This study presents a transcriptomic analysis of CRC cell-derived microvesicles, among which we identified 11,327 microvesicular transcripts involved in various tumor-related processes that reflect the physiology of the donor CRC cells. Among these transcripts, we found 241 mRNAs enriched in microvesicles beyond their levels in donor cells, of which 27 were involved in the cell cycle. Moreover, 20 of these 27 mRNAs were associated with cell cycle M-phase activities, and 15 of these showed differential expression patterns in CRC patient data. We then demonstrated that these mRNAs may modulate activities of M-phase processes during the cell cycle by reconstructing a cell cycle network, which showed that the corresponding proteins belong to and also interact with those involved in M-phase-related processes. To experimentally verify this network-driven hypothesis, we demonstrated that CRC cell-derived microvesicles are delivered to endothelial cells and stimulate the proliferation of endothelial cells. Since this proliferation is an essential step in neovascularization, our finding suggests that microvesicles have angiogenic activities.
The preparation of microvesicles that are not contaminated with cellular debris, bovine serum-derived microvesicles, or any non-microvesicular mRNA is critical for transcriptional profiling of microvesicles. As the half-life of individual microvesicular mRNA transcripts is unknown, the rapid analysis of microvesicular mRNA is also very important. We therefore followed our previously described purification methods with the following two major modifications [5
]. In particular, RNase A treatment was applied in order to degrade non-microvesicular RNA derived from damaged cells or leaky microvesicles (Figure ). The microvesicles were finally purified using iodixanol density gradient centrifugation, which is much faster than the conventional sucrose density gradient centrifugation [14
]. As a result, the high quality of the microvesicular RNA was obtained as indicated by the Bioanalyzer results (RIN: 8.7).
Growing evidence indicates that tumor-derived microvesicles are involved in tumor progression via the modulation of tumorigenesis, angiogenesis, immune response, invasion, and metastasis [1
]. Moreover, recent studies have reported that microvesicular mRNA can induce potent epigenetic changes in target cells, as microvesicular mRNA is delivered to target cells, where it is translated into functionally active proteins [11
]. Our findings that SW480-derived microvesicular mRNAs are involved in various tumor-related processes (Figure and Figure ) and SW480-derived microvesicles can initiate angiogenesis via the stimulation of endothelial cell proliferation (Figure ) also suggest that the microvesicular mRNAs play potential roles in progression of CRC. Furthermore, SW480-derived microvesicles are enriched with CENPE
, and CDK8
(Figure ) that are M-phase-related transcripts involved in tumorigenesis and tumor progression [26
]. Although we could not exclude the other possibility, our observations suggest that SW480-derived microvesicles promote the proliferation of endothelial cells by increasing their cell cycle activities via the horizontal transfer of these M-phase-related microvesicular mRNAs. Further studies on microvesicles derived from other colon cancer cells would provide valuable data. Thus, the inhibition of either microvesicle shedding or modulation of microvesicular function has been proposed as worthwhile approaches to cancer therapy. Recently, Al-Nedawi et al. (2009) showed that the treatment of Diannexin, which inhibits the uptake of the A431 (human squamous cell carcinoma cell line)-derived microvesicles into endothelial cells, to A431 tumor xenografts in mice led to a reduction of tumor growth rate and microvascular density [29
]. Thus, our findings above suggest that the inhibition of biogenesis, trafficking or function of the CRC-derived microvesicles also can offer a novel therapeutic approach for CRC.
Despite the progress in understanding and treating CRC, it remains a leading cause of mortality worldwide [30
]. A significant improvement in survival would be expected with early diagnosis of CRC. Several studies have reported that the level of tumor-derived microvesicles is elevated significantly in the serum or the ascites fluid of cancer patients [8
]. Recently, Taylor et al
. proposed that microRNA profiling of circulating tumor microvesicles could potentially be used to identify diagnostic markers for human ovarian cancer [33
]. Thus, our finding that the microvesicular mRNA reflects the mRNA signature of the parental CRC suggests that the microvesicular mRNA may be a useful diagnostic and/or prognostic marker of early stage cancer and could be evaluated in a non-invasive manner. Possible CRC marker candidates can be predicted as the microvesicle-enriched mRNAs that show colon-specific expression patterns (additional file 9