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/hypothesis MicroRNAs (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. Methods Microarray-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. Results Expression 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/interpretation Our 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.

Obesity 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. We 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. Of ~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. Our 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.