Changes In miRNA Expression Patterns Research Articles

Forced expression of microRNA-221-3p exerts protective effects against manganese-induced cytotoxicity in human lung epithelial cells

Manganese (Mn)-induced pulmonary toxicity and the underlying molecular mechanisms remain largely enigmatic. Further, in recent years, microRNAs (miRNAs) have emerged as regulators of several pollutants-mediated toxicity. In this context, our study aimed at elucidating whether miRNAs are involved in manganese (II) chloride (MnCl2) (Mn2+)-induced cytotoxicity in lung epithelial cells. Growth inhibition of Mn2+ towards normal human bronchial epithelial (BEAS-2B) and adenocarcinomic human alveolar basal epithelial (A549) cells was analyzed by MTT assay following 24 or 48 h treatment. Reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm), cell cycle arrest, and apoptosis were evaluated by flow cytometry. RT-qPCR and Western blot were performed to analyze the expression of cyclins, anti-oxidant genes, and miRNAs. We used small RNA sequencing to investigate Mn2+-induced changes in miRNA expression patterns. In both cell lines, Mn2+ treatment inhibited growth in a dose-dependent manner. Further, compared with vehicle-treated cells, Mn2+ (250 μM) treatment induced ROS generation, cell cycle arrest, apoptosis, and decreased ΔΨm as well as altered the expression of cyclins and anti-oxidant genes. Sequencing data revealed that totally 296 miRNAs were differentially expressed in Mn2+-treated cells. Among them, miR-221-3p was one of the topmost down-regulated miRNAs in Mn2+-treated cells. We further confirmed this association in A549 cells. In addition, transient transfection was performed to study gain-of-function experiments. Forced expression of miR-221-3p significantly improved cell viability and reduced Mn2+-induced cell cycle arrest and apoptosis in BEAS-2B cells. In conclusion, miR-221-3p may be the most likely target that accounts for the cytotoxicity of Mn2+−exposed lung epithelial cells.

Recent allopolyploidy alters Spartina microRNA expression in response to xenobiotic-induced stress.

Environmental contamination by xenobiotics represents a major threat for natural ecosystems and public health. In response, xenobiotic detoxification is a fundamental trait of organisms for developmental plasticity and stress tolerance, but the underlying molecular mechanisms remain poorly understood in plants. To decipher this process, we explored the consequences of allopolyploidy on xenobiotic tolerance in the genus Spartina Schreb. Specifically, we focused on microRNAs (miRNAs) owing to their central function in the regulation of gene expression patterns, including responses to stress. Small RNA-Seq was conducted on the parents S. alterniflora and S. maritima, their F1 hybrid S. x townsendii and the allopolyploid S. anglica under phenanthrene-induced stress (phe), a model Polycyclic Aromatic Hydrocarbon (PAH) compound. Differentially expressed miRNAs in response to phe were specifically identified within species. In complement, the respective impacts of hybridization and genome doubling were detected, through changes in miRNA expression patterns between S. x townsendii, S. anglica and the parents. The results support the impact of allopolyploidy in miRNA-guided regulation of plant response to phe. In total, we identified 17 phe-responsive miRNAs in Spartina among up-regulated MIR156 and down-regulated MIR159. We also describe novel phe-responsive miRNAs as putative Spartina-specific gene expression regulators in response to stress. Functional validation using Arabidopsis (L.) Heynh. T-DNA lines inserted in homologous MIR genes was performed, and the divergence of phe-responsive miRNA regulatory networks between Arabidopsis and Spartina was discussed.

A PHYLOGENETIC APPROACH TO EVALUATE THE RELATION BETWEEN miRNA EXPRESSIONS AND HEPATITIS C VIRUS IN HEPATOCELLULAR CARCINOMA

Objective: Hepatitis C virus(HCV) infection increases the risk for Hepatocellular carcinoma (HCC) development, which is the sixth most common cancer type worldwide. HCV-related HCC has high mortality rates and HCV genotype is shown to affect survival in HCC. Two molecules that are known to be associated with poor cancer prognosis in HCC are endoplasmic reticulum transmembrane stromal interaction molecule 1(STIM1) and plasma membrane calcium channel(Orai1). Materials and Methods: In this study, we have enhanced STIM1 and/or Orai1 expressions in subpopulation of cancer-stem-cell-like cells in Huh7 HCC cell line in order to mimic poor cancer prognosis and performed next generation sequencing on small RNA samples isolated from these cells in order to identify the changes in miRNA expression patterns. Multiple alignment and phylogenetic analysis were used on stem loop sequences of miRNAs with significant expression changes and tried to identify a possible evolutionary relation of these miRNAs with HCV genotypes. Results: Our results showed that there were 5 different regions in HCV genome where these miRNAs are mostly aligned. miR4521, miR3651, miR7974 and miR6715a showed closed relations with HCV-genotypes-1 and -7 within these regions. These miRNAs also known to be related with cancer progression in several cancer types including HCC. Conclusion: We propose that a phylogenetic approach can be helpful to evaluate the evolutionary strategies of cancer promoting viruses as well as possible viral origins of miRNAs that play role in cancer progression. This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) [Grant Number:113S399].

Molecular and docking studies of tetramethoxy hydroxyflavone compound from Artemisia absinthium against carcinogens found in cigarette smoke

Abstract Artemisia absinthium (AA) is an indigenous medicine used for treatment of inflammation of the liver and chronic fever, and is studied as an antimalarial and anticancer agent. The focus of the current investigation was to determine the action and effect of AA on microRNAs (miRNAs) from breast cancer cell lines. Molecular docking is a structure-based drug design process that studies the interaction of small molecule ligands with receptor biomacromolecules to predict binding mechanism and affinity. MiRNA expression profiling was done using microarray technology. Validation of transcripts with regulated expression pattern was done by SYBR-based quantitative real time PCR (qRT-PCR). AutoDock 4.2 programming allots polar hydrogens, bound together total Kollman charges, solvation borders, and fragmental volumes to the protein using auto dock devices in docking research (ADT). As confirmed by SYBR-based RT-PCR, our investigation discovered an upregulation of the miRNA-22 articulation and a downregulation of miRNA-199a*. These findings support and demonstrate the role of AA as a miRNA articulation-influencing factor in human breast cancer progression. AA’s tetramethoxy hydroxyflavone (p7F) molecule was found to be effective in the treatment of cancer. Changes in miRNA expression patterns could be a key pathogenic component in AA’s physiological action on cancer cells.

Circulating microRNAs as biomarkers of environmental exposure to polycyclic aromatic hydrocarbons: potential and prospects.

Exposure to polycyclic aromatic hydrocarbons (PAHs) produced from various pyrogenic and petrogenicsources in the environment has been linked to a variety of toxic effects in the human body. Genome-wide analyses have shown that microRNAs (miRNAs) can function as novel and minimally invasive biomarkers of environmental exposure to PAHs. The objective of this study is to explore miRNA signatures associated with early health effects in response to chronic environmental exposure to PAHs. We systematically searched Scopus and PubMed databases for studies related to exposure of PAHs with changes in miRNA expression patterns that represent early health effects in the exposed population. Based on previous studies, we included 15 cell-based and 9 each of animal model and human population-based studies for assessment. A total of 11 differentially expressed PAH-responsive miRNAs were observed eachin two or morecell-based studies (miR-181a and miR-30c-1), animal model studies (miR-291a and miR-292), and human population-based studies (miR-126, miR-142-5p, miR-150-5p, miR-24-3p, miR-27a-3p, miR-28-5p, and miR-320b). In addition, miRNAs belonging to family miR-122, miR-199, miR-203, miR-21, miR-26, miR-29, and miR-92 were found to be PAH-responsivein both animal model and cell-based studies; let-7, miR-126, miR-146, miR-30, and miR-320 in both cell-based and human population-based studies; and miR-142, miR-150, and miR-27 were found differentially expressedin both animal model and human population-based studies. The only miRNA whose expression was found to be altered in all the three groups of studies is miR-34c. Association of environmental exposure to PAHs with altered expression of specific miRNAs indicates that selective miRNAs can be used as early warning biomarkers in PAH-exposed population.

MiRNAs As Possible Predictors For Training Efficacy

Recent research suggests that genes encoding micro RNA (miRNA) molecules are differentially expressed in response to training. PURPOSE: To determine whether specific miRNAs serve as possible predictors for training efficacy. METHODS: Six healthy, sedentary female subjects (mean ± SD: age 23.2 ± 3.1 years, peak oxygen uptake (VO2peak) 31.7 ± 1.6 mL·kg-1·min-1, body mass index 23.0 ± 1.2 kg/m2), aged between 20 and 27 years, performed exercise training on a cycle ergometer, three times a week, for 6 weeks. Participants were randomly assigned to either moderate intensity continuous training (MICT, n=3) or high intensity interval training (HIIT, n=3). The MICT group performed 60 min of continuous cycling at the power output (PO) calculated as corresponding to 90% of the first lactate threshold (LT). The HIIT group performed a warm-up for 10 min at the PO calculated as corresponding to 70% of the maximal heart rate (HRmax), followed by four 4-min intervals at the PO calculated as corresponding to 90% of HRmax, with 4-min recovery periods at 30 W in between. Muscle biopsies were taken pre- and post-training from the vastus lateralis muscle, followed by isolation of total RNA. Samples were then analyzed using a commercial miRNA array. RESULTS: Participants increased their VO2peak after 6 weeks of training by 4.2 mL·kg-1·min-1 in mean (SD: 0.9). MICT and HIIT induced significant changes in miRNA expression patterns, part of which were specific for one of the two training regimens. For instance, there was a significant (p<0.006), 5.5-fold upregulation of the anti-apoptotic miRNA 21-5p in both training groups. Furthermore, we could identify distinct shift in miRNA patterns that correlated with exercise-induced changes in physiological parameters, such as changes in VO2peak (delta VO2peak vs delta expression of miRNA 503, r=-0.9, p=0.01) or microvascular properties. CONCLUSIONS: Our data suggest that MICT and HIIT exert distinct, but also overlapping effects on miRNA expression patterns. In addition, basal miRNA expression patterns might be associated with the individual response to training. Furthermore, the data are currently reproduced using semi-quantitative RT-PCR (qPCR). Our results might have important implications for the development of personalized exercise recommendations and therapeutic strategies.

Common miRNA Patterns of Alzheimer's Disease and Parkinson's Disease and Their Putative Impact on Commensal Gut Microbiota.

With the rise of Next-Generation-Sequencing (NGS) methods, Micro-RNAs (miRNAs) have achieved an important position in the research landscape and have been found to present valuable diagnostic tools in various diseases such as multiple sclerosis or lung cancer. There is also emerging evidence that miRNAs play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD). Apparently, these diseases come along with changes in miRNA expression patterns which led to attempts from researchers to use these small RNA species from several body fluids for a better diagnosis and in order to observe disease progression. Additionally, it became evident that microbial commensals might play an important role for pathology development and were shown to have a significantly different composition in patients suffering from neurodegeneration compared with healthy controls. As it could recently be shown that secreted miRNAs are able to enter microbial organisms, it is conceivable that the host’s miRNA might affect the gut microbial ecosystem. As such, miRNAs may inherit a central role in shaping the “diseased microbiome” and thereby mutually act on the characteristics of these neurodegenerative diseases. We have therefore (1) compiled a list of miRNAs known to be associated with AD and/or PD, (2) performed an in silico target screen for binding sites of these miRNA on human gut metagenome sequences and (3) evaluated the hit list for interesting matches potentially relevant to the etiology of AD and or PD. The examination of protein identifiers connected to bacterial secretion system, lipopolysaccharide biosynthesis and biofilm formation revealed an overlap of 37 bacterial proteins that were targeted by human miRNAs. The identified links of miRNAs to the biological processes of bacteria connected to AD and PD have yet to be validated via in vivo experiments. However, our results show a promising new approach for understanding aspects of these neurodegenerative diseases in light of the regulation of the microbiome.

Modeling Transcriptional Rewiring in Neutrophils Through the Course of Treated Juvenile Idiopathic Arthritis

Neutrophils in children with the polyarticular form of juvenile idiopathic arthritis (JIA) display abnormal transcriptional patterns linked to fundamental metabolic derangements. In this study, we sought to determine the effects of therapy on mRNA and miRNA expression networks in polyarticular JIA. Using exon and miRNA microarrays, we studied children with untreated active JIA (ADU, n = 35), children with active disease on therapy with methotrexate ± etanercept (ADT, n = 26), and children with inactive disease also on therapy (ID, n = 14). We compared the results to findings from healthy control children (HC, n = 35). We found substantial re-ordering of mRNA and miRNA expression networks after the initiation of therapy. Each disease state was associated with a distinct transcriptional profile, with the ADT state differing the most from HC, and ID more strongly resembling HC. Changes at the mRNA level were mirrored in changes in miRNA expression patterns. The analysis of the expression dynamics from differentially expressed genes across three disease states indicated that therapeutic response is a complex process. This process does not simply involve genes slowly correcting in a linear fashion over time. Computational modeling of miRNA and transcription factor (TF) co-regulatory networks demonstrated that combinational regulation of miRNA and TF might play an important role in dynamic transcriptome changes.

MiR-204-5p and miR-211-5p Contribute to BRAF Inhibitor Resistance in Melanoma.

Melanoma treatment with the BRAF V600E inhibitor vemurafenib provides therapeutic benefits but the common emergence of drug resistance remains a challenge. We generated A375 melanoma cells resistant to vemurafenib with the goal of investigating changes in miRNA expression patterns that might contribute to resistance. Increased expression of miR-204-5p and miR-211-5p occurring in vemurafenib-resistant cells was determined to impact vemurafenib response. Their expression was rapidly affected by vemurafenib treatment through RNA stabilization. Similar effects were elicited by MEK and ERK inhibitors but not AKT or Rac inhibitors. Ectopic expression of both miRNA in drug-naïve human melanoma cells was sufficient to confer vemurafenib resistance and more robust tumor growth in vivo Conversely, silencing their expression in resistant cells inhibited cell growth. Joint overexpression of miR-204-5p and miR-211-5p durably stimulated Ras and MAPK upregulation after vemurafenib exposure. Overall, our findings show how upregulation of miR-204-5p and miR-211-5p following vemurafenib treatment enables the emergence of resistance, with potential implications for mechanism-based strategies to improve vemurafenib responses.Significance: Identification of miRNAs that enable resistance to BRAF inhibitors in melanoma suggests a mechanism-based strategy to limit resistance and improve clinical outcomes. Cancer Res; 78(4); 1017-30. ©2017 AACR.

Effect of aberrant expression of miR-421, miR-29b_1_5p and miR-27b_5p on gastric adenoma development.

77 Background: Gastric cancer (GC) is the fourth leading cause of cancer-related death in the world. Some gastric adenomas may progress to adenocarcinoma in a short time. Several studies have examined adenomas the intermediate step of gastric carcinoma development. MicroRNAs have an important role in progression of gastric cancer but less is known about their role in premalignant adenomas. Changes in the expression pattern of miRNAs can be informative and highly significant in the gastric adenoma-carcinoma sequence progression as well. However, to date precancerous lesions (such as adenomas) have been investigated less frequently than cancers compared to healthy tissues. The aim of the present study was to perform a miRNA microarray analysis of normal, low-grade dysplasia, and high-grade dysplasia using fresh frozen tissues. Changes in miRNA expression patterns were also verified from samples obtained from the same patients using TaqMan MicroRNA Assays. Methods: Human tissue samples were obtained from nine patients who underwent endoscopic submucosal dissection (ESD) due to gastric adenoma or early gastric cancer at Severance Hospital of Yonsei University. Tumor and non-tumor mucosa tissue in each patient were taken just before ESD using biopsy forceps and frozen in the nitrogen tank. The miRNA expression profiles were analyzed by Affymetrix Gene-Chip miRNA 4.0 array (Homo sapiens). qRT-PCR was performed using TaqMan MicroRNA Assays (Applied Biosystems). Results: There were 28 (up-regulation) and 10 (down-regulation) miRNAs which showed significantly different expression between control group and low-/high-grade dysplasia group. miRNAs showing altered expression in normal and low-/high-grade dysplasia by microarrays were selected, and the expression tendencies were detected by real-time PCR validation. Three miRNAs (miR-421, miR-29b-1-5p, miR-27b-5p) were selected the continually upregulated from normal to low-/high-grade dysplasia based qRT-PCR in tissue samples. Conclusions: We identified new, less known miRNAs (miR-421, miR-29b-1-5p, miR-27b-5p), with altered expression in different grade dysplasia compare with normal samples.

Regulatory roles of micro-RNAs in T cell autoimmunity

ABSTRACTMicroRNAs (miRNAs) are short conserved non-coding RNA molecules that have recently emerged as post-transcriptional gene expression regulators. miRNAs degrade or repress target mRNAs by targeting the 3ʹ UTR region. miRNA-mediated gene regulation is crucial for regular biological processes such as immune cell lineage commitment, differentiation, maturation, and maintenance of immune homeostasis and regular function. Changes in miRNA expression patterns have been recorded in a wide range of human diseases, including autoimmunity, especially in T cell-related autoimmunity. This review discusses recent progress in miRNA-mediated regulation of T cell-related autoimmunity. Specifically, we discuss the modality of T cell-related autoimmunity regulation by miRNAs through affecting molecular and biological processes.

Tissue-Specific Expression Patterns of MicroRNA during Acute Graft-versus-Host Disease in the Rat.

MicroRNAs (miRNA) have emerged as central regulators of diverse biological processes and contribute to driving pathology in several diseases. Acute graft-versus-host disease (aGvHD) represents a major complication after allogeneic hematopoietic stem cell transplantation, caused by alloreactive donor T cells attacking host tissues leading to inflammation and tissue destruction. Changes in miRNA expression patterns occur during aGvHD, and we hypothesized that we could identify miRNA signatures in target tissues of aGvHD that may potentially help understand the underlying molecular pathology of the disease. We utilized a rat model of aGvHD with transplantation of fully MHC-mismatched T cell depleted bone marrow, followed by infusion of donor T cells. The expression pattern of 423 rat miRNAs was investigated in skin, gut, and lung tissues and intestinal T cells with the NanoString hybridization platform, in combination with validation by quantitative PCR. MHC-matched transplanted rats were included as controls. In the skin, upregulation of miR-34b and downregulation of miR-326 was observed, while in the intestines, we detected downregulation of miR-743b and a trend toward downregulation of miR-345-5p. Thus, tissue-specific expression patterns of miRNAs were observed. Neither miR-326 nor miR-743b has previously been associated with aGvHD. Moreover, we identified upregulation of miR-146a and miR-155 in skin tissue of rats suffering from aGvHD. Analysis of intestinal T cells indicated 23 miRNAs differentially regulated between aGvHD and controls. Two of these miRNAs were differentially expressed either in skin (miR-326) or in intestinal (miR-345-5p) tissue. Comparison of intestinal and peripheral blood T cells indicated common dysregulated expression of miR-99a, miR-223, miR-326, and miR-345-5p. Analysis of predicted gene targets for these miRNAs indicated potential targeting of an inflammatory network both in skin and in the intestines that may further regulate inflammatory cytokine production. In conclusion, comprehensive miRNA profiling in rats suffering from aGvHD demonstrate tissue-specific differences in the expression patterns of miRNA that may not be detected by profiling of peripheral blood T cells alone. These tissue-specific miRNAs may contribute to distinct pathologic mechanisms and could represent potential targets for therapy.

Hyperglycemia-induced changes in miRNA expression patterns in epicardial adipose tissue of piglets.

MicroRNAs (miRNAs) are a class of molecular posttranscriptional regulators found to participate in numerous biological mechanisms, such as adipogenesis, fat deposition, or glucose metabolism. Additionally, a detailed analysis on the molecular and cellular mechanisms of miRNA-related effects on metabolism leads to developing novel diagnostic markers and therapeutic approaches. To identify miRNA whose activity changed in epicardial adipose tissue in piglets during hyperglycemia, we analyzed the different miRNA expression patterns between control and hyperglycemia groups. The microarray analysis selected three differentially expressed microRNAs as potential biomarkers: hsa-miR-675-5p, ssc-miR-193a-3p, and hsa-miR-144-3p. The validation of miRNA expression with real-time PCR indicated an increased expression levels of ssc-miR-193a-3p and miR-675-5p, whereas the expression level of hsa-miR-144-3p was lower in epicardial adipose tissue in response to hyperglycemia (P<0.01). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses suggested that these miRNAs differentially expressed between hyperglycemic and control piglets are involved in insulin, adipocytokine, and phosphatidylinositol 3-kinase-Akt signaling pathways, and development of type 2 diabetes as well. The results suggested that hyperglycemia can significantly affect the expression patterns of miRNA in porcine adipose tissue.

Changes in miRNA expression patterns and vascular aging phenotypes in mice with developmental liver‐specific knockdown of IGF‐1

The endocrine changes that occur during development are highly conserved across mammalian species and include dramatic increases in the anabolic hormone IGF‐1 during adolescence. There is increasing experimental and clinical evidence that alterations in IGF‐1 levels during development regulate multiple aspects of the aging process and affect the incidence of multiple age‐related diseases, including cardiovascular and cerebrovascular diseases. The present study was designed to characterize the effect of developmental IGF‐1 deficiency on the vascular aging phenotype. To achieve that goal early‐onset, isolated endocrine IGF‐1 deficiency was induced mice by developmental knockdown of IGF‐1 specifically in the liver using Cre‐lox technology (Igf1f/f mice crossed with mice expressing albumin‐driven Cre recombinase). The animals were studied at an age representing ~75% of maximal lifespan potential, which corresponds to the biological age of a ~67 year old human. To assess vascular health endothelium‐dependent vasorelaxation and vascular ROS production were tested. Due to the emergence of miRNAs as important regulators of vascular aging phenotype, miRNA expression profile in the aorta of mice with developmental IGF‐1 deficiency was also tested. We found that aging was associated with endothelial dysfunction, which was unaffected by developmental IGF‐1 deficiency. We identified 19 miRNAs that were differentially expressed in the aortas of aged mice and 11 miRNAs that were differentially expressed miRNAs in aortas of mice with developmental IGF‐1 deficiency. GO terms enriched among predicted targets for miRNAs differentially expressed with age in the aorta include cytokine production, chromatin silencing, intracellular protein transport and regulation of apoptotic cell death. GO terms enriched among predicted targets for miRNAs differentially expressed with developmental IGF‐1 deficiency in the aorta include regulation of apoptotic cell death, blood vessel remodeling and intracellular protein transport. Collectively, developmental IGF‐1 deficiency is associated with a vascular miRNA expression profile later in life that likely plays a role in development of age‐related vascular pathologies.Support or Funding InformationThis work was supported by grants from the American Heart Association and the National Institute on Aging

T-cell Activation Induces Dynamic Changes in miRNA Expression Patterns in CD4 and CD8 T-cell Subsets.

T-cell activation affects microRNA (miRNA) expression in T-cell subsets. However, little is known about the kinetics of miRNA regulation and possible differences between CD4 and CD8 T cells. In this study we set out to analyze the kinetics of activation-induced expression regulation of twelve pre-selected miRNAs. The dynamics of the expression of these miRNAs was studied in sorted CD4 and CD8 CD45RO- T cells of healthy individuals stimulated with αCD3/αCD28 antibodies. Analysis of miRNA levels at day 3, 5, 7 and 10 showed significant activation-induced changes in expression levels of all twelve miRNAs. Expression levels of nine miRNAs, including miR-21, miR-146a and miR-155, were induced following activation, whereas expression of three miRNAs, including miR-31, were decreased following activation. The expression changes of miR-18a and miR-155 was relatively early, at day 3, whereas expression of miR-451, miR-21 and miR-146a was evident at day 5, 7 and 10, respectively. Four miRNAs showed a differential regulation between CD4 and CD8 T cells. Induction of miR-18a and miR-21 was more pronounced and occurred earlier in CD4 T cells compared to CD8 T cells. Downregulation of miR-223 and miR-451 was also more pronounced in CD4 T cells compared to CD8 T cells. In conclusion, we show a complex pattern of miRNA expression regulation upon T-cell activation with early and late as well as CD4 and CD8 T-cell specific changes. These differences might be the result of differences in kinetics and efficiency of CD4 and CD8 T cells in response to antigen priming.

Melanoma cells with acquired resistance to dabrafenib display changes in miRNA expression pattern and respond to this drug with an increase of invasiveness, which is abrogated by inhibition of NF-κB or the PI3K/mTOR signalling pathway

Melanoma cells with acquired resistance to dabrafenib display changes in miRNA expression pattern and respond to this drug with an increase of invasiveness, which is abrogated by inhibition of NFB or the PI3K/mTOR signalling pathway Simona Caporali, Ester Alvino, Adriana Amaro, Pedro Miguel Lacal, Lauretta Levati, Maria Grazia Atzori, Gian Carlo Antonini Cappellini, Federica Ruffini, Enzo Bonmassar, Ulrich Pfeffer, Stefania D’Atri

MiRNAs as novel therapeutic targets and diagnostic biomarkers for Parkinson’s disease: a patent evaluation of WO2014018650

Introduction: Parkinson’s disease (PD) requires novel means for early-stage recognition, enabling early therapeutic approaches. Since microRNAs (miRNAs) are important components of the gene regulatory networks in neurodegenerative diseases, there is huge effort to find correlation between expression patterns of miRNAs and disease.Areas covered: The current application claims the use of miRNA signatures to diagnose and treat PD also in early nonsymptomatic stages. Therefore, samples from putamen and CSF were collected to identify miRNA signatures in PD. Resulting microarrays provide expression patterns of miRNAs that were highly predictive for different stages of PD pathology. Different kits are provided to determine miRNA signatures and to classify the individual stage of disease. Furthermore, an oligonucleotide is suggested, with the aim to downregulate an miRNA, predicted to play a causal role in the development of PD pathology.Expert opinion: The asymptomatic early phase in PD is associated with specific changes in miRNA expression patterns. However, further work is required to assess the real diagnostic and therapeutic potential of this application’s findings. Targeting individual miRNAs is a strategy worth to be investigated, but many technical issues still need to be addressed.

Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms.

BackgroundMicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Next-generation sequencing (NGS) is increasingly employed in biomedical investigations. Although concordance between this platform and qRT-PCR based assays has been reported in high quality specimens, information is lacking on comparisons in biofluids especially urine. Here we describe the changes in miRNA expression patterns in a rodent model of renal tubular injury (gentamicin). Our aim is to compare RNA sequencing and qPCR based miRNA profiling in urine specimen from control and rats with confirmed tubular injury.ResultsOur preliminary examination of the concordance between miRNA-seq and qRT-PCR in urine specimen suggests minimal agreement between platforms probably due to the differences in sensitivity. Our results suggest that although miRNA-seq has superior specificity, it may not detect low abundant miRNAs in urine samples. Specifically, miRNA-seq did not detect some sequences which were identified by qRT-PCR. On the other hand, the qRT-PCR analysis was not able to detect the miRNA isoforms, which made up the majority of miRNA changes detected by NGS.ConclusionsTo our knowledge, this is the first time that miRNA profiling platforms including NGS have been compared in urine specimen. miRNAs identified by both platforms, let-7d, miR-203, and miR-320, may potentially serve as promising novel urinary biomarkers for drug induced renal tubular epithelial injury.

Microrna Expression In Patients With Myelodysplastic Syndromes Treated With Demethylating Agents

IntroductionMyelodysplastic syndromes (MDS) comprise a heterogeneous group of hematopoietic stem cell disorders characterized by bone marrow failure and dysplasia of one or more blood cell lineages resulting in peripheral blood cytopenias with frequent progression to AML. Treatment with azanucleosides improves survival and quality of life without the toxicities associated with intensive chemotherapy programs. The azanucleosides are DNA demethylating agents that may improve hematopoiesis in MDS by releasing tumor suppressor genes from silencing due to promoter hypermethylation. MicroRNAs (miRNAs) play key roles in cell growth and differentiation and oncogenic transformation. Dysregulation of miRNA expression may contribute to the development of MDS, as expression of several miRNAs has been reported to be downregulated in bone marrow cells from MDS patients. We performed the current study to test the hypothesis that treatment with demethylating agents modulates miRNA expression in hematopoietic tissue from MDS patients and to characterize the specific changes in miRNA expression. MethodsThis was a two-center, prospective cohort study to evaluate miRNA expression signatures in hematopoietic progenitor cells isolated from the bone marrow of subjects undergoing evaluation for peripheral blood cytopenias. Subjects were included for analysis if RNA of sufficient quality was available and the final pathologic diagnosis was one of the following: 1) normal (served as controls), 2) dyspoiesis without meeting criteria for MDS and without cytogenetic abnormalities, 3) MDS and 4) acute myeloid leukemia (AML) with myelodysplasia-related changes. Subjects who underwent treatment with a demethylating agent had bone marrow collected after treatment for before and after comparisons of miRNA signatures. Mononuclear cells were isolated from bone marrow aspirate specimens by density gradient centrifugation using a Ficoll-Paque technique. MicroRNA was isolated using the miRNeasy kit (Qiagen) and was analyzed using miScript miRNA PCR Array Human Cell Development & Differentiation (MIHS-103Z, Qiagen). Isolated microRNA was checked for quality control using miScript miRNA QC PCR array. Data were analyzed using Qiagen analysis software for microRNA, and miRNA levels are expressed as 2-ΔΔCT. P-values<0.05 were considered statistically significant. ResultsForty subjects were enrolled in the study. Eight subjects were excluded after the initial bone marrow biopsy for poor quality RNA (n = 3) or inappropriate diagnoses (lymphoid (n = 3) or myeloproliferative (n = 2) neoplasms). The remaining 32 subjects comprised 5 groups: 1) normal (n = 6), 2) dyspoiesis but not meeting criteria for MDS and normal cytogenetics (n = 5), 3) low-intermediate risk MDS (n = 8), 4) high-risk MDS (n = 6) and 5) AML with myelodysplasia-related changes (n = 7). Bone marrow samples were obtained from 5 patients after treatment with azanucleosides. The miRNA expression pattern in subjects with normal bone marrow and in those with no clear morphological evidence of MDS (groups 1 & 2) was similar. Conversely, subjects with low-intermediate risk MDS showed decreased expression of multiple miRNAs compared to controls (> 2 fold decrease in 22/84 miRNAs), most notably miR-126-3p (p = 0.02) and miR-20a-5p (p = 0.04). In contrast, high-risk MDS patients demonstrated a significant overall increase in microRNA expression (>2-fold increase in 33/84 miRNAs), especially miR-20b-5p (p = 0.04) and miR-125b-5p (p = 0.01). Treatment with demethylating agents led to decreased expression of most miRNAs (>2-fold decrease in 14/84 miRNAs), including miR-181a and let-7a, but an increase in others, such as miR-22 (2.3-fold increase). We obtained similar results in experiments conducted with primary bone marrow samples that were treated with demethylating agents ex vivo. SummaryOur study is the first to characterize the changes in miRNA expression patterns in patients with MDS treated with demethylating agents. We also identify novel miRNA candidates to be further evaluated as biomarkers for assessing response to treatment with demethylating agents (e.g., miR-22, which has been described as possibly having a role in regulating TET2). We also demonstrate the striking difference in the pattern of miRNA expression between low-risk and high-risk MDS patients, a feature that may one day be exploited to assist with making treatment decisions. Disclosures:No relevant conflicts of interest to declare.

Abstract 4082: Global miRNA expression changes induced by HDAC-42 and 5-azaCdr in lung cancer cells

Abstract Introduction: Epigenetic alterations are universally seen in lung and other cancers. These include DNA methylation and histone modifications, both pharmacologically targetable abnormalities by DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors. Despite preclinical evidence, epigenetic modifiers have, to date, been ineffective in the treatment of most solid tumors. MicroRNAs are small non-coding RNAs that impact RNA and protein expression through imperfect base pairing in the 3′UTRs of transcribed genes. We investigated the ability of epigenetic drugs to affect the miRNA expression from four lung cancer cell lines, A549, H1299, H719, H520 (representing adeno, large cell, small cell and squamous carcinomas respectively). Materials and Methods: MTT was used to evaluate cell growth, while Western immunoblot and immunofluorescence was used to detect histone H3 acetylation. Annexin-V by flow cytometry was used for apoptosis detection. NanoString was used for the global profiling of miRNAs and corresponding statistical methods was performed by the Biostatistical Core of OSU-CCC. Results: We evaluated the impact of 6 HDAC inhibitors on cell growth of A549 and H1299 cell lines. HDAC-42 was among the most potent drugs and inhibited growth at nM concentrations. Both HDAC-42 and 5-azaCdr increased apoptosis in all 4 cell lines. The most significant synergy between HDAC-42 and 5-azaCdr was in H719 in which apoptosis increased from 4.9 to 38.0% at 500 nM. As expected, synergistic histone H3 acetylation was also seen. Following 72 hour treatment, RNA was harvested and global miRNA expression by NanoString showed that the expression patterns of miRNAs are different among the 4 cell lines after treatment with 200 nM HDAC-42 and 3 µM 5-azaCdr. 5-azaCdr caused more miRNA difference than HDAC-42 (19/3 differential expressed miRNAs in 5-azaCdr treated and HDAC-42 treated H520 cells, 21/6 in H719, 29/9 in H1299, 9/8 in A549). We found that some miRNA changes were common across cell lines in the HDAC-42 treated group, (increased miR129-3p in HDAC-42 treated H1299, H719 and H520 and increased miR148a in HDAC-42 treated H1299, A549 and H719 cells). We also found that 5-azaCdr induced changes in miRNA expression patterns were similar between H520, H719 and H1299 (16 out of 20 miRNAs in H520, 16 out of 21 in H719, 11 out of 29 in H1299). The synergistic activity of 5-azaCdr and HDAC-42 was observed with several individual miRNAs like miR-129-3p and miR-515-5p. Conclusion: Both HDAC-42 and 5-azaCdr could regulate the expression of certain miRNAs in lung cancer cells. The effect of HDAC-42 and 5-azaCdr on four different lung cancer cell lines showed distinct miRNA expression patterns. The role of several candidate miRNAs such as miR-129-3p, miR-515-5p, miR-148a and miR-200c related to acetylation and methylation requires further study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4082. doi:1538-7445.AM2012-4082