1 aka CDK2AP1) tumor suppressor gene is downregulated in oral squamous cell carcinoma by multiple microRNAs

Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1; also known as deleted in oral cancer or DOC1) is a tumor suppressor gene known to play functional roles in both cell cycle regulation and in the epigenetic control of embryonic stem cell differentiation, the latter as a core subunit of the nucleosome remodeling and histone deacetylation (NuRD) complex. In the vast majority of oral squamous cell carcinomas (OSCC), expression of the CDK2AP1 protein is reduced or lost. Notwithstanding the latter (and the DOC1 acronym), mutations or deletions in its coding sequence are extremely rare. Accordingly, CDK2AP1 protein-deficient oral cancer cell lines express as much CDK2AP1 mRNA as proficient cell lines. Here, by combining in silico and in vitro approaches, and by taking advantage of patient-derived data and tumor material in the analysis of loss of CDK2AP1 expression, we identified a set of microRNAs, namely miR-21-5p, miR-23b-3p, miR-26b-5p, miR-93-5p, and miR-155-5p, which inhibit its translation in both cell lines and patient-derived OSCCs. Of note, no synergistic effects were observed of the different miRs on the CDK2AP1–3-UTR common target. We also developed a novel approach to the combined ISH/IF tissue microarray analysis to study the expression patterns of miRs and their target genes in the context of tumor architecture. Last, we show that CDK2AP1 loss, as the result of miRNA expression, correlates with overall survival, thus highlighting the clinical relevance of these processes for carcinomas of the oral cavity.

MicroRNA Expression Profile in Mycosis Fungoides – Preliminary Findings

Dear Editor, Mycosis Fungoides (MF) is the commonest cutaneous lymphoma, comprising about 12% of all T-cell lymphomas in India.[1] MF presents as patches, plaques or tumors. While the diagnosis of tumor stage MF is easy, lesions of patch and plaque stages pose problems in diagnosis.[2] There is a clinicopathologic overlap with several inflammatory conditions. Currently, there is no single diagnostic standard for early MF. It rests on a combination of clinical findings, histopathology, immunohistochemistry and T-cell receptor clonality testing.[3] miRNAs(miR) have great potential as biomarkers as they are robust, tissue-specific and can be obtained from both blood and tissues. Their value as diagnostic markers have been shown in several cancer types but is still nascent in MF.[4] We attempted to explore the role of miRs in patch and plaque stage MF in Indian patients. We selected 24 MF samples whose diagnosis was established based on clinical features, histopathology and follow up. We chose a set of 7miRs namely miR 203, miR205, miR223, miR155, miR 93, miR 92a, miR 21 along with two controls RNU48 and RUN44. The first three miRs have a tumor suppressor function, while the rest serve as OncomiRs. RNU44, 48 served as controls. Primer sequences designed using Primer3Plus software and then validated on Ensemble & NCBI Blast. After deparaffinisation and overnight incubation with proteinase K, RNA was extracted using RNAwiz and estimated by Ribogreen fluorimetry, the yield ranged from 5.06 to 49.6 ng. Real time PCR was done on the 26 samples on LightCycler 480 (Roche). Pre-incubation and initial denaturation of the template was at - 95°C for 10 min and amplification was done for 45 cycles at 95°C for 15 sec and 60°C for 1 min and Ct values were obtained. There were 24 samples from 21 patients. Their age ranged from 24-83 years and were predominantly men (14:7). There were 8 patch stage MF, 9 plaque stage and 7 were recurrent MF. There was no significant difference in the expression of any of the 7 miRs between the patch and plaque stages (p-values ranged from 0.423-0.963). There was no difference between patch/plaque stages and recurrent lesions. (p-values ranged from 0.481-0.963). We compared the average Ct values of the 3 tumor suppressor miRs versus the 4 OncomiRs, to look for any difference between patch, plaque and recurrent lesions. There was no significant difference in these either. (p-value ranged from 0.877 to 1.484) A few studies have focussed on the differential expression of miRs in distinguishing MF from inflammatory diseases. van Kester et al studied 19 tumor stage MF and compared their miR expression patterns with 12 benign skin conditions.[5] They found that 49 miR were differentially expressed in the two groups and miR 93, which has an anti-apoptotic function, showed the highest discrimination.[5] Ralfkiaer et al showed that early MF displays a miR profile distinct from advanced T-cell lymphomas.[6] They were also able to demonstrate different profiles in patients with progressive vs. non-progressive disease. McGirt et al demonstrated that miR223, which targets TOX gene, needed for T-cell development is overexpressed in early MF and diminishes with progression to advanced disease.[7] There is only one study which has compared miR profiles in patch vs. plaque stages of MF. Sorensen et al found 11 miRNAs were overexpressed in plaques as compared to patches, most significantly miR-142, miR-21 and miR-22.[8] We did not find any difference in miR (including miR21) expression between patch and plaque stages or in patients who had recurrences. Variation in results could be due to the methodology used (whole tissue vs. microarray), quantity and quality of RNA and density of the infiltrate.[8] Nevertheless, it seems that although clinically distinct, patch and plaque stage MF are more similar biologically. We will expand the scope to compare miR expression profiles of MF with various inflammatory skin diseases in the next part of our ongoing study. To summarise, miR expression does not appear to be useful in distinguishing patch vs. plaque stage MF. Our findings need to be validated in larger samples in Indian patients. Financial support and sponsorship Rajiv Gandhi University of Health Sciences, Bangalore for funding the project Conflicts of interest There are no conflicts of interest.

Identification of circulating miRNAs differentially expressed in patients with Limb-girdle, Duchenne or facioscapulohumeral muscular dystrophies

BackgroundLimb-girdle muscular dystrophy (LGMD) is a rare neuromuscular disease including a growing and heterogeneous number of subtypes with variable phenotype. Their clinical and histopathological characteristics frequently overlap with other neuromuscular dystrophies. Our goal was to identify, by a non-invasive method, a molecular signature including biochemical and epigenetic parameters with potential value for patient prognosis and stratification.ResultsCirculating miRNome was obtained by smallRNA-seq in plasma from LGMD patients (n = 6) and matched-controls (n = 6). Data, validated by qPCR in LGMD samples, were also examined in other common muscular dystrophies: Duchenne (DMD) (n = 5) and facioscapulohumeral muscular dystrophy (FSHD) (n = 4). Additionally, biochemical and clinical parameters were analyzed. miRNome analysis showed that thirteen differentially expressed miRs could separate LGMD vs control group by hierarchical clustering. Most of differentially expressed miRs in LGMD patients were up-regulated (miR-122-5p, miR-122b-3p, miR-6511a-3p, miR-192-5p, miR-574-3p, mir-885-3p, miR-29a-3p, miR-4646-3p, miR-203a-3p and miR-203b-5p) whilst only three of sequenced miRs were significantly down-regulated (miR-19b-3p, miR-7706, miR-323b-3p) when compared to matched controls. Bioinformatic analysis of target genes revealed cell cycle, muscle tissue development, regeneration and senescence as the most affected pathways. Four of these circulating miRs (miR-122-5p, miR-192-5p, miR-19b-3p and miR-323b-3p), together with the myomiR miR-206, were further analysed by qPCR in LGMD, DMD and FSHD. The receiver operating characteristic curves (ROC) revealed high area under the curve (AUC) values for selected miRs in all groups, indicating that these miRs have good sensitivity and specificity to distinguish LGMD, DMD and FSHD patients from healthy controls. miR-122-5p, miR-192-5p and miR-323-3p were differentially expressed compared to matched-controls in all groups but apparently, each type of muscular dystrophy showed a specific pattern of miR expression. Finally, a strong correlation between miRs and biochemical data was only found in LGMD patients: while miR-192-5p and miR-122-5p negatively correlated with CK, miR-192-5p positively correlated with vitamin D3 and ALP.ConclusionsAlthough limited by the small number of patients included in this study, we propose here a specific combination of circulating miR-122-5p/miR-192-5p/miR-323-3 and biochemical parameters as a potential molecular signature whose clinical value for LGMD patient prognosis and stratification should be further confirmed in a larger cohort of patients.

Differences in MicroRNA Expression in Pericoronary Adipose Tissue in Coronary Artery Disease Compared to Severe Valve Dysfunction.

Pericoronary adipose tissue (PCAT) is a source of microRNAs (miRs) that act as messengers for intercellular communication. We investigated whether the PCAT surrounding significant coronary atherosclerotic lesions shows specific miR expression patterns compared with PCAT surrounding plaque-free segments. We included 49 patients with 3-vessel coronary artery disease (CAD) and 19 patients with severe valvular disease but no CAD, who underwent elective cardiac surgery. The PCAT was harvested from two sites: adjacent to a significant atherosclerotic coronary lesion and from plaque-free segments. miR-133a, miR-21, miR-26b, miR-9, and miR-143 levels in PCAT cells were quantified by real-time reverse transcription polymerase chain reaction (data expressed as arbitrary units). Expression of miR-133, miR-21, and miR-26b in adipose tissue at a site without atherosclerotic lesion was much lower in patients with CAD than in those without CAD (0.82 ± 1.37 vs 1.86 ± 0.52, P < .001, 0.45 ± 1.3 vs 1.51 ± 1.11, P < .001, 0.3 ± 1.25 vs 1.2 ± 0.73, P = .02, respectively). In addition, miR-133, miR-21, and miR-143 in CAD patients showed significantly greater expression in PCAT from atherosclerotic lesion compared with plaque-free segments (1.32 ± 0.96 vs 0.82 ± 0.37 (P = .011), 0.91 ± 1.7 vs 0.3 ± 1.25 (P = .012), 1.2 ± 1.59 vs 0.43 ± 0.54 (P < .001), respectively). Our findings open new perspectives for the role of PCAT in the pathophysiology of atherosclerosis and should be further investigated.

Differences in microRNA expression in pericoronary tissue adjacent to atherosclerotic plaque and from lesion-free coronary artery

Abstract Background Pericoronary adipose tissue (PCAT) plays a key role in the pathogenesis of atherosclerosis. PCAT is a source of microRNAs (miRs) that acts as messengers for intercellular communication. In this study, we investigated whether the PCAT surrounding coronary occlusive atherosclerotic lesions shows specific miR expression patterns compared to PCAT surrounding plaque-free segments. Purpose To evaluate the expression of miRs that are known to be implicated in the pathophysiology of atherosclerosis. Methods We included 49 patients (38 men, age 65±9 years) with 3-vessel coronary artery disease, who underwent elective coronary bypass surgery. The PCAT was harvested from two sites: adjacent to an occlusive atherosclerotic coronary lesion, and a plaque-free segment. miR-133a, miR-21, miR-26b, miR-9 and miR-143 levels in PCAT cells were quantified by real-time reverse transcription polymerase chain reaction. Results miR-26b, miR-21 and miR-143 showed significantly greater expression in PCAT samples taken from around atheromatous plaque (154±295 versus 37±96, p=0.014, for miR-26b; 122±265 versus 29±79, p=0.014, for miR-21; and 181±356 versus 31±50, p=0.03, for miR-143) (Figure 1). In addition, the expression of miR-143, miR-26b and miR-21 in PCAT from atherosclerotic plaque showed robust positive correlations with body.mass index (r=0.451, p=0.001, r=0.41, p=0.03 and r=0.416, p=0.003 respectively), Conclusions Analysis of PCAT located in proximity to atherosclerotic plaque showed a different pattern of miR expression compared to PCAT in a plaque-free segment. Our findings open new perspectives for the role of PCAT in the pathophysiological mechanisms of atherosclerotic disease and should be further investigated. Funding Acknowledgement Type of funding sources: None.

Integrative small and long RNA omics analysis of human healing and nonhealing wounds discovers cooperating microRNAs as therapeutic targets.

MicroRNAs (miR), as important epigenetic control factors, reportedly regulate wound repair. However, our insufficient knowledge of clinically relevant miRs hinders their potential therapeutic use. For this, we performed paired small and long RNA-sequencing and integrative omics analysis in human tissue samples, including matched skin and acute wounds collected at each healing stage and chronic nonhealing venous ulcers (VUs). On the basis of the findings, we developed a compendium (https://www.xulandenlab.com/humanwounds-mirna-mrna), which will be an open, comprehensive resource to broadly aid wound healing research. With this first clinical, wound-centric resource of miRs and mRNAs, we identified 17 pathologically relevant miRs that exhibited abnormal VU expression and displayed their targets enriched explicitly in the VU gene signature. Intermeshing regulatory networks controlled by these miRs revealed their high cooperativity in contributing to chronic wound pathology characterized by persistent inflammation and proliferative phase initiation failure. Furthermore, we demonstrated that miR-34a, miR-424, and miR-516, upregulated in VU, cooperatively suppressed keratinocyte migration and growth while promoting inflammatory response. By combining miR expression patterns with their specific target gene expression context, we identified miRs highly relevant to VU pathology. Our study opens the possibility of developing innovative wound treatment that targets pathologically relevant cooperating miRs to attain higher therapeutic efficacy and specificity.

Comprehensive Analysis of microRNA Expression During the Progression of Colorectal Tumors

BackgroundNo effective early diagnostic biomarkers are available for colorectal cancer (CRC). Therefore, we sought to identify new biomarkers that could identify CRC from progression as a pre-cancerous lesion to its invasive form. Recent studies have shown that microRNAs (miRs) are associated with the onset of cancer invasion and progression.AimsWe hypothesized that the identification of miRs associated with CRC might be useful to detect this disease at early stages.MethodsWe conducted an integrated analysis of 79 isolated colorectal tumor glands, including adenomas, intramucosal cancers, and invasive CRCs that showed a microsatellite stable phenotype using GeneChip miRNA 4.0 microarray assays. The colorectal tumors we examined were divided into 2 cohorts (42 in the first cohort and 37 in the second cohort).ResultsFirst, cluster analysis was performed to stratify expression patterns of multiple miRs that were pooled according to the following criteria: fold change in expression (< −2.0 or > 2.0), p < 0.05, and mature miRs. As a result, the expression patterns of pooled miRs were subdivided into 3 subgroups that were correlated with tumor grade. Each subgroup was characterized by specific miRs. In addition, we found that specific miRs, including miR-140-3p and miR-378i, were closely associated with cancer invasion. Finally, we analyzed paired dysregulated miRs between adenomatous and cancerous components present within the same tumor.DiscussionWe showed that several miRs were dysregulated during progression from adenoma to intramucosal cancer. Specific miRs may have key roles in progression from intramucosal tumor to invasive CRC.

Abstract 12403: Type 1 Diabetes Alters Estrogen-Stimulated microRNA Expression and Impairs Nitric Oxide Production in Human Coronary Artery Endothelial Cells

Introduction: Women developed CAD about 1 decade after men. This advantage is erased in T1DM women, and the mechanisms remain unclear. Estrogen (E2) stimulates endothelial NO production via estrogen receptors, and microRNA (miR) have emerged as critical regulators of endothelial function. Whether E2 stimulation of NO production is impaired in T1DM and whether E2 stimulates different miR expression patterns are unknown. Hypothesis: E2-stimulated NO production will be impaired in T1DM endothelial cells which will also express differences in miR expression with E2 exposure. Methods: All experiments were done with and without incubation with 1 nM 17β-estradiol for 24 hours. MicroRNA was isolated from culture human coronary artery endothelial cells (HCAECs) from a woman with and a woman without T1DM. MicroRNA was isolated from HCAECs with four different exposure conditions (T1DM ± 1 nM 17β-estradiol for 24 hr and non-T1DM± 1 nM 17β-estradiol for 24 hr, N=4 biological replicates for all exposures). Fold changes in expression comparing T1DM and non-T1DM cells with and without estradiol stimulation were generated at significance testing performed using DESeq2 with FDR set at 5%. NO production was measured in HCAECs under the same 4 conditions using DAF2-DA and HPLC. Results: 249 miRs were differentially expressed inT1DM HCAECs compared to non-T1DM HCAECs in non-E2 stimulated samples. In E2-stimulated samples, 201 miRs were differentially expressed. Cross-referencing these comparisons showed 16 miRs differentially expressed in T1DM HCAECs relative to non-T1DM HCAECs both with and without E2 stimulation. 4 of these (miRs 548-ar-3p, 625-3p, 369-3p, and 376a-5p) were significantly changed in the opposite direction with estrogen stimulation compared to unstimulated HCAECs, suggesting changes in the expression were due to changes in E2 signaling in T1DM HCAECs. Further, estradiol stimulation of T1DM HCAECS demonstrates a significantly blunted NO production compared to non-T1DM HCAECs (N=6, P&lt;0.05). Conclusions: E2-stimulated NO production is impaired and E2-stimulated changes in miR expression differ in the T1DM endothelium. Further work is necessary to determine whether these differences in miR expression impact E2-stimulated NO production in T1DM.

Uncovering the Roles of MicroRNAs in Major Depressive Disorder: From Candidate Diagnostic Biomarkers to Treatment Response Indicators.

Major depressive disorder (MDD) is a recurrent debilitating illness that represents a major health burden due to its increasing worldwide prevalence, unclear pathological mechanism, nonresponsive treatment, and lack of reliable and specific diagnostic biomarkers. Recently, microRNA species (miRs) have gained particular interest because they have the ability to post-transcriptionally regulate gene expression by modulating mRNA stability and translation in a cohesive fashion. By regulating entire genetic circuitries, miRs have been shown to have dysregulated expression levels in blood samples from MDD patients, when compared to healthy subjects. In addition, antidepressant treatment (AD) also appears to alter the expression pattern of several miRs. Therefore, we critically and systematically reviewed herein the studies assessing the potential biomarker role of several candidate miRs for MDD, as well as treatment response monitoring indicators, in order to enrich the current knowledge and facilitate possible diagnostic biomarker development for MDD, which could aid in reducing both patients’ burden and open novel avenues toward a better understanding of MDD neurobiology.

Transcriptomics and Other Omics Approaches to Investigate Effects of Xenobiotics on the Placenta.

The conceptus is most vulnerable to developmental perturbation during its early stages when the events that create functional organ systems are being launched. As the placenta is in direct contact with maternal tissues, it readily encounters any xenobiotics in her bloodstream. Besides serving as a conduit for solutes and waste, the placenta possesses a tightly regulated endocrine system that is, of itself, vulnerable to pharmaceutical agents, endocrine disrupting chemicals (EDCs), and other environmental toxicants. To determine whether extrinsic factors affect placental function, transcriptomics and other omics approaches have become more widely used. In casting a wide net with such approaches, they have provided mechanistic insights into placental physiological and pathological responses and how placental responses may impact the fetus, especially the developing brain through the placenta-brain axis. This review will discuss how such omics technologies have been utilized to understand effects of EDCs, including the widely prevalent plasticizers bisphenol A (BPA), bisphenol S (BPS), and phthalates, other environmental toxicants, pharmaceutical agents, maternal smoking, and air pollution on placental gene expression, DNA methylation, and metabolomic profiles. It is also increasingly becoming clear that miRNA (miR) are important epigenetic regulators of placental function. Thus, the evidence to date that xenobiotics affect placental miR expression patterns will also be explored. Such omics approaches with mouse and human placenta will assuredly provide key biomarkers that may be used as barometers of exposure and can be targeted by early mitigation approaches to prevent later diseases, in particular neurobehavioral disorders, originating due to placental dysfunction.

Sputum microRNA-screening reveals Prostaglandin EP3 receptor as selective target in allergen-specific immunotherapy.

Several microRNAs (miRs) have been described as potential biomarkers in liquid biopsies and in the context of allergic asthma, while therapeutic effects on the airway expression of miRs remain elusive. In this study, we investigated epigenetic miR-associated mechanisms in the sputum of grass pollen-allergic patients with and without allergen-specific immunotherapy (AIT). Induced sputum samples of healthy controls (HC), AIT-treated and -untreated grass pollen-allergic rhinitis patients with (AA) and without asthma (AR) were profiled using miR microarray and whole-transcriptome microarray analysis of the same samples. miR targets were predicted in silico and used to identify inverse regulation. Local PGE2 levels were measured using ELISA. Two hundred and fifty ninemiRs were upregulated in the sputum of AA patients compared with HC, while only one was downregulated. The inverse picture was observed in induced sputum of AIT-treated patients: while 21miRs were downregulated, only 4miRs were upregulated in asthmatics upon AIT. Of these 4miRs, miR-3935stood out, as its predicted target PTGER3, the prostaglandin EP3 receptor, was downregulated in treated AA patients compared with untreated. The levels of its ligand PGE2 in the sputum supernatants of these samples were increased in allergic patients, especially asthmatics, and downregulated after AIT. Finally, local PGE2 levels correlated with ILC2 frequencies, secreted sputum IL-13levels, inflammatory cell load, sputum eosinophils and symptom burden. While profiling the sputum of allergic patients for novel miR expression patterns, we uncovered an association between miR-3935 and its predicted target gene, the prostaglandin E3 receptor, which might mediate AIT effects through suppression of the PGE2 -PTGER3 axis.

A Set of 17 microRNAs Common for Brain and Cerebrospinal Fluid Differentiates Primary Central Nervous System Lymphoma from Non-Malignant Brain Tumors.

The diagnosis of primary central nervous system (CNS) lymphoma, which is predominantly of the diffuse large B-cell lymphoma type (CNS DLBCL), is challenging. MicroRNAs (miRs) are gene expression-regulating non-coding RNAs that are potential biomarkers. We aimed to distinguish miR expression patterns differentiating CNS DLBCL and non-malignant CNS diseases with tumor presentation (n-ML). Next generation sequencing-based miR profiling of cerebrospinal fluids (CSFs) and brain tumors was performed. Sample source-specific (CSF vs. brain tumor) miR patterns were revealed. Even so, a set of 17 miRs differentiating CNS DLBCL from n-ML, no matter if assessed in CSF or in a tumor, was identified. Along with the results of pathway analyses, this suggests their pathogenic role in CNS DLBCL. A combination of just four of those miRs (miR-16-5p, miR-21-5p, miR-92a-3p, and miR-423-5p), assessed in CSFs, discriminated CNS DLBCL from n-ML samples with 100% specificity and 67.0% sensitivity. Analyses of paired CSF-tumor samples from patients with CNS DLBCL showed significantly lower CSF levels of miR-26a, and higher CSF levels of miR-15a-5p, miR-15b-5p, miR-19a-3p, miR-106b-3p, miR-221-3p, and miR-423-5p. Noteworthy, the same miRs belonged to the abovementioned set differentiating CNS DLBCL from non-malignant CNS diseases. Our results not only add to the basic knowledge, but also hold significant translational potential.

Identification of unique microRNA expression patterns in bone marrow hematopoietic stem and progenitor cells after hemorrhagic shock and multiple injuries in young and old adult mice.

After severe trauma, the older host experiences more dysfunctional hematopoiesis of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs), and dysfunctional differentiation of circulating myeloid cells into effective innate immune cells. Our main objective was to compare BM HSPC microRNA (miR) responses of old and young mice in a clinically relevant model of severe trauma and shock. C57BL/6 adult male mice aged 8 to 12 weeks (young) and 18 to 24 months (old) underwent multiple injuries and hemorrhagic shock (polytrauma [PT]) that engenders the equivalent of major trauma (Injury Severity Score, >15). Pseudomonas pneumonia (PNA) was induced in some young and old adult mice 24 hours after PT. MicroRNA expression patterns were determined from lineage-negative enriched BM HSPCs isolated from PT and PT-PNA mice at 24 and 48 hours postinjury, respectively. Genome-wide expression and pathway analyses were also performed on bronchoalveolar lavage (BAL) leukocytes from both mouse cohorts. MicroRNA expression significantly differed among all experimental conditions (p < 0.05), except for old-naive versus old-injured (PT or PT-PNA) mice, suggesting an inability of old mice to mount a robust early miR response to severe shock and injury. In addition, young adult mice had significantly more leukocytes obtained from their BAL, and there were greater numbers of polymorphonuclear cells compared with old mice (59.8% vs. 2.2%, p = 0.0069). Despite increased gene expression changes, BAL leukocytes from old mice demonstrated a more dysfunctional transcriptomic response to PT-PNA than young adult murine BAL leukocytes, as reflected in predicted upstream functional pathway analysis. The miR expression pattern in BM HSPCs after PT (+/-PNA) is dissimilar in old versus young adult mice. In the acute postinjury phase, old adult mice are unable to mount a robust miR HSPC response. Hematopoietic stem and progenitor cell miR expression in old PT mice reflects a diminished functional status and a blunted capacity for terminal differentiation of myeloid cells.

In Vivo Stimulation of α- and β-Adrenoceptors in Mice Differentially Alters Small RNA Content of Circulating Extracellular Vesicles.

When myocardial function is compromised as in heart failure (HF), there is activation of the sympathetic nervous system with elevated circulating catecholamine levels. These catecholamines activate cardiac and extra-cardiac adrenergic receptors (ARs). Interest in secreted extracellular vesicles (EVs) from the heart is growing and in HF, it is not known whether excessive activation of α- or β-adrenergic receptors (ARs) could induce specific changes in EV content. In this study, we have evaluated, by next generation sequencing, the small RNA content, including micro-RNAs (miRs), of circulating EVs of mice exposed to chronic selective α- or β- AR stimulation. EVs from mouse blood were purified by differential ultracentrifugation resulting in EVs with an average size of 116.6 ± 4.8 nm that by immunoblotting included protein markers of EVs. We identified the presence of miRs in blood EVs using miR-21-5p and -16-5p real-time PCR as known constituents of blood exosomes that make up a portion of EVs. We next performed next generation sequencing (NGS) of small non-coding RNAs found in blood EVs from mice following 7 days of chronic treatment with isoproterenol (ISO) or phenylephrine (PE) to stimulate α- or β-ARs, respectively. PE increased the percent of genomic repeat region reads and decreased the percent of miR reads. In miR expression analysis, PE and ISO displayed specific patterns of miR expression that suggests differential pathway regulation. The top 20 KEGG pathways predicted by differential expressed miRs show that PE and ISO share 11 of 20 pathways analyzed and reveal also key differences including three synapse relative pathways induced by ISO relative to PE treatment. Both α-and β-AR agonists can alter small RNA content of circulating blood EVs/exosomes including differential expression and loading of miRs that indicate regulation of distinct pathways. This study provides novel insight into chronic sympathetic nervous system activation in HF where excessive catecholamines may not only participate in pathological remodeling of the heart but alter other organs due to secretion of EVs with altered miR content.

Valproic acid combined with cisplatin-based chemoradiation in locally advanced head and neck squamous cell carcinoma patients and associated biomarkers.

BackgroundCisplatin-based chemoradiation (CCRT) offers locally advanced head and neck squamous cell carcinoma (LAHNSCC) patients high local control rate, however, relapses are frequent. Our goal was to evaluate if association of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, with CCRT improved response rate (RR) and associated biomarkers.MethodsThis phase II trial included patients with unresectable locally advanced (LA) oropharynx (OP) squamous cell carcinoma. CCRT began after 2 weeks of VPA (P1). Primary goal was RR at 8 weeks after chemoradiation (CRT)+VPA (P2). Biomarkers included microRNA (miR) polymerase chain reaction (PCR)-array profiling in plasma compared to healthy controls by two-sample t-test. Distribution of p-values was analysed by beta-uniform mixture. Findings were validated by real-time PCR quantitative polymerase chain reaction (qPCR) for selected miRs in plasma and saliva. p16, HDAC2 and RAD23 Homolog B, Nucleotide Excision Repair Protein (HR23B) tumour immunohistochemistry were evaluated.ResultsGiven significant toxicities, accrual was interrupted after inclusion of ten LA p16 negative OP patients. All were male, smokers/ex-smokers, aged 41–65 and with previous moderate/high alcohol intake. Nine evaluable patients yielded a RR of 88%. At false discovery rate of 5%, 169 miRs were differentially expressed between patients and controls, including lower expression of tumour suppressors (TSs) such as miR-31, -222, -let-7a/b/e and -145. miR-let-7a/e expression was validated by qPCR using saliva. A HDAC2 H-score above 170 was 90% accurate in predicting 6-month disease-free survival.ConclusionsVPA and CRT offered high RR; however, with prohibitive toxicities, which led to early trial termination. Patients and controls had a distinct pattern of miR expression, mainly with low levels of TS miRs targeting Tumor protein P53 (TP53). miR-let-7a/e levels were lower in patients compared to controls, which reinforces the aggressive nature of such tumours (NCT01695122).

Abstract 17239: miRNAs Secreted by Exosomes Derived From Adipose Tissue Mesenchymal Stem Cells Overexpressing GATA-4 Increase Endothelial Cell Survival and Promote Angiogenesis

Exosomes (EXO) secreted from stem cells play a critical role in cell based therapy and appear to have greater salutary therapeutic effects than whole cells. Our previous study showed that EXO produced by GATA-4-overexpressing mesenchymal stem cells (MSCs) significantly promoted angiogenesis and protected cardiomyocytes resulting in improved ischemic myocardial function. In the present studies, we compared the miR expression pattern and determined whether Exo GATA-4 delivered miRs particularly promoted angiogenesis. Methods and Results: EXO were collected from conditioned medium of adipose tissue derived MSCs that were transfected with GATA-4 (Exo GATA-4 ) or with vector-empty (Exo null ) using ultracentrifugation ( Fig A ). Functional analysis showed that Exo GATA-4 significantly increased tube formation, migration, and proliferation of human umbilical vein endothelial cells (HUVECs) compared to Exo null . MicroRNA-seq data showed that 7 of the 230 miRs found in EXO, were significantly upregulated in Exo GATA-4 ( Fig B ). To determine the effect of upregulated miRs on Exo GATA-4 mediated angiogenesis, miR-155 and miR-6240 mimic, inhibitor, and negative control were directly transferred into HUVECs. After 48 hours, HUVECs were harvested for measuring endothelial cell behavior. Transfection of miR-6240-mimic significantly increased HUVECs proliferation, tolerance against H 2 O 2 induced injury ( Fig C ), tube-like structure formation ( Fig D ) and cell migration ( Fig E ) compared to those transfected with negative control. Concurrently, no significant differences were found in proliferation, survival, and tube-like structure formation among the cells transfected with miR-155-mimic and negative control, although miR-155-mimic significantly increased cell migration. Conclusion: The upregulated miRs in Exo GATA-4 may variably regulate endothelial cell behavior and the role of various miRs needs to be investigated individually.

Identification of Unique mRNA and miRNA Expression Patterns in Bone Marrow Hematopoietic Stem and Progenitor Cells After Trauma in Older Adults.

Older adults have significantly worse morbidity and mortality after severe trauma than younger cohorts. The competency of the innate immune response decreases with advancing age, especially after an inflammatory insult. Subsequent poor outcomes after trauma are caused in part by dysfunctional leukocytes derived from the host's hematopoietic stem and progenitor cells (HSPCs). Our objective was to analyze the bone marrow (BM) HSPC transcriptomic [mRNA and microRNA (miR)] responses to trauma in older and younger adults. BM was collected intraoperatively <9 days after initial injury from trauma patients with non-mild injury [ISS ≥ 9] or with shock (lactate ≥ 2, base deficit ≥ 5, MAP ≤ 65) who underwent operative fixation of a pelvic or long bone fracture. Samples were also analyzed based on age (<55 years and ≥55 years), ISS score and transfusion in the first 24 h, and compared to age/sex-matched controls from non-cancer elective hip replacement or purchased healthy younger adult human BM aspirates. mRNA and miR expression patterns were calculated from lineage-negative enriched HSPCs. 924 genes were differentially expressed in older trauma subjects vs. age/sex-matched controls, while 654 genes were differentially expressed in younger subjects vs. age/sex-matched control. Only 68 transcriptomic changes were shared between the two groups. Subsequent analysis revealed upregulation of transcriptomic pathways related to quantity, function, differentiation, and proliferation of HSPCs in only the younger cohort. miR expression differences were also identified, many of which were associated with cell cycle regulation. In summary, differences in the BM HSPC mRNA and miR expression were identified between older and younger adult trauma subjects. These differences in gene and miR expression were related to pathways involved in HSPC production and differentiation. These differences could potentially explain why older adult patients have a suboptimal hematopoietic response to trauma. Although immunomodulation of HSPCs may be a necessary consideration to promote host protective immunity after host injury, the age related differences further highlight that patients may require an age-defined medical approach with interventions that are specific to their transcriptomic and biologic response. Also, targeting the older adult miRs may be possible for interventions in this patient population.

An omic approach to congenital diaphragmatic hernia: a pilot study of genomic, microRNA, and metabolomic profiling.

The omic approach can help identify a signature that can be potentially used as biomarkers in babies with congenital diaphragmatic hernia (CDH). To find a specific microRNA (miR) and metabolic fingerprint of the tracheal aspirates (TA) of CDH patients. We conducted a genetic analysis from blood samples. TA samples collected in the first 48 h of life in patients with CDH, compared with age-matched controls. Metabolomics done by a mass spectroscopy-based assay. Genomics done using chromosomal microarray analysis. CDH (n = 17) and 16 control neonates enrolled. miR-16, miR-17, miR-18, miR-19b, and miR-20a had an increased expression, while miR-19a had a twofold decreased expression in CDH patients, compared with age-matched control patients. Specific metabolites separated neonates with CDH from controls. A genetic mutation found in a small subset of patients. Specific patterns of metabolites and miR expression can be discerned in TA samples in infants with CDH.