Impact of miRNA expression on an invasive phenotype

Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA and analyzed for changes in invasive cell behaviour by matrigel invasion chamber assays or video microscopy.Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA. Changes in predicted target gene expression potentially linked to the invasive phenotype will be monitored by qPCR, Western Blotting and confocal immunofluorescence microscopy (WWU, CONICET). Regulation of selected targets will be confirmed by 3’UTR luciferase reporter assays and plasmid-based complementation analysis using target constructs devoid of the endogenous 3’UTR (WWU).Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA.Altered signal transduction processes will be analyzed by phosphokinase array screening (R&D systems) and phosphokinase blotting (WWU, CONICET). Finally, we will employ proprietary nanoscale topography analysis (nanostic TM) provided by SEREND-IP to determine cell surface properties (filopodia formation, cell shape, etc.) in cooperation with partners WWU and CONICET. This technique has the potential to develop a marker-free diagnostic tool for invasive endometriotic cell behavious based on quantitative atomic force microscopy.