In the present study, we performed an enrichment analysis using IPA to observe the pathways regulated by the HIPARCO-score miRNAs (miR-378a-3p, miR-100-5p and miR-486-5p), a set of miRNAs that identify patients more likely to exhibit a favorable BP response to CPAP. Our main objective was to investigate the main biological functions regulated by these three miRNAs that are involved in the favorable response to CPAP treatment in patients with OSA and RH.
We observed the contribution of miR-378a-3p, miR-100-5p and miR-486-5p in the regulation of a variety of genes and pathways, especially in those related to cardiovascular function and cancer. Specifically, we observed a role for these miRNAs in heart hypertrophy, heart failure, apoptosis, and heart tissue injury. This finding is corroborated by a variety of studies in the literature demonstrating an association between miRNAs and cardiovascular disease and hypertension(18, 25). Similarly, studies have shown a strong relation between OSA and these events. Therefore, it has been reported that OSA is associated with hypertension and with hypertension-associated end-stage organ diseases such as stroke, coronary heart disease, and arrhythmia(33). Moreover, previous studies suggest that OSA has a role in the initiation and progression of several cardiovascular diseases(28). Considering this, the effect of CPAP in RH patients could be mediated by these miRNAs through their actions on cardiovascular-associated genes and functions. In fact, we observed that the studied miRNAs are associated with the NF-kB signaling pathway, which is known to upregulate the transcription of genes involved in endothelin signaling(23). Accordingly, several NF-kB binding sites were identified in the endothelin gene promoter region(10). Thus, the mechanism by which CPAP treatment decreases BP may be associated with the involvement of these miRNAs with the NF-kB signaling pathway, ultimately affecting the endothelin signaling. Further studies will be necessary to confirm this.
Cardiac failure is often associated with prolonged and maladaptive cardiac hypertrophy(11, 21). Considering that cardiomyocytes lose their ability to divide soon after birth, cardiac hypertrophy is an important adaptive response to maintain or increase the cardiac output of the organism(19). We observed that miR-378a-3p, miR-100-5p and miR-486-5p are involved in the regulation of several molecules related to activation pathways that lead to a hypertrophic response at the cellular level. For instance, the miR-378a-3p-mediated induction of the cytokine receptor GP130 activates the MAPK, PI3K and signal transducer and activator of transcription 3 (STAT3) pathways, which leads to the activation of genes involved in hypertrophy and survival pathways(32). Consequently, the translation of various signals through the GRB2-SOS complex and Akt activation that targets mTOR also occurs. mTOR speeds up the process of protein synthesis by activating its downstream targets p70S6K and eIF4E, resulting in protein synthesis, cell growth and cell proliferation. In turn, these kinases mediate cellular responses to stress, such as DNA damage and nutrient deprivation(26). Interestingly, Wen et al demonstrated that intermittent hypoxia, which is one of the main features of OSA, led to the inhibition of mTOR phosphorylation and activation of AMPK phosphorylation, inducing cellular apoptosis(31). On the other hand, a variety of studies reported an association between cardiac hypertrophy and the severity of OSA(20). Considering these findings, it is possible to observe the role of the studied miRNAs in the cardiovascular-related pathways that are also affected by OSA. In addition, it is important to address that RH may lead to cardiac hypertrophy(6). Thus, the studied miRNAs associated with a favorable response to CPAP treatment could be involved in the regulation of such adverse outcome in the long-term. This highlights the importance of these miRNAs not only as biomarkers but also as possible therapeutic targets. This relationship should be further explored in future studies.
miR-378a-3p, miR-100-5p and miR-486-5p also seem to regulate the effect of proinflammatory cytokines in beta cells via NF-kB(2, 24). miR-378a regulates TAK1, a kinase that mediates the signal transduction induced by TGF-beta and bone morphogenetic protein (BMP), and controls a variety of cell functions, including transcription regulation and apoptosis. Additionally, in response to interleukin-1 (IL-1), TAK1 forms a kinase complex that includes TRAF6 (also regulated by miR-486), MAP3K7P1/TAB1 and MAP3K7P2/TAB2; this complex is required for the activation of NF-kB(1). Therefore, these miRNAs modify NF-kB activity, which is pivotal for activating or preventing the overstimulation of the toll-like receptor (TLR) pathway(2). On the other hand, rapid reoxygenation at the end of apneas/hypopneas leads to the production of free radicals, inducing oxidative stress and the upregulation of NF-kB. In addition, there is evidence indicating that CPAP treatment reduces the levels of inflammatory mediators, such as interleukin-6, tumor necrosis factor-α, and C-reactive protein(3). Thus, there is considerable compatibility between the molecules and pathways affected by the studied miRNAs and those affected by OSA or CPAP treatment regarding inflammatory mechanisms.
A substantial number of studies have reported the oncogenic and tumor suppressing roles of miRNAs. Additionally, several miRNAs are markers for the early diagnosis of cancer(12, 30). In fact, the present study suggests a role for the studied miRNAs in a variety of cancer-related pathways. This leads to a possibility for which there is still no evidence that the phenotype of good CPAP responders in terms of BP might be associated with other characteristics, such as different prognosis in tumor-related diseases.
It is important to note that there are still considerable limitations in miRNA studies, and more investigations are needed to confirm the role of miR-378a-3p, miR-100-5p, and miR-486-5p in cardiovascular diseases. Considering that these miRNAs were measured in plasma, no distinction was made between miRNAs derived from exosomes and those derived from other events, such as cell death. Thus, the results discussed herein should be interpreted with caution. Regardless, the observed significant change in the circulating profile of miRNAs is sufficient as a biomarker to be interpreted by itself.