Changes In microRNA Expression Research Articles

Exploiting microRNA and mRNA profiles generated in vitro from carcinogen-exposed primary mouse hepatocytes for predicting in vivo genotoxicity and carcinogenicity.

The well-defined battery of in vitro systems applied within chemical cancer risk assessment is often characterised by a high false-positive rate, thus repeatedly failing to correctly predict the in vivo genotoxic and carcinogenic properties of test compounds. Toxicogenomics, i.e. mRNA-profiling, has been proven successful in improving the prediction of genotoxicity in vivo and the understanding of underlying mechanisms. Recently, microRNAs have been discovered as post-transcriptional regulators of mRNAs. It is thus hypothesised that using microRNA response-patterns may further improve current prediction methods. This study aimed at predicting genotoxicity and non-genotoxic carcinogenicity in vivo, by comparing microRNA- and mRNA-based profiles, using a frequently applied in vitro liver model and exposing this to a range of well-chosen prototypical carcinogens. Primary mouse hepatocytes (PMH) were treated for 24 and 48h with 21 chemical compounds [genotoxins (GTX) vs. non-genotoxins (NGTX) and non-genotoxic carcinogens (NGTX-C) versus non-carcinogens (NC)]. MicroRNA and mRNA expression changes were analysed by means of Exiqon and Affymetrix microarray-platforms, respectively. Classification was performed by using Prediction Analysis for Microarrays (PAM). Compounds were randomly assigned to training and validation sets (repeated 10 times). Before prediction analysis, pre-selection of microRNAs and mRNAs was performed by using a leave-one-out t-test. No microRNAs could be identified that accurately predicted genotoxicity or non-genotoxic carcinogenicity in vivo. However, mRNAs could be detected which appeared reliable in predicting genotoxicity in vivo after 24h (7 genes) and 48h (2 genes) of exposure (accuracy: 90% and 93%, sensitivity: 65% and 75%, specificity: 100% and 100%). Tributylinoxide and para-Cresidine were misclassified. Also, mRNAs were identified capable of classifying NGTX-C after 24h (5 genes) as well as after 48h (3 genes) of treatment (accuracy: 78% and 88%, sensitivity: 83% and 83%, specificity: 75% and 93%). Wy-14,643, phenobarbital and ampicillin trihydrate were misclassified. We conclude that genotoxicity and non-genotoxic carcinogenicity probably cannot be accurately predicted based on microRNA profiles. Overall, transcript-based prediction analyses appeared to clearly outperform microRNA-based analyses.

Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells

Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke.

Processing of miR17-92 Cluster in Hepatic Stellate Cells Promotes Hepatic Fibrogenesis During Alcohol-Induced Injury.

Exposure to alcohol and its metabolites can initiate hepatic injury and fibrogenesis. Fibrosis is mediated through hepatic stellate cell (HSC) activation, leading to global changes in mRNA and microRNA (miR) expression. miRs are expressed in cells or shuttled to exosomes which can be detected in tissue culture media (TCM) and biological fluids. The mechanisms and function underlying the differential expression and processing of miRs and their downstream effects during hepatic injury remain poorly understood. Expression of primary (pri)-miR17-92. and individual members of this cluster, miR17a, 18a, 19a, 20a, 19b, and 92, were examined in primary HSCs and human LX2 cells exposed to alcohol-conditioned media (CM), liver tissue from a rodent model of alcoholic injury, and in exosomes from TCM and plasma of rodent models and patients with alcoholic liver disease (ALD). miR expression was examined in HSCs transduced with an AAV2 vector carrying GFP-miR19b or GFP-control transgene under the collagen promoter. Profibrotic markers were enhanced in primary HSCs and LX2 cells exposed to alcohol-CM, concomitant with decreased miR19b expression and a significant increase in pri-miR17-92. Increased pri-miR17-92 was confirmed in a rodent model of alcohol-induced liver injury. Individual members of the cluster were inversely proportionate in cells and exosomes. AAV2-mediated miR19b overexpression inhibited miR17-92 and altered expression of individual cluster members in cells and exosomes. Expression of individual miR17-92 cluster members in plasma exosomes isolated from patients with ALD was similar to that seen in a rodent model of alcoholic injury and invitro. Reintroduction of miR19b inhibits HSC activation and modulates expression of pri-miR17-92 and the inverse expression of individual cluster members in cells and exosomes. Better understanding of miR17-92 processing may provide mechanistic insights into the role of individual miRs and exosomes during hepatic injury, revealing new therapeutic targets.

A microarray study of chronic unpredictable mild stress rat blood serum with electro-acupuncture intervention

In the present study, we investigated the changes of microRNA (miRNA) expression upon depression and electro-acupuncture (EA) intervention in chronic unpredictable mild stress (CUMS) rats using microarray analysis. Results showed that EA intervention remarkably improved behavioral indexes in terms of crossing number, rearing number, sucrose preference and body weight of CUMS rats. Microarray analysis revealed that a total of 153 differentially expressed miRNAs were regulated by CUMS, and the expression of 180 differentially expressed miRNAs was changed after EA intervention. Among these miRNAs, two miRNAs were significantly up-regulated and four miRNAs were significantly down-regulated by CUMS. Moreover, four miRNAs were significantly up-regulated and 12 miRNAs were significantly down-regulated after EA intervention. The expressions of miR-383-5p and miR-764-5p were up-regulated after CUMS, while their expressions were down-regulated by EA intervention. Further analysis showed that 1260 possible target genes were predicted for miR-383-5p and miR-764-5p, and 97 pathways and 137 gene ontology (GO) were involved. Among these pathways and GO, about 20 pathways and 21 GO were related to depression. Changes of miR-383-5p and miR-764-5p indicated that EA might exert its therapeutic effect on depression through promoting the neurotrophy and inhibiting the abnormal apoptosis of neurons as well as other correlative signal pathways. In conclusion, our present study enriched the understanding of pathological process of depression and revealed possible mechanisms of EA on depression.

Long-term exposure of MCF-12A normal human breast epithelial cells to ethanol induces epithelial mesenchymal transition and oncogenic features.

Alcoholism is associated with breast cancer incidence and progression, and moderate chronic consumption of ethanol is a risk factor. The mechanisms involved in alcohol's oncogenic effects are unknown, but it has been speculated that they may be mediated by acetaldehyde. We used the immortalized normal human epithelial breast cell line MCF-12A to determine whether short- or long-term exposure to ethanol or to acetaldehyde, using in vivo compatible ethanol concentrations, induces their oncogenic transformation and/or the acquisition of epithelial mesenchymal transition (EMT). Cultures of MCF-12A cells were incubated with 25 mM ethanol or 2.5 mM acetaldehyde for 1 week, or with lower concentrations (1.0–2.5 mM for ethanol, 1.0 mM for acetaldehyde) for 4 weeks. In the 4-week incubation, cells were also tested for anchorage-independence, including isolation of soft agar selected cells (SASC) from the 2.5 mM ethanol incubations. Cells were analyzed by immunocytofluorescence, flow cytometry, western blotting, DNA microarrays, RT/PCR, and assays for miRs. We found that short-term exposure to ethanol, but not, in general, to acetaldehyde, was associated with transcriptional upregulation of the metallothionein family genes, alcohol metabolism genes, and genes suggesting the initiation of EMT, but without related phenotypic changes. Long-term exposure to the lower concentrations of ethanol or acetaldehyde induced frank EMT changes in the monolayer cultures and in SASC as demonstrated by changes in cellular phenotype, mRNA expression, and microRNA expression. This suggests that low concentrations of ethanol, with little or no mediation by acetaldehyde, induce EMT and some traits of oncogenic transformation such as anchorage-independence in normal breast epithelial cells.

Identification and Analysis of Novel Viral and Host Dysregulated MicroRNAs in Variant Pseudorabies Virus-Infected PK15 Cells.

Pseudorabies (PR) is one of the most devastating diseases in the pig industry. To identify changes in microRNA (miRNA) expression and post-transcriptional regulatory responses to PRV infection in porcine kidney epithelial (PK15) cells, we sequenced a small RNA (sRNA) library prepared from infected PK15 cells and compared it to a library prepared from uninfected cells using Illumina deep sequencing. Here we found 25 novel viral miRNAs by high-throughput sequencing and 20 of these miRNAs were confirmed through stem-loop RT-qPCR. Intriguingly, unlike the usual miRNAs encoded by the α-herpesviruses, which are found clustered in the large latency transcript (LLT), these novel viral miRNAs are throughout the PRV genome like β-herpesviruses. Viral miRNAs are predicted to target multiple genes and form a complex regulatory network. GO analysis on host targets of viral miRNAs were involved in complex cellular processes, including the metabolic pathway, biological regulation, stimulus response, signaling process and immune response. Moreover, 13 host miRNAs were expressed with significant difference after infection with PRV: 8 miRNAs were up-regulated and 5 miRNAs were down-regulated, which may affect viral replication in host cell. Our results provided new insight into the characteristic of miRNAs in response to PRV infection, which is significant for further study of these miRNAs function.

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME).

BackgroundChronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients.MethodsmiRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets.ResultsMicroarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology.ConclusionThis study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function.

Peripubertal Stress With Social Support Promotes Resilience in the Face of Aging.

The peripubertal period of development is a sensitive window, during which adverse experiences can increase the risk for presentation of cognitive and affective dysfunction throughout the lifespan, especially in women. However, such experiences in the context of a supportive social environment can actually ameliorate this risk, suggesting that resilience can be programmed in early life. Affective disorders and cognitive deficits commonly emerge during aging, with many women reporting increased difficulty with prefrontal cortex (PFC)-dependent executive functions. We have developed a mouse model to examine the interaction between peripubertal experience and age-related changes in cognition and stress regulation. Female mice were exposed to peripubertal chronic stress, during which they were either individually housed or housed with social interaction. One year after this stress experience, mice were examined in tasks to access their cognitive ability and flexibility in stress reactive measures. In a test of spatial memory acquisition and reversal learning where aged females normally display a decreased performance, the females that had experienced stress with social interaction a year earlier showed improved performance in reversal learning, a measure of cognitive flexibility. Because peripuberty is a time of major PFC maturation, we performed transcriptomic and biochemical analysis of the aged PFC, in which long-term changes in microRNA expression and in myelin proteins were found. These data suggest that stress in the context of social support experienced over the pubertal window can promote epigenetic reprogramming in the brain to increase the resilience to age-related cognitive decline in females.

The functional consequences of age-related changes in microRNA expression in skeletal muscle.

A common characteristic of ageing is disrupted homeostasis between growth and atrophy of skeletal muscle resulting in loss of muscle mass and function, which is associated with sarcopenia. Sarcopenia is related to impaired balance, increased falls and decline in quality of life of older people. Ageing-related transcriptome and proteome changes in skeletal muscle have been characterised, however the molecular mechanisms underlying sarcopenia are still not fully understood. microRNAs are novel regulators of gene expression known to modulate skeletal muscle development and homeostasis. Expression of numerous microRNAs is disrupted in skeletal muscle with age however, the functional consequences of this are not yet understood. Given that a single microRNA can simultaneously affect multiple signalling pathways, microRNAs are potent modulators of pathophysiological changes occurring during ageing. Here we use microRNA and transcript expression profiling together with microRNA functional assays to show that disrupted microRNA:target interactions play an important role in maintaining muscle homeostasis. We identified miR-181a as a regulator of the sirtuin1 (Sirt1) gene expression in skeletal muscle and show that the expression of miR-181a and its target gene is disrupted in skeletal muscle from old mice. Moreover, we show that miR-181a:Sirt1 interactions regulate myotube size. Our results demonstrate that disrupted microRNA:target interactions are likely related to the pathophysiological changes occurring in skeletal muscle during ageing.Electronic supplementary materialThe online version of this article (doi:10.1007/s10522-016-9638-8) contains supplementary material, which is available to authorized users.

MicroRNA and gene expression changes in unruptured human cerebral aneurysms.

OBJECTIVE The molecular mechanisms behind cerebral aneurysm formation and rupture remain poorly understood. In the past decade, microRNAs (miRNAs) have been shown to be key regulators in a host of biological processes. They are noncoding RNA molecules, approximately 21 nucleotides long, that posttranscriptionally inhibit mRNAs by attenuating protein translation and promoting mRNA degradation. The miRNA and mRNA interactions and expression levels in cerebral aneurysm tissue from human subjects were profiled. METHODS A prospective case-control study was performed on human subjects to characterize the differential expression of mRNA and miRNA in unruptured cerebral aneurysms in comparison with control tissue (healthy superficial temporal arteries [STA]). Ion Torrent was used for deep RNA sequencing. Affymetrix miRNA microarrays were used to analyze miRNA expression, whereas NanoString nCounter technology was used for validation of the identified targets. RESULTS Overall, 7 unruptured cerebral aneurysm and 10 STA specimens were collected. Several differentially expressed genes were identified in aneurysm tissue, with MMP-13 (fold change 7.21) and various collagen genes (COL1A1, COL5A1, COL5A2) being among the most upregulated. In addition, multiple miRNAs were significantly differentially expressed, with miR-21 (fold change 16.97) being the most upregulated, and miR-143-5p (fold change -11.14) being the most downregulated. From these, miR-21, miR-143, and miR-145 had several significantly anticorrelated target genes in the cohort that are associated with smooth muscle cell function, extracellular matrix remodeling, inflammation signaling, and lipid accumulation. All these processes are crucial to the pathophysiology of cerebral aneurysms. CONCLUSIONS This analysis identified differentially expressed genes and miRNAs in unruptured human cerebral aneurysms, suggesting the possibility of a role for miRNAs in aneurysm formation. Further investigation for their importance as therapeutic targets is needed.

CD46 Activation Regulates miR-150–Mediated Control of GLUT1 Expression and Cytokine Secretion in Human CD4+ T Cells

CD46 is a cell surface complement inhibitor widely expressed in human tissues, in contrast to mice, where expression is limited to the testes. In humans, it has been identified as an important T cell costimulatory receptor, and patients deficient in CD46 or its endogenous ligands are unable to mount effective Th1 T cell responses. Stimulation of human CD4(+) T cells with CD3 and CD46 also leads to the differentiation of a "switched" Th1 population, which shuts down IFN-γ secretion and upregulates IL-10 and is thought to be important for negative feedback regulation of the Th1 response. In the present study, we show that CD46 costimulation leads to amplified microRNA (miR) expression changes in human CD4(+) T cells, with associated increases in activation more potent than those mediated by the "classic" costimulator CD28. Blockade of cell surface CD46 inhibited CD28-mediated costimulation, identifying autocrine CD46 signaling as downstream of CD28. We also identify a downregulation of miR-150 in CD46-costimulated T cells and identify the glucose transporter 1 encoding transcript SLC2A1 as a target of miR-150 regulation, connecting miR-150 with modulation of glucose uptake. We also investigated microRNA expression profiles of CD46-induced switched IL-10-secreting Th1 T cells and found increased expression of miR-150, compared with IFN-γ-secreting Th1 cells. Knockdown of miR-150 led to a reduction in IL-10 but not IFN-γ. CD46 therefore controls both Th1 activation and regulation via a miR-150-dependent mechanism.

Changes in MicroRNA Expression During Maturation of the Bovine Corpus Luteum: Regulation of Luteal Cell Proliferation and Function by MicroRNA-34a.

The corpus luteum (CL) develops from the remnants of the ovulatory follicle and produces progesterone, required for maintenance of pregnancy in mammals. The differentiation of granulosal and thecal cells into luteal cells is accompanied by hypertrophy and hyperplasia of cells. As the CL matures, growth ceases and in ruminants, the tissue acquires the ability to undergo regression in response to prostaglandin F2alpha. The regulators of this transition are poorly understood. MicroRNA, which are posttranscriptional regulators of tissue development and function, are expressed in the CL. However, the pattern of their expression and their function during the transition from developing to functional CL is not known. The objectives of this study were to profile the expression of miRNA in developing versus mature bovine CL and determine effects of miRNA on bovine luteal cell survival and function. Knockdown of Drosha in midcycle (MC) luteal cells decreased progesterone and increased luteal cell apoptosis in the presence or absence of proinflammatory cytokines. Microarray analysis demonstrated that a greater number of miRNA were expressed in MC compared to D4 CL. Ingenuity pathway analysis (IPA) predicted that D4-specific miRNA regulate pathways related to carbohydrate metabolism, while MC-specific miRNA regulate pathways related to cell cycle and apoptosis signaling. Both predictions are consistent with a switch in the CL from a growing phase to a maintenance phase. One of the MC specific miRNA, miR-34a, was selected for further analysis. Increased concentrations of miR-34a in MC luteal cells resulted in decreased luteal cell proliferation, increased progesterone production, and inhibition of Notch1 and YY1 translation, but had no effect on luteal cell apoptosis. In conclusion, these data support a role for miRNA in general, and miR-34a in particular, in luteal formation and function.

Global MicroRNA Expression Profiling of Mouse Livers following Ischemia-Reperfusion Injury at Different Stages.

Hepatic ischemia-reperfusion injury is a dynamic process consisting of two stages: ischemia and reperfusion, and triggers a cascade of physiological and biochemical events. Given the important role of microRNAs in regulating gene expression, we analyzed gene expression changes in mouse livers at sham control, ischemia stage, and reperfusion stage. We generated global expression profiles of microRNA and mRNA genes in mouse livers subjected to ischemia-reperfusion injury at the three stages, respectively. Comparison analysis showed that reperfusion injury had a distinct expression profile whereas the ischemia sample and the sham control were clustered together. Consistently, there are 69 differentially expressed microRNAs between the reperfusion sample and the sham control whereas 28 differentially expressed microRNAs between the ischemia sample and the sham control. We further identified two modes of microRNA expression changes in ischemia-reperfusion injury. Functional analysis of both the differentially expressed microRNAs in the two modes and their target mRNAs revealed that ischemia injury impaired mitochondrial function, nutrient consumption, and metabolism process. In contrast, reperfusion injury led to severe tissue inflammation that is predominantly an innate-immune response in the ischemia-reperfusion process. Our staged analysis of gene expression profiles provides new insights into regulatory mechanisms of microRNAs in mouse hepatic IR injury.

Changes in the Expression of MicroRNA in the Buildup Phase of Wasp Venom Immunotherapy: A Pilot Study

Background: Allergen-specific immunotherapy is the most effective method of treatment in allergy to wasp venom. However, its mechanism of action is still not fully understood. The aim of this study is to describe changes in microRNA (miRNA) expression in patients undergoing the buildup phase of venom immunotherapy. Methods: The study group comprised 7 adult patients with a history of severe systemic reactions after stinging by a wasp. In all patients, sensitization to wasp venom had been confirmed by skin tests and serum IgE. The buildup phase of wasp venom immunotherapy (VIT) was conducted according to an ultrarush protocol. In blood samples collected before and 24 h after completing the VIT buildup phase, 740 miRNAs were assessed. Results: Of the 740 miRNAs, 440 were detected in the study group, and in 5 expression was significantly changed after the buildup phase of VIT: miR-370, miR-539, miR-502-3p, miR-299, and miR-29c. Another 62 miRNAs changed 2-fold in some patients (nonsignificant), including increases in miR-143 (stimulating FOXp3 expression) and let-7d (reducing expression of IL-13, IL-6, and TLR4), and decreases in proinflammatory miR-301, miR-146b, miR-106, and miR-485. Conclusions: Several changes in miRNA expression have been found as a result of the buildup phase of wasp VIT, with lower expression of some miRNAs involved in allergic inflammation and higher expression of those possibly involved in tolerance induction. However, the role of the most significant changes is uncertain.

Bioinformatics Analysis of microRNA Time-Course Expression in Brown Rat (Rattus norvegicus): Spinal Cord Injury Self-Repair.

This is a bioinformatic study designed to investigate the time-course expression changes of microRNAs (miRNAs) after spinal cord injury (SCI). To investigate the mechanism of SCI self-repair at miRNAs level and target genes level. SCI results in loss of sensory and locomotor function, and SCI self-repair might provide clinical therapies; however, the mechanism of SCI self-repair remains unclear. The miRNA expression profile (GSE19890) of adult female Wistar brown rats (Rattus norvegicus) in SCI (laminectony and contusion), sham (laminectony but no contusion), and control (untreated) groups was downloaded from Gene Expression Omnibus. Totally, 35 chips were available, including five controls, five SCI-1-day, five SCI-3-day, five SCI-7-day, five sham-1-day, five sham-3-day, and five sham-7-day. Betr and limma package were used to screen time-course differentially expressed miRNAs (DEmiRNAs), followed by Bayesian hierarchical clustering (BHC), synergetic and functional enrichment analysis through BHC and cluster Profiler packages, respectively. Furthermore, STRING database and Cytoscape software were used to construct interaction networks between time-course DEmiRNAs, and GenCLip2.0 software was applied to pathway enrichment for key genes associated with nervous system. Totally, 68 time-course DEmiRNAs were identified and divided into 15 BHC clusters. Then, 100 time-course DEmiRNA pairs with synergetic function were identified, and time-course DEmiRNAs and target genes interaction networks were constructed, in which 10 genes (AKT1, VEGFA, CTNNB1, IGF1, APP, PTEN, CDC42, BDNF, SOD2, and IFNG) with highest degrees were found. Furthermore, key genes were significantly enriched in neurotrophin signaling pathway. After SCI, miRNAs might collectively regulate target genes, facilitating or inhibiting self-repair. Modulation of these miRNAs might provide novel therapies for SCI treatment. N/A.

Expression of microRNA let-7a, miR-155, and miR-205 in tumor and tumor-adjacent histologically normal tissue in patients with non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a main group of lung malignancies. Epigenetic changes are as important as genome structural changes in carcinogenesis. MicroRNA (miRNA) is a class of non-coding single-stranded RNAs that play an important role in the regulation of matrix RNA (mRNA) translation and degradation. MicroRNA expression changes occur in many cancers. According to the field cancerization theory, tumor-adjacent histologically normal tissue takes part in tumor progression by triggering cell transformation. The important clinical implication is that the fields may serve as the basis for a recurrence after surgery. Thus, the aim of our study was to determine the expression levels of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues to evaluate these changes as potential prognostic markers in NSCLC patients. The expression of miRNAs let-7a, miR-155, and miR-205 in tumor and tumor-adjacent apparently normal tissues at 2 and 5 cm was determined by real-time PCR with subsequent quantification using a 2-ΔΔСt method. The findings were then analyzed to reveal possible associations with clinical and morphological parameters, such as age, cancer stage, and tumor grade. The expression of miRNA let-7a was found to be significantly lower in tumor than that in tumor-adjacent apparently normal tissue at 2 and 5 cm. In groups of patients older than 63 years with Stage III-IV NSCLC, the expressions of microRNA let-7a and miR-155 in tumor tissue were substantially lower than that in the adjacent normal tissue. Beyond that point, patients with high-grade tumors had also a significantly lower expression of miRNA let-7a in relatively adjacent apparently normal tissue. The findings suggest that miRNA let-7a and miR-155 may be used as poor prognostic markers for patients with NSCLC.

MiR-181b in Chronic Lymphocytic Leukemia B Cells Is Regulated By Cellular Interaction with CD4+ T Cells and Increases the CTL Toxicity Versus the Leukemic Clone

Abstract Clinical progression of chronic lymphocytic leukemia (CLL) is characterized by gradual reduction of the ratio T/B cells, along with immune cell dysfunction due, at least in part, to T cell defects, such as decreased expression of CD40L and reduced signaling via the TCR CD3. This compromise the ability of T cells to respond and to eliminate leukemic cell from CLL patients. Enhanced activation of either allogenic or autologous T cells can drive the death of CLL cells in vitro and in human subjects. Changes in microRNAs expression also characterize clinical progression of CLL with a strong decrease of miR-181b/a and miR-130a associated with the more aggressive phase of the disease. The miR-181b targets anti-apoptotic proteins, such as BCL-2 and MCL1 and its expression correlates with those protein levels in CLL. In this study we demonstrate that the expression of those microRNAs in CLL-B cells, are regulated by T cells. We co-cultured allogenic pure CLL-B cells with either activated (CD2, CD3 and CD28 antibodies, used to mimic antigen-presenting cells) or not activated CD4+ T cells from healthy donors. We observed a significant increase of mir-181b/a and miR-130a expression in CLL B-cells after co-culture with activated CD4+ T cells in 8 out of 11 cases. A significant increase of these miRs was also determined in purified CLL B-cells after 4 days activation of peripheral blood mononuclear cells (PBMCs) from CLL patients, even if in minor rate. By the use of specific antibodies, co-culture with Hela CD40 expressing cells and transwell experiments, we established that this effect is a T/B contact-dependent signaling mediated through CD40L-CD40 interaction. We determine that increased expression of the 3 miRs occurs at the transcriptional level. Since the expression of miR-181b showed the most significant variation in previous experiments it was selected for further analyses. We next investigated the in vivo role of the miR-181b in highly immunodeficient mice. The CLL cell line, MEC-01, infected with either the LV-miR-181b_coGFP or the LV-CTRL_coGFP was intravenously inoculated in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were sacrificed after 4 weeks and assayed for percentage of GFP+ cells in bone marrow and spleen compartments. The miR-181b did not show any specific effect into the leukemic clone. However when the same cells were inoculated in an environment hosting mature T cells, miR-181b consistently influences the death of leukemic cells (Fig 1B), suggesting that T cells are required to potentiate the apoptotic role of this miRNA. To explain what we observed in vivo, we mixed in vitro MEC-01 infected with either the LV-miR-181b or the LV-CTRL and CD8+ T cells from healthy donors. After few hours of contact T cells showed stronger cytotoxic effect on MEC-01 carrying miR-181b as compared to the control. Mixed lymphocyte reaction CD40L-activated CLL and T cells is used to generate effector CTLs. Therefore we grew T cell with CD40L-activated MEC-01 in which the expression of miR-181b was either shut down by lentiviral vector or unchanged as control. After one week, we monitored by cytofluorimetry the CD38 surface marker on T cells since its expression has been associated with more active CTLs and, by ELISA, the release of IL-10, the inhibitor of the potent inducer of CTLs INF-g. We demonstrate that activated MEC-01 with higher expression of miR-181b leads to an increase of the cell number expressing CD38 and this was accompanied by a reduced release of IL-10 from B cells through down-regulation of c-FOS, which we show to be target of the miR-181b and to promote the transcription of the IL-10. In conclusion, our data suggest a role of the miR-181b in the immune response against CLL-B cells. We show that an efficient activation of CD4+ T cells through CD3-complex pathway and a right CD40L-CD40 interaction lead to a significant increase of the some miRNAs deregulated over the progression of chronic lymphocytic leukemia, namely miR-181b. This miRNA potentiates the cytotoxicity of T cells favoring the killing of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

Radiofrequency ablation can reverse the abnormal circulating microRNA expression changes in patients with atrial fibrillation

To observe the change of circulating microRNAs(miRNAs), regulatory mechanism in patients with atrial fibrillation (AF) before and after radiofrequency ablation (RFA). From January 2011 to December 2013, peripheral blood samples were taken from 30 AF patients (10 paroxysmal, 10 persistent and 10 permanent AF) before and 3 months after RFA. The total RNA was extracted and hybridized with the miRNA chips, and the differential expression of miRNA and clustering analysis in whole genome were made with Volcano Plot and tMEV software respectively, and validated by real-time PCR. The target gene analysis of miRNAs was predicted through the Mirbase, Miranda and Targetscan databases. Results were compared with those from 10 healthy subjects (control group). Compared with control group, the expressions of 25 miRNAs were down-regulated before RFA and up-regulated after RFA in AF group, while other 40 miRNAs expression changed in the opposite way; among them, the expressions of 7 miRNAs including miR-199a-3p/miR-199b-3p were down- regulated >1.5-fold before RFA and up-regulated>100-fold after RFA; oppositely, 6 miRNAs including miR-BART8-3p were up-regulated>1.5-fold before RFA and down-regulated>10-fold after RFA. Interestingly, 6 miRNAs including miR-30b-5p, which were involved in AF-related electrical and structural remodeling, were down-regulated>5-fold before RFA, but up-regulated>50-fold after RFA. Four miRNAs including miR-377-5p, which were involved in the regulation of CACNA1C ICaL channel protein, were different before and after RFA. miRNAs regulate the occurrence and development of AF. RFA can change the expression of miRNAs in AF patients, which may be important for reversing the electrical and structural remodeling and maintaining sinus rhythm after RFA. miRNAs, such as miR-30b-5p, miR-377-5p and miR-199a-3p/miR-199b-3p etc., might become the target markers for early diagnosis and intervention of AF in future.

Chemical composition and “in vitro” anti-inflammatory activity of Vitis vinifera L. (var. Sangiovese) tendrils extract

Grapevine (Vitis vinifera L.) is currently used for wine production, herbal medicine and food supplements. In this study we investigated the phenolic composition of grape tendril extract (TVV), because these compounds are probably responsible for the observed biological effects of TVV. We performed our investigation on cells implicated in the inflammatory process and in vascular damage, such as monocyte (U937) and senescent endothelial (HUVEC) cells. We analysed changes in microRNA (miRNA) expression during a pro-inflammatory condition in LPS (lipopolysaccharide) stimulated cells. MiR-126, defined as angiomiR for its ability in the maintenance of endothelial cell functions, and miR-146a, defined as inflamma-miR, for its ability to down-modulate (NF-kB nuclear factor-kB) pro-inflammatory pathway, were analysed along with their targets. TVV extract (40 µg/mL) was able to prevent the LPS stimulus and may play an important role in the prevention of endothelial dysfunction when the accumulation of senescent endothelial cells occurs.

MicroRNA expression changes after atrial fibrillation catheter ablation.

Atrial fibrillation (AF) is most common arrhythmia in general population, with increasing trend in mortality and morbidity. Electrophysiological and structural abnormalities, promoting abnormal impulse formation and propagation, lead to this disease. AF catheter ablation is related to a not small percentage of nonresponder patients. microRNAs (miRs) have been used as AF fibrotic and electrical alterations biomarkers. miRs may differentiate responders patients to ablative approach. Selective miR target therapy, as upregulation by adenovirus transfection and/or miR downregulation by antagomiR, may be used to treat AF patients. Catheter ablation of triggering electrical pulmonary veins activity or fibrotic areas defragmentation may be upgraded by miR therapy to prevent cardiac electrical and fibrotic remodeling after AF ablation.