This study demonstrates that MPs serve as vehicles for intercellular communication and potentially as cancer biomarkers through their discrete miRNA signatures. RT-PCR analysis showed that MPs carry the transcripts of their vesiculation machinery (floppase and scramblase) (Figure 1A and B) together with transcripts encoding miRNA biogenesis enzymes (Drosha, Dicer and Argonaute) (Figure 1C, D and E). The presence of these phospholipid enzymes (floppase and scramblase) may help in intracellular vesicle trafficking either by inducing membrane vesiculation or by providing an environment favorable for binding of vesicle coat proteins . This finding suggests that MPs are self-sufficient and possess the capacity to potentially induce vesiculation in the recipient cell. Although, the presence of scramblase 1 and 3 in exosomes has been previously reported [20, 35], to our knowledge, this is the first demonstration of the presence of transcripts encoding the vesiculation machinery in MPs. The presence of Drosha, Dicer and Argonaute transcripts within the shed cargo potentially implicates MPs as key intercellular regulators of miRNA biogenesis in recipient cancer cell populations.
Affymetrix miRNA microarray was used to explore the miRNA expression profiles of MPs and their donor cells from both leukaemia and breast cancer cells in this study (Figure 5). This data was validated using RT-PCR studies where we observed almost similar trends for most miRNAs examined (miR-107, miR-125b and miR-210) except for miR-150 (Figure 4). The basis of this difference in currently unknown and may be attributed to the differences in assays. Our analysis showed that several human miRNAs are selectively packaged into MPs. Upon coculture with recipient cells, we observe an increase in select miRNAs, inlcuding miR-1246, miR-1308, miR-1228*, miR-149*, miR-638 and miR-923 (Figure 7).These miRNAs displayed a 2-12-fold increase in expression levels in the MPs relative to their donor cells (Figure 7A). These observations are consistent with selective packaging, which we have previously shown for miR-451 and miR-326 in MPs shed from MDR resistant breast and leukaemia cells . In addition, our findings are consistent with previous reports demonstrating that these same miRNAs are also selectively packaged into exosomes [24, 36–38].
We also demonstrate that following MP transfer to recipient breast cancer and leukaemia cells, the same miRNAs were significantly increased in the acquired cells, with levels ranging from 2-15-fold increase (Figure 7B). Once again the expression level of the miRNAs in the MP compartment was directly correlated to the levels observed in the acquired population following MP transfer (Figure 5). In saying this however, we cannot exclude the possibility that increased miRNA levels in the recipient cells are caused by either/both direct or indirect MP-mediated effects on the transcription of the miRNA. Interestingly, the acquired population displayed miRNA expression trends reflective of the donor cells (Figure 7B). This demonstrates that the recipient cell reflects the donor trait after MP-mediated transfer of cargo. These findings are consistent with our previous report where we have shown the “re-templating ” of recipient cells to reflect donor cell traits following MP-mediated transfer of MDR transporter transcripts .
The miRNAs identified in our study play crucial roles in cancer cell biology. Using the miRDB target prediction program , NKIRAS1, which is a NFKB inhibitor was identified as one of the targets of miR-1308. Nuclear factor kappa B (NFκB) is a family of transcription factors that play important roles in regulating cell differentiation, proliferation, immune response and blocking apoptosis [40, 41]. This family of transcription factors have been reported to chemosensitize P-gp overexpressing cancer cells . This miRNA has also been shown to be upregulated in cancerous tissues and also in the more aggressive inflammatory breast cancer (IBC) in comparison to the non-IBC tissues [43, 44]. Similarly, miR-1246 targets the NKF3 kinase family member gene, SGK269 (miRDB database). NKF3 or PEAK1 promotes anchorage independent growth and tumour progression in pancreatic cancer cells transplanted in mice . miR-149* is known to induce apoptosis by the direct inhibition of Akt1 and E2F1 in neuroblastoma cells . Akt is the key kinase of the signal pathway, which mediates the regulation of divergent cellular processes including apoptosis, proliferation, differentiation and metabolism .
miR-638 has been found to be consistently, highly expressed in human plasma and its presence in the plasma may be physiologically necessary . As such, the ratio of miR-92a/miR-638 in blood is associated with diagnosis in acute leukaemia patients . Gene targets of miR-638, cyclin G2 and transcription elongation regulator 1-like factor (miRDB database), were involved in p53 and platelet-derived growth factor (PDGF) signalling pathways . miR-638 was one of the downregulated miRNAs in colorectal liver metastases with respect to the adjacent liver tissues that have the potential to serve as a prognostic and predictive marker of colorectal liver metastases . Likewise, miR-1228* has been previously shown to be highly expressed in malignant mesothelioma tumour samples compared to normal samples .
The role of the miRNAs and their targeted pathways in the cells examined in this study are currently unknown. It is feasible that the same miRNAs may serve similar functions in these cells as is the case in the other reported cancers. However, the pathway analysis of the predicted targets of the 7 identified miRNAs in this study showed the maximum percentage of target genes to be significantly related to “pathways in cancer” and at least seven other pathways as well that were cancer related (Figure 8). The malignancy-related upregulated expression of these miRNAs may serve as potential biomarker in the treatment of cancer.
Chemotherapy comprises the major therapeutic strategy for clinical cancer treatment. However, chemotherapy fails to eliminate all tumour cells because of intrinsic or acquired drug resistance, which is the most common cause of tumour recurrence [52, 53]. The role of miRNAs in the regulation of resistance mediated by multidrug transporters has only been examined recently . Interestingly, we found that some of the significantly expressed miRNAs (like miR-455-3p) identified in this study target the multidrug resistant protein, P-glycoprotein (P-gp). For example, miRDB target prediction shows MDR member 1 or (P-gp) and HIF1AN (hypoxia-inducible factor 1, alpha subunit inhibitor) as miR-455-3p targets. HIF-1 alpha has been shown to induce MDR in hepatocellular carcinoma cell line . In this study, the microarray analysis showed that the resistant leukaemia cell line has a lower expression level of this miRNA relative to its drug sensitive cells, which is consistent with its overexpression of P-gp. In the acquired cells we observed a suppression of miR-455-3p implicating potentially increased P-gp levels after MP transfer. Hence, our previous observations of P-gp protein transfer in the drug sensitive recipient cells after MP coculture  may be due to the transfer of these regulatory miRNAs together with protein via the MP cargo. In breast cancer, the overexpression of miR-923 was shown to be upregulated in the Taxol resistant cancer cells relative to the normal cells .
Other than pathways related to malignancies and MP vesiculation, “ABC transporters” was identified as a significant biological pathway with the highest percentages of the identified miRNA target genes. Previous studies have also reported on the role of miRNAs involved in MDR in cancer. These include miR-27a and miR-451, whose expressions were shown to induce MDR1/P-gp expression in resistant human ovarian cancer cells . The overexpression of MRP1 and miR-326 levels was inversely related in breast cancer tissues and leukaemia [33, 57]. Recently, miR-345 and miR-7 have shown to target MRP1 in MDR breast cancer cells relative to parental cells . Apart from MDR, the ABC transporter, ABCA1 has also been shown to possess floppase activity and is related to MP production . In addition, of the significant predicted pathways identified for the miRNAs in this study, two of them namely: “calcium signalling pathway” and “regulation of actin cytoskeleton” were related to MP vesiculation (Figure 8). This suggests that MPs not only carry the enzymes for its release but also carry miRNAs and genes which may be potentially involved in its production and release.
Our study gives an implication of the role miRNAs contained within the MP cargo may play in contributing to the emergence of MDR and in regulating transporter expression in recipient cancer cells. Previous studies have shown miRNA transferred by other vesicular bodies like microvesicles to be functional in the recipient cells [60, 61]. However, the functional role of the identified miRNAs in this study needs further exploration to have any clinical relevance.