Postgraduate research position – Probing the role of long non-coding RNA in myofibroblast differentiation

Background: Recent advances in RNA sequencing technology and epigenetics have allowed us to investigate the transcriptomic landscape of a cell in greater detail than ever before. This has revealed that the proportion of transcripts expressed by a cell that do not code for proteins (non-coding RNA) far exceeds previous estimations. The expression of the most numerous of these, long non-coding RNA, is closely linked to complexity of the organism and recently has been shown to play a key role in cellular differentiation. In this project, we propose to examine the role of long non-coding RNA in myofibroblast differentiation, a key process in wound healing, fibrosis and cancer. The presence of myofibroblasts in the microenvironment of a number of tumours is associated with aggressive disease, suggesting that targeting the mechanisms underlying this aberrant differentiation may be a viable novel therapeutic approach. In silico analysis reveals the existence of two novel putative lncRNA, VCAN-AS1 and VCAN–AS2, transcribed antisense to the mRNA coding for a key extracellular matrix protein, versican (VCAN), known to be involved in myofibroblast differentiation and tumour progression. As other antisense lncRNA are known to regulate their protein coding counterparts, in this project we will examine the expression of this transcript, and potentially other lncRNA of interest, and determine their role in modulating myofibroblast differentiation.

Hypothesis: LncRNA regulate myofibroblast differentiation and thereby influence stromal-tumour interactions

Methodology: A range of techniques will be employed including qPCR, qPCR arrays, tissue culture, immunocytochemistry, in situ hybridisation, siRNA and transfections and western blotting. You will work in a thriving research group with core technical support. Full training will be provided.

Experimental outline: The following studies will be performed:

1. Examine the expression of VCAN AS1 and VCAN AS2 in a panel of cancer cell lines and fibroblasts.
2. Carry out lncRNA expression profiling, including VCAN AS1/2 if detected, in fibroblasts and myofibroblasts.
3. Examine subcellular localisation of lncRNA of interest using in situ hybridisation
4. Knock down lncRNA of interest using siRNA and examine effects on protein coding genes involved in myofibroblast transdifferentiation such as alpha smooth muscle actin and VCAN.
5. Test effects of modulating lncRNA expression in either tumour cells or fibroblasts (as appropriate) on cancer cell migration using mono- or co-culture models.
6. Test the effects of agents known to promote myofibroblast differentiation, such as TGFβ, on VCAN AS and VCAN expression.

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