The Role of Noncoding RNAs in Brain Cells during Rat Cerebral Ischemia

*Ivan B. Filippenkov, Lyudmila V. Dergunova and Svetlana A. Limborska*

## **Abstract**

Ischemic brain stroke is one of the most serious and socially important medical conditions. Transcriptome analysis is a prospective approach to the study of the mechanisms of brain functioning, both under normal conditions and in ischemia. In addition to mRNA encoding proteins, the study of noncoding RNAs in ischemia has exceptional importance for the development of new strategies for neuroprotection. Of greatest interest are microRNAs (miRNAs) and circular RNAs (circRNAs). circRNAs have a closed structure and predominantly brain-specific expression. They can interact with miRNAs, diminish their activity, and thereby inhibit miRNA-mediated repression of mRNA. Recently, it has become clear that the analysis of circRNA-miRNA-mRNA interactions is an important requirement for the detailed study of the mechanisms of damage and regeneration during ischemia. This chapter reviews the most recent data on the role of circRNAs, miRNAs, mRNAs, and their interactions in brain cells under normal conditions and in cerebral ischemia.

**Keywords:** functional genomics, experimental rat brain ischemia, mRNAs, noncoding RNAs, circular RNAs, microRNAs

#### **1. Introduction**

Ischemic stroke is a serious condition and is one of the leading causes of disability and death worldwide. It arises as a consequence of a critical decrease in blood flow in the brain tissues, which leads to the death of neurons and glial cells. Therapy aimed at treating or preventing ischemic stroke is one of the most significant problems of modern medicine. Molecular genetic approaches using experimental models of ischemia based on small laboratory animals are of great importance and provide perspectives for studying the mechanisms underlying the damage to nerve cells and their ability to recover. Events occurring in ischemic stroke in humans caused by the formation of a thrombus are best reflected by the permanent middle cerebral artery occlusion (pMCAO) model. Additionally, the transient middle cerebral artery occlusion (tMCAO) model best reflects the events occurring in ischemic stroke in humans caused by subsequent treatment with thrombolytic drugs. The results of clinical studies suggest that thrombolysis is among the most effective and affordable methods of treating ischemic stroke. At the same time, it is known that reperfusion

after thrombolysis not only contributes to the restoration of penumbra cells but also causes additional damage to brain cells, including disruption of endothelial microvascular cells, the excess oxygen radicals, and activation of apoptosis.

Ischemic brain damage in combination with reperfusion damage is a complex process resulting from changes in the levels of transcripts of genes in response to pathological effects. Currently it has been shown that informational RNA and various types of noncoding RNA (ncRNA), in particular, microRNA (miRNA) and long ncRNA, are actively involved in the response to the pathology. Recently, the idea that long ncRNAs can interact with miRNAs and diminish their activity has been actively developed. Such functions are attributed to circular RNA (circRNA), which is a new and actively studied type of RNA. circRNAs can also participate in the pathogenesis of various neurodegenerative and inflammatory diseases and cancer. These properties of circRNAs can be exploited in medicine to develop technologies to correct pathological processes caused by disruption of gene expression. This chapter will examine the most recent data on the roles of circRNAs, miRNAs, mRNAs, and their interactions in brain cells under normal conditions and in cerebral ischemia.

#### **2. Ischemic stroke**

According to the latest data from the World Health Organization, ischemic stroke, which is the result of a permanent or temporary decrease in cerebral blood flow, is in most cases caused by occlusion of cerebral arteries by a thrombus or embolus and is of particular importance among vascular conditions [1–3]. This serious condition is the second most common cause of the general mortality rate of the population in Russia and is the most common cause of impaired brain function [4]. Long-term studies of ischemic stroke have proven the existence of necrosis and penumbra zones in the first hours and days after the development of ischemic stroke. The penumbra is the tissue located around the ischemic nucleus in conditions of limited access of oxygen and glucose, and cells in the penumbra are capable of recovery. The concept of a "therapeutic window" was developed in which this window is a period during which the restoration of penumbra cells is still possible and most effective. The duration of the therapeutic window may vary depending on the organism and model of ischemia, but for most cells, it is limited to 3–6 hours [4–9].

Cerebral ischemia results from biochemical changes in brain tissues after ischemic damage. During ischemia, following the occlusion of the vessel, the glutamate-calcium cascade is activated, contributing to an influx of Ca2+ ions, the formation of intracellular mediators (phosphoinositol and diacylglycerol), membrane depolarization, accumulation of glutamate, and further influx of Ca2+ leading to damage to the cell macromolecules and ultimately to cell death [4, 10]. Among the factors affecting the development of ischemic stroke, it is important to consider the effects of molecular genetic parameters. High hopes of clinicians are placed on identifying and developing systems of genetic markers, which are an important step toward the development of personalized medicine and individualized prevention. It is extremely important to study the genetic systems that determine the mechanisms underlying the events during the therapeutic window, the death of neurons during ischemic damage, and the restoration of neurological functions.

#### **3. Transcriptomics of ischemic stroke**

Recently, as a result of the rapid development of genome-wide analysis and multi-omics technologies, it has become clear that tissue damage and regeneration

**85**

*The Role of Noncoding RNAs in Brain Cells during Rat Cerebral Ischemia*

during ischemia is a complex process resulting from a change in transcript levels of a significant number of genes in response to pathological effects. Thus, early-response genes such as *c-fos* and *c-jun* [11] and zinc finger genes trigger cell proliferation and differentiation [12, 13], while genes that encode heat-shock proteins are involved in the inflammatory response and cytoskeleton organization [14], and others are predominantly activated after the onset of ischemia. Of great importance and perspective in molecular genetic studies are the models based on small laboratory animals that best reflect certain features of the development of the ischemic process. Study of the molecular mechanisms of cell death using pMCAO and tMCAO models conducted by Ford et al. revealed molecular functions and biological processes unique for each model [15]. Genes unique to tMCAO were predominantly involved in the induction of inflammatory and oxidative stress, while pMCAO resulted in the expression of genes that were more associated with metabolic activity and cellular signaling [15]. A study of the dynamics of changes in gene expression in rat brain a day after pMCAO revealed a substantial number of genes that changed expression significantly and are involved in the development of ischemic damage, including those determining cell survival and death, the immune response, functioning of the vascular system, and also processes associated with hematopoiesis, immune cells, lymphocytes, leucocytes, and other cells [16].

The most frequently used tMCAO model showed a reorganization of the functioning of many genes in various areas of rodent brains, including the infarction center, during the first day after the transient occlusion [15, 17–19]. In particular, activation of the transcription factor Nf-κb was shown. An increase of the mRNA level of *Cox2*, which encodes one of the key enzymes for the synthesis of the pro-inflammatory prostaglandin E2 (PGE2), was accompanied by an increase in the level of the corresponding protein, not only at the source but also in adjacent regions, and accompanied by increased concentration of PGE2 [20–22]. At the same time, as a result of the opening of the blood-brain barrier in brain sections, extensive leucocyte infiltration was observed [21, 23, 24]. An increase of the mRNA level of the gene for INOS, encoding an enzyme for the synthesis of NO, also participating in the development of the inflammatory response in the lesion, was also noted [22, 25]. In the ischemia-reperfusion model, it was also shown that cytokines (IL-1β, IL6), adhesion molecules (ICAM1, E-selectin, MMP-9), MAPK kinase, and c-fos transcription factors were involved in the development of inflammation [17, 20, 23, 26–29]. Wang et al. studied the molecular mechanism of ischemia-reperfusion pathogenesis using genome-wide transcriptome analysis (RNA-Seq) in the hippocampus of rats at 24 h after tMCAO. These investigators detected 182 differentially expressed genes (DEGs), most of which were upregulated [17]. A Gene Ontology analysis showed that these DEGs were mainly associated with inflammation, stress, immune response, glucose metabolism, and apoptosis [17]. Our analysis of gene expression under tMCAO conditions using RNA-Seq confirmed these results. However, in the subcortical structures of the brain that contained the focus of ischemic damage and the penumbra, we identified hundreds of genes that changed expression 24 h after tMCAO using RNA-Seq. Among these, we found activation of genes involved in inflammatory and immune reactions. There were gene encoding chemokines (*Ccl2* and *Ccl3*), heat-shock proteins (*Hspa1* and *Hspb1*), macrophage receptors (*Msr1*), secreted phosphoprotein 1 (*Spp1*), cytokine 3 suppressor (*Socs3*), and other proteins. Mass suppression of genes that ensure the functioning of neurotransmitter systems (*Chrm1*, *Chrm4*, *Cplx2*, *Drd2*, *Gabra5*, and *Gng7*) was also shown [19]. A study of the dynamics of changes of gene expression in rat brain a day after tMCAO conditions revealed a significant activation of the expression of genes involved in biosynthetic cell systems (ribosome, proteasome, DNA replication, and purine metabolism functional categories). The effect obtained indicated

*DOI: http://dx.doi.org/10.5772/intechopen.88402*

#### *The Role of Noncoding RNAs in Brain Cells during Rat Cerebral Ischemia DOI: http://dx.doi.org/10.5772/intechopen.88402*

*Non-Coding RNAs*

**2. Ischemic stroke**

after thrombolysis not only contributes to the restoration of penumbra cells but also causes additional damage to brain cells, including disruption of endothelial micro-

Ischemic brain damage in combination with reperfusion damage is a complex process resulting from changes in the levels of transcripts of genes in response to pathological effects. Currently it has been shown that informational RNA and various types of noncoding RNA (ncRNA), in particular, microRNA (miRNA) and long ncRNA, are actively involved in the response to the pathology. Recently, the idea that long ncRNAs can interact with miRNAs and diminish their activity has been actively developed. Such functions are attributed to circular RNA (circRNA), which is a new and actively studied type of RNA. circRNAs can also participate in the pathogenesis of various neurodegenerative and inflammatory diseases and cancer. These properties of circRNAs can be exploited in medicine to develop technologies to correct pathological processes caused by disruption of gene expression. This chapter will examine the most recent data on the roles of circRNAs, miRNAs, mRNAs, and their

vascular cells, the excess oxygen radicals, and activation of apoptosis.

interactions in brain cells under normal conditions and in cerebral ischemia.

According to the latest data from the World Health Organization, ischemic stroke, which is the result of a permanent or temporary decrease in cerebral blood flow, is in most cases caused by occlusion of cerebral arteries by a thrombus or embolus and is of particular importance among vascular conditions [1–3]. This serious condition is the second most common cause of the general mortality rate of the population in Russia and is the most common cause of impaired brain function [4]. Long-term studies of ischemic stroke have proven the existence of necrosis and penumbra zones in the first hours and days after the development of ischemic stroke. The penumbra is the tissue located around the ischemic nucleus in conditions of limited access of oxygen and glucose, and cells in the penumbra are capable of recovery. The concept of a "therapeutic window" was developed in which this window is a period during which the restoration of penumbra cells is still possible and most effective. The duration of the therapeutic window may vary depending on the organ-

ism and model of ischemia, but for most cells, it is limited to 3–6 hours [4–9]. Cerebral ischemia results from biochemical changes in brain tissues after ischemic damage. During ischemia, following the occlusion of the vessel, the glutamate-calcium cascade is activated, contributing to an influx of Ca2+ ions, the formation of intracellular mediators (phosphoinositol and diacylglycerol), membrane depolarization, accumulation of glutamate, and further influx of Ca2+ leading to damage to the cell macromolecules and ultimately to cell death [4, 10]. Among the factors affecting the development of ischemic stroke, it is important to consider the effects of molecular genetic parameters. High hopes of clinicians are placed on identifying and developing systems of genetic markers, which are an important step toward the development of personalized medicine and individualized prevention. It is extremely important to study the genetic systems that determine the mechanisms underlying the events during the therapeutic window, the death of neurons during

ischemic damage, and the restoration of neurological functions.

Recently, as a result of the rapid development of genome-wide analysis and multi-omics technologies, it has become clear that tissue damage and regeneration

**3. Transcriptomics of ischemic stroke**

**84**

during ischemia is a complex process resulting from a change in transcript levels of a significant number of genes in response to pathological effects. Thus, early-response genes such as *c-fos* and *c-jun* [11] and zinc finger genes trigger cell proliferation and differentiation [12, 13], while genes that encode heat-shock proteins are involved in the inflammatory response and cytoskeleton organization [14], and others are predominantly activated after the onset of ischemia. Of great importance and perspective in molecular genetic studies are the models based on small laboratory animals that best reflect certain features of the development of the ischemic process. Study of the molecular mechanisms of cell death using pMCAO and tMCAO models conducted by Ford et al. revealed molecular functions and biological processes unique for each model [15]. Genes unique to tMCAO were predominantly involved in the induction of inflammatory and oxidative stress, while pMCAO resulted in the expression of genes that were more associated with metabolic activity and cellular signaling [15]. A study of the dynamics of changes in gene expression in rat brain a day after pMCAO revealed a substantial number of genes that changed expression significantly and are involved in the development of ischemic damage, including those determining cell survival and death, the immune response, functioning of the vascular system, and also processes associated with hematopoiesis, immune cells, lymphocytes, leucocytes, and other cells [16].

The most frequently used tMCAO model showed a reorganization of the functioning of many genes in various areas of rodent brains, including the infarction center, during the first day after the transient occlusion [15, 17–19]. In particular, activation of the transcription factor Nf-κb was shown. An increase of the mRNA level of *Cox2*, which encodes one of the key enzymes for the synthesis of the pro-inflammatory prostaglandin E2 (PGE2), was accompanied by an increase in the level of the corresponding protein, not only at the source but also in adjacent regions, and accompanied by increased concentration of PGE2 [20–22]. At the same time, as a result of the opening of the blood-brain barrier in brain sections, extensive leucocyte infiltration was observed [21, 23, 24]. An increase of the mRNA level of the gene for INOS, encoding an enzyme for the synthesis of NO, also participating in the development of the inflammatory response in the lesion, was also noted [22, 25]. In the ischemia-reperfusion model, it was also shown that cytokines (IL-1β, IL6), adhesion molecules (ICAM1, E-selectin, MMP-9), MAPK kinase, and c-fos transcription factors were involved in the development of inflammation [17, 20, 23, 26–29]. Wang et al. studied the molecular mechanism of ischemia-reperfusion pathogenesis using genome-wide transcriptome analysis (RNA-Seq) in the hippocampus of rats at 24 h after tMCAO. These investigators detected 182 differentially expressed genes (DEGs), most of which were upregulated [17]. A Gene Ontology analysis showed that these DEGs were mainly associated with inflammation, stress, immune response, glucose metabolism, and apoptosis [17]. Our analysis of gene expression under tMCAO conditions using RNA-Seq confirmed these results. However, in the subcortical structures of the brain that contained the focus of ischemic damage and the penumbra, we identified hundreds of genes that changed expression 24 h after tMCAO using RNA-Seq. Among these, we found activation of genes involved in inflammatory and immune reactions. There were gene encoding chemokines (*Ccl2* and *Ccl3*), heat-shock proteins (*Hspa1* and *Hspb1*), macrophage receptors (*Msr1*), secreted phosphoprotein 1 (*Spp1*), cytokine 3 suppressor (*Socs3*), and other proteins. Mass suppression of genes that ensure the functioning of neurotransmitter systems (*Chrm1*, *Chrm4*, *Cplx2*, *Drd2*, *Gabra5*, and *Gng7*) was also shown [19]. A study of the dynamics of changes of gene expression in rat brain a day after tMCAO conditions revealed a significant activation of the expression of genes involved in biosynthetic cell systems (ribosome, proteasome, DNA replication, and purine metabolism functional categories). The effect obtained indicated

a large-scale reorganization of nucleic acid and protein biosynthesis that was apparently related to the adaptive response of brain cells to the damage caused by ischemia-reperfusion.
