Abstract 947: Polycomb protein SUZ12 is regulated by a novel miRNA like RNA (miR-G655A) present in extracellular vesicles secreted by mesenchymal stem/tromal cells
Authors: Patrice Penfornis, Joseph D. Fernandes, Radhika R. Pochampally
Journal: Cancer Research (2016)

Abstract Introduction: Role of human mesenchymal stem/stromal cells (hMSCs) in cancer progression is well studied. Previously, we showed that hMSCs use autophagy to survive in adverse environment (e.g. serum deprivation). Using this model to mimic the solid tumor core, we1ve shown that the secretome of these stressed hMSCs is also tumor supportive. Furthermore, we showed that extracellular vesicles (EVs) secreted by cells contain small RNAs, proteins and lipids that are tumor supportive. In this study, we focused on a new and unknown small RNA contained in EVs, its potential mRNA targets in recipient cancer cells and its regulation in parent hMSCs and other cancer cells. Methods: EVs were isolated from serum deprived human mesenchymal stem/stromal cells (hMSCs) using a concentrator followed by serial ultracentrifugation. RNA molecules less that 200 bases were isolated and a purified cDNA library was used for cluster generation then sequenced on Illumina GAIIx. Human cancer cells (osteosarcoma, fibrosarcoma and breast cancer) miRNA knockdown models were obtained using agomirs and shRNA strategies against miRNA-G655A. Cell proliferation assays were performed using trypan blue and Cyquant DNA detection. In vivo tumor progression in nude mice was tracked by caliper measurement and relative GFP signal detected with in vivo imaging station. Small RNA targets were revealed by Affimetrix Human gene 2.0 ST array analysis and confirmed by 31UTR luciferase assays. Cross linked-ChIP assays for EZH2 and SUZ12 were used to identify interactions with the new small RNA promoter region. Results: Next generation sequencing of EVs from hMSCs identified an unknown small RNA, temporarily named miR-G665A. We report here that miR-G665A is conserved in mammals and its expression is confirmed, but varies between tissues or cell line1s. EVs secreted by hMSCs are notably enriched with miR-G665A. Both in vitro and in vivo knockdown studies using agomirs and shRNA strategies indicate that miR-G665A participate in cell proliferation and tumor progression. Its expression is also regulated through cell cycle. Microarray data from these knockdown studies revealed potential end targets of miR-G665A (e.g. interleukins,MMPs). Some direct targets are validated using 31UTR luciferase assays. One of the direct target is SUZ12, a component of the polycomb group complex PRC2, a master epigenetic regulator which contributes to the neoplastic phenotype. We also show that PRC2 complex (SUZ12 and EZH2) targets the regulation of the expression of this novel small RNA in a feedback loop mechanism. Conclusion: The role of this novel small RNA on the regulation of an essential transcription factor could lead to a better understanding of the crosstalk mechanisms between mesenchymal stem/stromal and cancer cells. This work is supported by NIH grant CA1515851 and the UMMC Cancer Institute startup funds. Citation Format: Patrice Penfornis, Joseph D. Fernandes, Radhika R. Pochampally. Polycomb protein SUZ12 is regulated by a novel miRNA like RNA (miR-G655A) present in extracellular vesicles secreted by mesenchymal stem/tromal cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 947.