New project 2

1. Applicant

2. Topic

The role of microRNAs in migration of melanoma cells

3. Short summary

Fig.: Invasion assay of a melanoma cell line after treatment with miRNA mimic (lower panel) or control mimic (upper panel).

Malignant melanoma is a fast progressing tumor which tends to metastasize already at small size and early time point. Thus it is utmost important to unravel mechanisms of invasion and migration in this tumor. The aim of this study is to investigate the role of microRNAs (miRNAs) in melanoma migration and progression. Our starting material is composed of cell lines and tumor tissues from patients with varying median survival after diagnosis of stage IV (6 versus 53 months) and a large number of normal human epidermal melanocytes (NHEM). We have performed miRNA expression profiling in these three groups and identified a number of deregulated miRNAs which are presently further analyzed.

Since published profiling data are not very consistent, functional assays are needed to comprehensively screen for relevant miRNAs. We will use a high-throughput scratch assay and melanoma cells transfected with a large miRNA mimic library to search for miRNAs regulating migration. From our preliminary experiments we have two positive control miRNAs for these experiments. The expression of validated miRNAs will be determined by qPCR in the cell lines of different aggressiveness, as well as in NHEM, tumor specimen and nevi.

Selected miRNAs will be further analyzed with respect to their target specificity on the protein level (comparative proteome analysis; SILAC). Putative targets will be validated by Western blotting and specificity will be tested in reporter assays. Finally, melanoma cell lines stably transfected with the miRNAs of interest will be tested in comparison to their untransfected counterparts for tumor growth and metastasizing potential in NOD/SCID mice. A comparative expression analysis of these miRNAs in mouse and fish tumor models will give information on whether or not these genes are conserved in melanoma of different species.

The results of this study will identify miRNAs which down-regulate migration and might be used in future as therapeutics to counteract tumor spread.

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