Abstract
IntroductionExosomes are 30-100 nm 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. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme encoded by the smpd3 gene that triggers exosome secretion. However, whether exosome-mediated microRNA transfer plays any role in cell invasion remains poorly understood. Thus, the aim of this study was to identify the exosomal microRNAs involved in breast cancer invasion.MethodsThe expression level of endogenous and exosomal miRNAs were examined by real time PCR and the expression level of target proteins were detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes and to study its uptake and transfer. Luciferase reporter plasmids and its mutant were used to confirm direct targeting. Furthermore, the functional significance of exosomal miR-10b was estimated by invasion assay.ResultsIn this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes. In particular, nSMase2 or ceramide promotes the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppresses this secretion. Moreover, upon uptake, miR-10b can suppress the protein level of its target genes such as HOXD10 and KLF4, indicating its functional significance. Finally, treatment with exosomes derived from MDA-MB-231 cells could induce the invasion ability of non-malignant HMLE cells.ConclusionTogether, our results 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-256) contains supplementary material, which is available to authorized users.
Highlights
Exosomes are 30-100 nm 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
In this study, we demonstrate that microRNA carrying exosomes can be transferred among different cell lines through direct uptake. miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cancer cells or non-malignant breast cells; it is actively secreted into medium via exosomes
The overexpression of miR-21, miR10b, and miR-19a can be utilized as serum biomarkers for aggressive breast cancer and inflammatory breast cancer as they are associated with the acquisition of malignant characteristics like increased tumor cell proliferation, migration, invasion, dissemination, and metastasis [9]
Summary
Exosomes are 30-100 nm 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. This includes raft-lipids such as cholesterol, sphingolipids, ceramide and glycerophospolipids with long and saturated fatty-acyl chains Due to their endosomal origin, exosomes from different cell types contains common proteins such as Rab GTPases, Annexins, flotillin, Alix, TSG101, hsp and hsp, integrins and tetraspanins (CD63, CD9, CD81 and CD82). The overexpression of miR-21, miR10b, and miR-19a can be utilized as serum biomarkers for aggressive breast cancer and inflammatory breast cancer as they are associated with the acquisition of malignant characteristics like increased tumor cell proliferation, migration, invasion, dissemination, and metastasis [9] In another case involving 89 patients with primary breast cancer (n =59) and metastatic disease (n =30), and 29 healthy women, breast cancer-associated miR-10b, miR-34a, miR-141 and miR-155 were elevated in the blood of breast cancer patients and are associated with tumor progression [10]. Overexpression of miR-105 in nonmetastatic breast cancer cells can induce metastasis, whereas inhibition of miR-105 in highly metastatic tumors has the opposite effect [11]
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