Reports of research work funded by grants prior to 2013
Institute of Environmental Science & Research Ltd
A further investigation of candidate miRNAs involved in obesity and type 2 diabetes mellitus in adipose tissue and plasma.
AM Jones, R Stubbs and DP MacCartney-Coxson
Type two diabetes mellitus (T2DM) and obesity are diseases of epidemic proportions in New Zealand. Gastric bypass surgery results in significant weight-loss and in many cases effective resolution of insulin resistance and T2DM. As a result of real-time PCR validation (WMRF grant awarded to M Hayes/D Macartney-Coxson/R Stubbs) of our miRNA array data we identified three microRNA (miRNA) candidates as having a potential role in these diseases. We aimed to further investigate these candidates by to:
- Determining the localization of candidate miRNA expression in adipose tissue in conjunction with investigating adipose tissue inflammation, and macrophage polarization.
- Investigating candidate miRNA levels in plasma,
Due to the level of funding achieved we decided to concentrate on objective one.
Since 2004 The Wakefield Gastroenterology Research Trust has collected subcutaneous abdominal (SbA) and omental (Om) adipose tissue from 22 patients pre and post Roux-en Y Gastric bypass surgery (RYGB). These samples include 17 females and 5 males. Patients can be clinically subdivided into T2DM (n=7), insulin resistant (n=8) and insulin sensitive (n=7).
In order to perform immunohistochemistry and in situ hybridisation, frozen tissue samples (22 pre SbA, 22 post SbA, 22 pre Om and 22 post Om) were fixed and embedded into paraffin. Then from each paraffin block, 15 adjacent sections were cut on to poly-L-lysine slides. This was carried out off-site at The University of Otago Wellington Medical School as they have the appropriate histological facilities.
In situ hybrdisation of miRNA in formalin fixed tissue is a relatively new and challenging technique. Thus far has we have concentrated on one candidate miRNA (mir-A) to optimize the technique. We performed the hybridisation according to the manufacturers (Exiqon) recommendation using a LNA-based miRNA antisense oligonucleotides labelled with digoxigenin at the 5’ end. A scrambled miRNA-LNA labelled negative control probe and a U6 snRNA positive control was included in each assay. From the literature we decided to use colon cancer sections as a positive control to optimize the in situ protocol. Figure 1: shows expression of mir-A in a) colon cancer and b) expression of the positive control in adipose tissue.
Figure 1. a) mir-223 expression in colon cancer using 60nM of probe hybridised at 60°C. b) U6 snRNA control in adipose tissue. Staining was performed with NBT-BCIP (blue). Sections were counterstained with nuclear red.
Initial indications suggest that mir-A expression is specific to one cell type. However, we need to perform double staining to confirm this.
We also propose to study adipose tissue morphology in more detail with respect to macrophage subtypes M1 and M2 and interpret the expression of our candidate miRNA in this context. We have optimized antibody concentrations for immunohistochemical analyses of CD68, CD163 and 4 other proteins. The immunohistochemistry protocol for CD68 and CD163 works well on adipose tissue sections when di-amino benzidine (DAB) is used as the chromogen. However, immunofluorescent analyses have proved more challenging due to the auto-fluorescence exhibited by adipose tissue. We have identified one possible solution from the literature which we are actively pursuing.
The majority of the initial preparatory work (ie tissue fixing, embedding and sectioning, technique optimization) is complete and we are almost ready to process all the samples. We will initially concentrate on those patient groups in which we saw the greatest expression change of our candidate miRNA. Once complete this work will be written up in conjunction with the initial array and real-time PCR validation and submitted as a manuscript to a peer reviewed journal.