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Cheng et al. Vessel Plus 22020;4:17 I http://dx.doi.org/10.20517/2574-1209.2020.08 Page 9 of 15

Within the groups of non-coding RNAs, miRNA are now considered promising biomarkers in many
pathological conditions [99,100] due to their stability in various bodily fluids such as saliva, serum, plasma and
CSF, and their role in gene expression regulation [101-106] . One of the advantages of studying EV-associated
miRNAs is their particular stability as they are protected within a plasma membrane [107] . These short, non-
coding RNA molecules display critical regulatory functions as they are involved in nearly all physiological
processes such as cellular differentiation, proliferation, metabolism, development, and homeostasis [108,109] .

As previously mentioned, a large portion of EV miRNA studies are focused on cancers, which have shown
the significance of identifying cargo miRNA. In a 2019 clinical cancer study, miRNA from whole plasma,
EVs and EV-free plasma from lung adenocarcinoma and granuloma patients were evaluated. The study
determined that whole plasma, EVs and EV-free plasma had differing miRNA expression profiles and the
prediction performance of EVs was better than EV-free plasma. Plasma was the best predictor however,
due to the lack of knowledge in storage and processing techniques of EVs [110] . Elevated levels of plasma
EVs have since been observed in patients affected by various forms of cancer compared to healthy subjects
and, interestingly, these levels decreased upon removal of the tumour, simultaneously decreasing tumour
specific miRNA profiles within the plasma EVs [111,112] , which provides a further link between cancer and
increased EV production. Similar results were demonstrated in patients with autoimmune, infectious and
cardiovascular diseases, and neurological disorders [113-116] .

Next-generation sequencing technology is the recommended, standard approach when investigating the
miRNA content of EVs for novel biomarker identification [117] . Accumulated sequencing data suggest the
potential for miRNAs as diagnostic and prognostic markers, as well as for parasitic diseases caused by
platyhelminths, arthropods, and protozoa, including Plasmodium spp. [118] . When analysing EVs derived
from helminth parasites (Trichuris muris), the content was sequenced using the HiSeq 500 system,
identifying 56 miRNA, 22 of which were novel [119] . A similar study looking at hookworms using the
NextSeq 500 system identified 52 miRNA, many of which were found to be involved in inflammation
regulation when mapped to mouse genes [120] .

One of the first studies on miRNA in Plasmodium infection suggested that this parasite did not have
specific miRNAs but rather, takes advantage of the transcriptional machinery within RBCs for the
activation and suppression of gene expression [121] . This study also identified miR-451 as highly expressed in
iRBCs, although its accumulation was not associated with malaria infection [121] .

A study from Thailand observed, for the first time, lower expression of miR-451 and miR-16 in the plasma
of adults infected with malaria, suggesting their role as biomarkers for malaria infection, especially in
Plasmodium vivax infected individuals [122] . However, a large portion of transcriptomic studies have been
performed using murine models focusing on the host’s response to infection [123-125] , or using in vitro systems
to target a specific cell-type. For instance, let-7i, miR-27a, and miR-150 were found to be over-expressed
in the brain of CM-mice but not in non-CM animals [124] . Overexpression of these miRNA during infection
may be essential for the instigation of neurological syndromes by regulating their downstream targets,
thus having a potential regulatory role in the pathogenesis of severe malaria, as well as being targets for
therapeutic intervention [124] .

Similar to iRBCs, EVs from iRBCs display higher levels of miR-451a and let-7b when compared to nRBCs,
and once miR-451a within EVs is engulfed by endothelial cells, their gene expression and barrier properties
are affected, which may then lead to vascular dysfunction, making the miRNA a possible target for
[52]
therapeutic intervention . Using a more complex model, our group analysed plasma EVs from mice with
CM and found that the miRNAs from malaria EVs played a regulatory role in severe malaria pathogenesis.
miR-146a levels were higher and miR-193b levels lower in plasma-derived EVs while miR-205, miR-215,