RNAi as a tool to inhibit the angiogenic potential of human Mesenchymal Stem/Stromal Cells in malignancy

Mesenchymal Stem/Stromal Cells (MSCs) have an active role in supporting the maintenance of a dynamic and homeostatic tissue microenvironment by secretion of a broad range of biologically active molecules. Upon interaction with cancer cells, MSCs become active participants in tumour development namely by promoting angiogenesis through the secretion of pro-angiogenic molecules, such as vascular endothelial growth factor (VEGF). Since tumour angiogenesis is one of the hallmarks of cancer progression, targeting this mechanism by using a genetic engineering approach, such as by RNA interference (RNAi) technology, seems a promising strategy to slow down tumour growth. In this regard, this project involves the transfection of bone marrow-derived MSCs (BM-MSCs) with small-interfering RNA (siRNA), chemically synthetized or expressed as a short-hairpin RNAs (shRNAs) from a minicircle (MC) vector, that target VEGF expression. Overall, RT-qPCR results revealed a VEGF-mRNA knockdown of approximately 50% after synthetic siRNA transfection of BM-MSCs. Similarly, ELISA results indicate a decrease in VEGF secretion. Regarding transfection results with MC vector, similar results were expected, however, increased VEGF expression was consistently found at mRNA and protein levels after BM-MSCs transfection. Although further studies are required, these discrepancies appear to be related to the incorrect processing of the transcribed shRNA into a functional RNAi. In conclusion, this study provides important insights regarding the implementation of an RNAi-based system that targets VEGF expression, aiming at slowing down tumour angiogenesis.