Abstract 4154: Exosomal microRNAs: mediators for cell-cell communications

Abstract Background: Exosomes are 30-100nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor-cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs to neighboring or distant cells. MicroRNAs are small regulatory non-coding RNA molecules that regulate protein-coding genes. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme expressed by smpd3 gene that triggers exosome secretion. In this study, we investigated the exosome-mediated microRNA transfer among various types of cells, the regulation of exosome-mediated microRNA secretion and functional role of these exosomal microRNAs inside the recipient cell after uptake. Methods: Exosomes were isolated from culture media of cells by serial centrifugation, filtration and ultracentrifugation method. Isolated exosomes were stained with PKH26 dye and then observed for cellular uptake in different cells by confocal microscopy after counterstaining with Hoechst stain. In addition, this uptake was also shown by exosome isolated from stable 293T cell line overexpressing the pCDH-CD63-GFP fusion protein. To determine the effect of nSMase2 on exosome secretion, stable cell lines were established with smpd3 gene. Levels of microRNAs secreted in media were determined by real time PCR. To validate the function of exosomal microRNA in the recipient cells, we determined the protein level of microRNA target genes by western blot. Results: We first showed that exosomes can be transferred among different cell lines through direct uptake. We found that miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cells; it is actively secreted into medium. For example, nSMase2 promoted the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppressed this secretion. Importantly, western blot revealed that upon uptake, miR-10b can still reduce the protein level of its target genes such as Bim, indicating its functional significance. Conclusion: We found that the exosomes secreted from cancer cells containing microRNA can be taken up by other cells and have functional role in the recipient cells suggesting the importance of exosome-mediated microRNA transfer in tumor microenvironment. Our results collectively suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4154. doi:1538-7445.AM2012-4154