Investigating the role of miR-21 in autophagy and VMP1
O'Leary, Kate Marie
Previous work has identified a differentially methylated region (DMR) around the miR-21/VMP1 locus in IBD patients compared to HC and elevated levels of VMP1 and miR-21 have been detected in patients. MiR-21 is a microRNA that is associated with immune related disorders, and VMP1 Is a key regulator of autophagy, a pathway heavily implicated in CD pathogenesis. The aim of this project was to elucidate the role of miR-21 in IBD pathogenesis and whether this is affected through VMP1. There were three main approaches to this question, firstly to establish the genetics of the region in CD, UC and HC and to correlate this to the expression of miR-21, VMP1 and methylation of the region. Secondly, using pyrosequencing to study methylation of this region, and 8 others to develop a diagnostic model for IBD. And thirdly to manipulate miR-21 levels in cell lines to investigate the effects on autophagy and VMP1. Results: Investigating the genetics of the region so far has not found any mutations, possibly as this is a highly conserved region. Further sequencing is required before this can be fully established. Pyrosequencing assays have been developed and performed on 8 CpG sites and a diagnostic model has been developed to distinguish IBD from HC, although it is not effective at distinguishing UC from and CD. And finally, a VMP1 antibody for western blotting and miR-21 mimic and inhibitor for cell lines have been optimised. An interaction between VMP1 and NOD2 has been established by Co-IP.