Spontaneous (non-traumatic) intracerebral hemorrhage (ICH) or hemorrhagic stroke is a common and severe illness of central nervous system with high rates of morbidity and mortality. The etiology of ICH is closely associated with hypertension, atherosclerosis and vascular malformations [1]. Data on ICH and its subsequent effects on central nervous system are limited compared with molecular genetic researches of pathogenesis of ischemic stroke. ICH triggers a number of pathophysiological processes including apoptosis and necrosis, oxidative stress, violation of blood-brain barrier (BBB), cerebral edema, inflammation and remodeling of extracellular matrix. These processes can lead to severe neurological deficiency or death (Fig. 1) [2].

MicroRNAs (miRNAs) are small non-coding RNA molecules composed of 18—22 nucleotides. These molecules function as post-transcriptional regulators of gene expression in mammalian cells. Their action is realized through pairing with 3'-untranslated regions (3'-UTR) in mRNA molecules (mRNAs) that inhibits gene activity through translational repression. MiRNAs are involved in the most of fundamental biological processes such as cell cycle control, cellular metabolism, apoptosis, proliferation and differentiation [3].

The purpose of this work is to analyze current knowledge about the role of miRNAs in pathogenesis of hemorrhagic stroke.

MicroRNA and ICH pathogenesis

ICH is a multifactorial disease with multiple established causes [4]. Endothelial dysfunction and significant changes in the walls of cerebral vessels under the influence of chronic hypertension and atherosclerosis are essential in pathogenesis of ICH [5,6]. Hemorrhage from the altered artery results certain pathophysiological processes including nerve cell death, inflammation, oxidative stress, BBB disturbance, cerebral edema, inflammation. MicroRNAs regulate target genes in the aforementioned processes via binding to 3’-UTR of mRNA to suppress gene expression (Table 1).

Apoptosis of neurons

Apoptosis is a programmed cell death and vital for normal metabolism. This mechanism supports cellular homeostasis via removal of aging or damaged cells. However, some pathological processes are associated with disturbed apoptosis and programmed cell death becomes uncontrolled [7]. It has been recently shown that changed miRNA expression can modulate survival of neurons after hemorrhagic stroke via regulation of the target genes [8]. Liu et al. reported increased expression of miR-298 in neurons and blood samples in both experimental models of ischemic and hemorrhagic stroke in vivo [9]. Further studies have shown that increased expression of endogenous miR-298 after ischemic stroke contributes to brain damage in vitro and in vivo by inhibition of RAC-alpha serine/threonine protein kinase/N-terminal kinase/nuclear factor Kappa-B signaling cascade (Akt1/JNK/NF-κB) and subsequent pathway of autophagy. The same may be assumed for hemorrhagic stroke [10]. Akt genes are involved in pathways ensuring cellular survival by inhibition of apoptosis. Xi et al. demonstrated inhibition of neuronal apoptosis via regulation of signal pathway of phosphoinositide-3-kinase/Akt (PIK3R2/Akt) using miR-126-3p mimic after experimental ICH in vivo. Mimics are synthetic short double-stranded oligonucleotides which mimic the target microRNA and increase their expression [11].

It was proved that injection of anti-miR-132 into the lateral ventricles of mice after experimental ICH (antagomirs — synthetic oligonucleotides inhibiting the target microRNA and reducing its expression) had neuron-protective effect, reduced neuronal death and led to regression of neurological deficit [12]. Caspases are known to be essential in apoptotic processes. MiR-155 has important role in modulation of cellular apoptosis via regulation of caspase-3 expression. Reduced expression of miR-155 significantly decreased apoptosis of endothelial cells of cerebral microvasculature [13]. Moreover, overexpression of endogenous miR-126 via miR-126 mimic delivered by lentiviruses has a protective role by inhibition of neuronal apoptosis in hemorrhagic stroke through decrease of caspase-3 levels in vitro and in vivo [14]. Shen et al. found reduced expression of miR-133b in brain tissues of Sprague — Dawley (SD) rats in 24 hours after ICH. Peak values were observed in 72 hours after experimental ICH. However, systemic administration of exosomes modified with mimic miR-133b was followed by significantly increased expression of miR-133b. As a result, reduced apoptosis of brain cells was observed. Western blotting showed that overexpression of miR-133b significantly suppressed expression of Ras-genes (RhoA) and activated signaling pathway of extracellular signal-regulated kinase-1/2/CREB (ERK1/2/CREB) in brain tissues. Thus, this study showed that miR-133b is characterized by neuroprotective anti-apoptotic effect in ICH [15].

Kim et al. reported that experimental ICH in SD rats was followed by more than 4-fold increase of let-7c miRNAs expression in the basal ganglia cells on the side of hematoma compared to contralateral cells [16]. They also created a model of neuronal damage after ICH by applying thrombin to pheochromocytoma cell line in rats (PC12). Basal ganglia are one of the most frequent locations of hypertensive hematomas. Therefore, it was advisable to assume the role of let-7c in pathogenesis of ICH. Blood degradation product, such as thrombin, may also be important for activation of let-7c. Subsequently, intranasal administration of anti-let-7c was accompanied by reduced expression of this microRNA and neuronal apoptosis around the hematoma. Anti-let-7f (let-7c and let-7f are microRNAs from the same let-7 family) was used in a model of ischemic stroke in vivo. The authors supposed that insulin-like growth factor receptor (IGF1R) is a key target for let-7c. IGF1R overexpression reduced cellular apoptosis [17]. It also follows that let-7c miRNA may be a potential therapeutic target with subsequent activation of IGF1R with neuroprotective effect in ICH.

BBB disturbances and cerebral edema

BBB is a physical barrier between blood and tissue of the brain and spinal cord. This structure strictly regulates molecular exchange between the blood and nerve tissue. Therefore, BBB has an important role in homeostasis and maintains normal microenvironment for complex neural activity. ICH is followed by interstitial edema as a result of vascular rupture and violation of BBB permeability. These processes result vasogenic cerebral edema. Water transport channels (aquaporins, AQP) are involved in development of edema [18]. AQPs 1, 4 and 9 are the main types of AQP found in central nervous system [19, 20]. Aquaporins are also involved in cellular apoptosis in central nervous system in addition to their role in formation of edema [21]. It was shown that AQP 4 expression is increased after ICH [22]. Zheng et al. revealed that miR-145 has a protective role for astrocytes in ischemic conditions via inhibition of AQP 4 expression and decrease of edema. These data may be continued in experiments in vivo with hemorrhagic stroke model for more accurate analysis of the relationship between miR-145 and AQP-4 [23]. Extracellular matrix of the brain also undergoes changes in response to damage. Disturbed BBB permeability with subsequent vasogenic edema lead to early expression of metalloproteinases (MMP) 2 and 9 with damage and impaired integrity of extracellular matrix [24]. Degradation of extracellular matrix causes hemorrhagic transformation of ischemic focus that worsens the outcome of stroke.

Some authors demonstrated significantly increased expression of miR-130a in blood serum of patients after ICH and in nerve tissue samples around the hematoma in rats after ICH. These data positively correlate with changes in hematoma volume [25]. The use of anti-miR-130a can significantly reduce cerebral edema, BBB permeability and improve neurological functions via increased expression of caveolin-1 and decreased expression of MMP-2,9 [24]. In addition to inhibition of apoptosis, miR-132 and miR-126-3p significantly reduced cerebral edema, inflammatory process and BBB permeability after hemorrhagic stroke in vivo [9,12]. Among other factors, enlargement of hematoma usually occurs in early phase of ICH and is closely associated with poor outcome [26]. Zhu et al. have shown a positive correlation between serum miR-126 and enlargement of hematoma after ICH. Prediction based on the levels of circulating miR-126, enlargement of hematoma and severity of edema around the hematoma could be useful to choose treatment strategy in patients with hemorrhagic stroke such as dehydration or surgical intervention [27].

Edema around the hematoma occurs mainly due to impaired integrity of BBB caused by cell apoptosis. Hu et al. showed that proliferation and apoptosis of endothelial cells of cerebral microvasculature are regulated by miR-23a-3p. The authors found that ZO-1 is a potential target for miR-23a-3p. ZO-1 is an important protein of tight adhesive contacts and essential element of BBB. This protein was first identified in 1986 [28]. ZO-1 is involved in barrier function, regulation of cell transport, cell polarity, proliferation and differentiation. In most cases, damaged structure of ZO-1 changes tight contacts. Increased or reduced expression of ZO-1 in endothelial cells of cerebral microvasculature also significantly influenced apoptosis and proliferation. Thus, miR-23a-3p contributes to edema around the hematoma via regulation of ZO-1 protein translation. Positive correlation of increased expression of serum miRNA-23a-3p and severity of edema around the hematoma in patients with ICH was also demonstrated in this study [29].

Xi et al. reported low expression of circulating serum miR-27a-3p in patients with ICH. However, these data may be non-specific because this level of serum miR-27a-3p expression is also found in other CNS diseases, such as traumatic brain injury. At the same time, further data of these authors revealed reduced expression in serum, hematoma and cells around the hematoma in rats after hemorrhagic stroke. Moreover, injection of miR-27a-3p mimic into the lateral ventricles ultimately reduced edema and neuronal apoptosis around the hematoma. These data confirm that reduced expression of miR-27a-3p is not an accidental event after ICH. Destruction of BBB and subsequent activation of microglia exacerbate ICH-induced brain damage and neurological deficit [30]. Thus, preservation of BBB integrity is essential to minimize secondary brain damage after ICH. In this context, increased expression of miR-27a-3p mimic after hemorrhagic stroke restored BBB function, decreased neuronal apoptosis and activity of microglia and leukocyte infiltration around the hematoma. These processes were followed by improvement of neurological functions in rats [31].

Thus, further studies of the mechanisms of BBB disruption and possible role of microRNAs in this process can lead to development of new treatment methods to prevent secondary brain damage after hemorrhagic stroke.

Inflammatory process

Inflammation is a complex immune response after various injuries. Inflammatory process contributes to sanation and repair of tissues under normal conditions. However, excessive activation of immune responses is harmful and can lead to damage. Hemorrhagic stroke results activation of microglia and release of inflammatory mediators including tumor necrosis factor alpha (TNF-α). These mechanisms aggravate central nervous system damage. In addition, there are also some cytokines released by mononuclear phagocytes, T-lymphocytes and neutrophils. These mediators are also involved in neuronal inflammation [32].

It was said that miR-132 overexpression after mimic injection in mice after ICH improves prognosis compared to the control group. Overexpression of miR-132 inhibits activation of microglia and expression of pro-inflammatory cytokines [12]. Overexpression of endogenous miR-367 inhibits inflammatory response via reducing the expression of interlekin-1 receptor-associated kinase 4 (IRAK4) in vitro and in vivo. In addition, miR-367 also inhibits activation of NF-kB and release of its pro-inflammatory factors, such as interleukin-6 (IL-6), interleukin -1 β (IL-1β) and TNF-α 33 [26]. MiR-223 has been reported to inhibit cryopyrin expression (NLRP3) and inflammatory process via caspase-1 and IL-1β. Thus, neurological function is improved in mice with ICH [34]. Vascular cell adhesion molecule 1 (VCAM-1) is an important molecule promoting localization of immune cells at the site of inflammation. Expression of this molecule is regulated by miR-126 [35]. The role of microRNAs in activation of microglia and polarization of macrophages is also important in pathogenesis of inflammation after ICH. Some authors reported that miR-155 promotes a shift of polarization towards M1 phenotype by targeting genes associated with M2. MiR-155 is clearly targeted at some genes associated with M2 phenotype and reduces expression of pro-inflammatory factors associated with M2 such as arginase-1 (Arg-1), interleukin-10 (IL-10), receptor for interleukin-13 alpha (1IL13Ra1) and mannose receptor (CD206) [36, 37].

The possible role of let-7c and IGF1R as its target in apoptosis was discussed in previous chapter. The authors also suggested that anti-let-7c will probably have anti-inflammatory effect in ICH. There are several reports confirming association of IGF1R with inflammation [17, 38]. Higashi et al. reported that IGF1R signaling reduces progression of atherosclerotic lesion in ApoE deficient mice via decrease of release of inflammatory cytokines and suppression of accumulation of macrophages and foam cells in lesion foci [38]. Selvamani et al. analyzed MiRNA let-7f in an in vivo model of cerebral infarction using hybridization in situ combined with immunohistochemistry. The authors found predominant activation of let-7f in microglia that was associated with reduced IGF signaling [17]. Probably, there are other targets for let-7c which control inflammatory cascade after ICH accompanied by neuronal injury. On the other hand, reduced cellular apoptosis associated with activation of IGF1R can indirectly reduce recruitment of inflammatory cells to the area around the hematoma. Further researches devoted to release of inflammatory cytokines and regulation of immune cells via modulation of miRNAs let-7c will be valuable to understand its role in inflammatory process after ICH.

Release of pro-inflammatory cytokines including interferon-β (IFN-β), IL-6 and TNF-α is important component of inflammation after ICH. Xu et al. found increased expression of IFN-β, IL-6 and TNF-α mRNA accompanied by increased expression of miR-155 and reduced expression of SOCS-1 in an in vivo model of hemorrhagic stroke. The authors indicated possible role of miR-155/SOCS-1 signaling cascade in inflammatory process. In addition, expression of miR-155 and pro-inflammatory cytokines was significantly reduced after dexamethasone administration in mice with ICH. Thus, glucocorticoids attenuate inflammation via targeting miR-155/SOCS-1 signaling pathway in mice [39].

Oxidative stress

Neuronal injury and death from oxidative stress occurs both in cerebral ischemia and intracerebral hemorrhage. Nerve tissue is very sensitive to oxidative injury considering highly oxygenated environment with high content of lipids and iron [40]. Oxidative injury is predominantly mediated by lysis of red blood cells after intracerebral hemorrhage. Red blood cells in hematoma are lysed by membrane-attacking complex (MAC) from the complement cascade [41]. Hemoglobin released from red blood cells is cleaved to iron by hemoxygenase 2 (HO-2) [42]. Subsequently, iron will undergo a Fenton reaction with formation of reactive oxygen species (ROS). Similar to ischemic stroke, inflammation is important for generation of ROS during intracerebral hemorrhage. Pogue et al. reported reduced expression of complementary factor H under increased expression of miR-146a in neurons under oxidative stress [43]. Xu et al. found that inhibition of miR-27b by anti-27b can reduce brain damage and increase expression of nuclear factor 2 (Nrf2), enzymatic hemoxygenase-1 (Hmox1), superoxide dismutase-1 (SOD1), and quinone oxidoreductase-1 (Nqo1) after ICH through Nrf2/ARE signaling pathway [44]. Nevertheless, there is only a small number of studies regarding miRNAs and oxidative stress in hemorrhagic stroke.