The role of microRNA 146b in modulating inflammation in human adipose tissue

In obesity fat becomes more inflamed, which contributes to the development of complications of obesity such as diabetes and heart disease. MicroRNAs are small molecules which modulate gene expression within cells. Our group has shown that the levels of one microRNA, mir146b, increase in fat with obesity. Mir146b has been suggested to increase in response to inflammation and then act to suppress inflammation in other tissues, and so I hypothesise that it regulates inflammation within fat tissue, and therefore may be a treatment target for the complications of obesity. This study will explore how mir146b works and the size of its effect. Experiments will aim to confirm that mir146b suppresses inflammation in cell cultures, identify the targets that mir146b acts on, and assess whether it has a clinical effect by looking at mir146b levels in obese people with or without metabolic complications of obesity.

Obesity and its associated metabolic complications represent a major public health issue. There is evidence that inflammation within adipose tissue in obesity predisposes to complications including insulin resistance and diabetes, with the toll-like signaling pathway playing a key role in this. MicroRNAs (miRNAs) are small non-coding RNAs which post-transcriptionally regulate gene expression. Based on strong and consistent pilot data derived from multiple microarray experiments with confirmatory RT-PCR, we have identified microRNA-146b (mir146b) as being strongly regulated in human adipose tissue. Mir146b is postulated to negatively regulate the toll-like signalling pathway. I will test the hypothesis that mir146b modulates inflammation within adipose tissue, and that the magnitude of this effect is clinically significant.
1. In vitro work will study the impact of mir146b on inflammatory signalling in adipocytes and macrophages using mir146b transfection and inhibition.
2. Traditional bioinformatics based miRNA target prediction algorithms have limited sensitivity and specificity, so I will complement these with an experimental approach in order to predict the mRNA targets of mir146b. A variant of ribonucleoprotein immunoprecipitation-microarray (RIP-Chip) will be used. The miRNA, mRNA and Argonate protein within the RISC complex will be covalently cross linked using UV irradiation within cells in which mir146b is inhibited or in control cells. This will then be immunoprecipitated and the mRNA fraction purified and subjected to microarray analysis. This approach will be complemented by the validation of a selection of targets with luciferase reporter assays.
3. To verify the clinical significance of mir146b I will examine targeted cohorts of obese subjects with low and high adipose tissue inflammation phenotypes recruited from the general population using the Oxford Biobank. I will characterise subjects in terms of adipose tissue and whole body inflammation and inflammatory signalling, metabolic parameters including insulin resistance and mir146b expression, in order to test the hypothesis that mir146b negatively regulates adipose tissue inflammation, and therefore the complications of obesity, in vivo.