MicroRNAs (miRNAs) contribute to both neuronal and immune cell fate, but their involvement in intertissue communication remained unexplored. The brain, via vagal secretion of acetylcholine (ACh), suppresses peripheral inflammation by intercepting cytokine production; therefore, we predicted that microRNAs targeting acetylcholinesterase (AChE) can attenuate inflammation. Here, we report that inflammatory stimuli induced leukocyte overexpression of the AChE-targeting miR-132. Injected locked nucleic acid (LNA)-modified anti-miR-132 oligonucleotide depleted miR-132 amounts while elevating AChE in mouse circulation and tissues. In transfected cells, a mutated 3'UTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice overexpressing 3'UTR null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 upregulation in brain and bone marrow. Our findings identify the AChE mRNA-targeting miR-132 as a functional regulator of the brain-to-body resolution of inflammation, opening avenues for study and therapeutic manipulations of the neuro-immune dialog.

Mental stress modifies both cholinergic neurotransmission and alternative splicing in the brain, via incompletely understood mechanisms. Here, we report that stress changes brain microRNA (miR) expression and that some of these stress-regulated miRs regulate alternative splicing. Acute and chronic immobilization stress differentially altered the expression of numerous miRs in two stress-responsive regions of the rat brain, the hippocampal CA1 region and the central nucleus of the amygdala. miR-134 and miR-183 levels both increased in the amygdala following acute stress, compared to unstressed controls. Chronic stress decreased miR-134 levels, whereas miR-183 remained unchanged in both the amygdala and CA1. Importantly, miR-134 and miR-183 share a common predicted mRNA target, encoding the splicing factor SC35. Stress was previously shown to upregulate SC35, which promotes the alternative splicing of acetylcholinesterase (AChE) from the synapse-associated isoform AChE-S to the, normally rare, soluble AChE-R protein. Knockdown of miR-183 expression increased SC35 protein levels in vitro, whereas overexpression of miR-183 reduced SC35 protein levels, suggesting a physiological role for miR-183 regulation under stress. We show stress-induced changes in miR-183 and miR-134 and suggest that, by regulating splicing factors and their targets, these changes modify both alternative splicing and cholinergic neurotransmission in the stressed brain.

Aims/hypothesisMicroRNAs (miRNAs) are short endogenous RNAs that regulate multiple biological processes including adipogenesis and fat metabolism. We sought to identify miRNAs that correlate with BMI and to elucidate their upstream regulation and downstream targets.MethodsMicroarray-based expression profiling of 233 miRNAs was performed on subcutaneous abdominal adipose tissue biopsies from 29 non-diabetic Pima Indian participants. Correlation of the expression levels of eight miRNAs with BMI was assessed by quantitative reverse transcription (QRT) PCR in adipose samples from 80 non-diabetic Pima Indians with a BMI of 21.6–54.0 kg/m2. The upstream regulation of one of these miRNAs, miR-221, was tested by treating cultured human pre-adipocytes with leptin, TNF-α and insulin. Predicted targets of miR-221 were validated using QRT-PCR, immunoblots and luciferase assays. The downstream effects of miR-221 overexpression were assayed by proteomic analysis.ResultsExpression levels of miR-221 were positively correlated with BMI (particularly in women) and fasting insulin concentrations, while the levels of miR-193a-3p and miR-193b-5p were negatively correlated with BMI; other miRNAs did not show significant associations in the 80 samples. miR-221 was downregulated by leptin and TNF-α treatment in cultured human pre-adipocytes. Conversely, miR-221 overexpression upregulated several proteins involved in fat metabolism, mimicking peroxisome proliferator-activated receptor (PPAR) activation. Furthermore, miR-221 directly downregulated the adiponectin receptor 1 (ADIPOR1) and the transcription factor v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1). Adiponectin signalling is known to promote insulin sensitivity, and ETS1 is crucial for angiogenesis.Conclusions/interpretationOur data suggest that miR-221 may contribute to the development of the insulin resistance that typically accompanies obesity, by affecting PPAR signalling pathways and by directly downregulating ADIPOR1 and ETS1.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-2950-9) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Harpophora maydis, a phytopathogenic fungus, causes late wilt, a severe vascular maize disease characterized by relatively rapid wilting of maize plants near fertilization. The disease is currently controlled using resistant varieties. Here, we evaluated seed coating efficiency with azoxystrobin against H. maydis in a series of in vitro and in vivo trials. A real-time polymerase chain reaction (qPCR)-based method was developed and proved to be a sensitive, accurate tool for monitoring H. maydis DNA inside infected seeds, sprouts, and tissues of mature plants. In the early growth stages, the chemical coating drastically reduced the pathogen DNA prevalence in host tissues and minimized the suppressing effect on the plants’ biomass and development. In an infested field, the qPCR assay identified the pathogen 20 days after seeding, up to a month before conventional PCR detection. In the resistant fodder maize cultivar 32D99, which showed only minor disease symptoms, the seed coating blocked fungal progression and increased cob and plant weight by 39 and 60%, respectively. Nevertheless, this treatment was unable to protect a sensitive maize hybrid, cultivar Prelude, at the disease wilting breakout (60 days after sowing). These results encourage further examination of azoxystrobin and other fungicides in the field using the qPCR detection method to evaluate their efficiency.

BackgroundObesity is a risk factor for colorectal cancer (CRC). Normal and tumor cells respond to metabolic hormones, such as leptin and insulin. Thus, obesity-associated resistance to these hormones likely leads to changes in gene expression and behavior of tumor cells. However, the mechanisms affected by leptin and insulin signaling in CRC cells remain mostly unknown.MethodsWe hypothesized that microRNAs (miRNAs) are involved in the regulation of tumorigenesis-related gene expression in CRC cells by leptin and insulin. To test this hypothesis, miRNA levels in the CRC-derived cell lines HCT-116, HT-29 and DLD-1 were profiled, following leptin and insulin treatment. Candidate miRNAs were validated by RT-qPCR. Predicted miRNA targets with known roles in cancer, were validated by immunoblots and reporter assays in HCT-116 cells. Transfection of HCT-116 cells with candidate miRNA mimic was used to test in vitro effects on proliferation and invasion.ResultsOf ~800 miRNAs profiled, miR-4443 was consistently up-regulated by leptin and insulin in HCT-116 and HT-29, but not in DLD-1, which lacked normal leptin receptor expression. Dose response experiments showed that leptin at 100 ng/ml consistently up-regulated miR-4443 in HCT-116 cells, concomitantly with a significant decrease in cell invasion ability. Transfection with miR-4443 mimic decreased invasion and proliferation of HCT-116 cells. Moreover, leptin and miR-4443 transfection significantly down-regulated endogenous NCOA1 and TRAF4, both predicted targets of miR-4443 with known roles in cancer metastasis. miR-4443 was found to directly regulate TRAF4 and NCOA1, as validated by a reporter assay. The up-regulation of miR-4443 by leptin or insulin was attenuated by the inhibition of MEK1/2.ConclusionsOur findings suggest that miR-4443 acts in a tumor-suppressive manner by down-regulating TRAF4 and NCOA1 downstream of MEK-C/EBP-mediated leptin and insulin signaling, and that insulin and/or leptin resistance (e.g. in obesity) may suppress this pathway and increase the risk of metastatic CRC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2938-1) contains supplementary material, which is available to authorized users.

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AIMTo identify the effect of hydrogen-rich water (HRW) and electrolyzed-alkaline water (EAW) on high-fat-induced non-alcoholic fatty acid disease in mice.METHODSMice were divided into four groups: (1) Regular diet (RD)/regular water (RW); (2) high-fat diet (HFD)/RW; (3) RD/EAW; and (4) HFD/EAW. Weight and body composition were measured. After twelve weeks, animals were sacrificed, and livers were processed for histology and reverse-transcriptase polymerase chain reaction. A similar experiment was performed using HRW to determine the influence and importance of molecular hydrogen (H2) in EAW. Finally, we compared the response of hepatocytes isolated from mice drinking HRW or RW to palmitate overload.RESULTSEAW had several properties important to the study: (1) pH = 11; (2) oxidation-reduction potential of -495 mV; and (3) H2 = 0.2 mg/L. However, in contrast to other studies, there were no differences between the groups drinking EAW or RW in either the RD or HFD groups. We hypothesized that the null result was due to low H2 concentrations. Therefore, we evaluated the effects of RW and low and high HRW concentrations (L-HRW = 0.3 mg H2/L and H-HRW = 0.8 mg H2/L, respectively) in mice fed an HFD. Compared to RW and L-HRW, H-HRW resulted in a lower increase in fat mass (46% vs 61%), an increase in lean body mass (42% vs 28%), and a decrease in hepatic lipid accumulation (P < 0.01). Lastly, exposure of hepatocytes isolated from mice drinking H-HRW to palmitate overload demonstrated a protective effect from H2 by reducing hepatocyte lipid accumulation in comparison to mice drinking regular water.CONCLUSIONH2 is the therapeutic agent in electrolyzed-alkaline water and attenuates HFD-induced nonalcoholic fatty liver disease in mice.

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