Dysregulated miRNAs have been associated with the etiopathogenesis of many cerebrovascular disorders, and altered plasma miRNA expression has been previously reported in aSAH (Jamaluddin et al. 2011). In our study, using miRNA microarray analysis we identified 70 miRNAs with significantly altered expression (log fold-change greater or less than 2) in aneurysm tissues compared to control tissues: 67 were down-regulated, and 3 were up-regulated. The top 10 dysregulated miRNAs were further validated individually using qPCR, and these miRNAs were observed to be significantly downregulated in aneurysm tissues.
Recently, limited miRNA expression studies have identified several differentially expressed miRNA in aneurysm tissue (Bekelis et al. 2016a; Liu et al. 2014a). However, their results are not readily comparable, which may be due to the use of different analytical methods (different microarray-based methodologies, qPCR and sequencing), validation strategies (independent testing vs no validation vs validation in the same cohort) and control tissues. Mean while, we further explored the association of the differentially expressed miRNA with the clinical characteristics of patients. Expression levels of 2 miRNAs (miR-125b-5p and miR-143-3p) were considerably lower in patients with vasospasm compared to patients without vasospasm. Moreover, expression of miR-125b-5p, miR-143-3p and miR-199a-5p was significantly down-regulated in patients with WFNS grades 3 & 4 compared to those with WFNS grade 1 & 2. Previous studies report that miR-143/miR-145 is highly expressed in vascular smooth muscle cells, endothelial cells, and inflammatory cells (Cheng et al. 2009). Also, it has been suggested that miR-143/mir-145 are critical modulators of vascular smooth cell phenotype in response to shear stress in atherosclerosis and hypertension (Santovito et al. 2013). In addition, a prior study by Bekelis et al in patients with unruptured aneurysms, reported that miR-143 is significantly downregulated and contributed to the modulation of vascular smooth muscle cell phenotype (Bekelis et al. 2016b). A recent study showed that miR-125b-5p regulates both the innate immune response and the inflammatory process by directly targeting the expression of a gene encoding 5-lipoxygenase enzyme involved in the biosynthesis of leukotrienes (Busch et al. 2015). Furthermore, miR-125b is reported to be associated with cell proliferation, apoptosis, and vascular smooth cell phenotyping in aneurysms (Liu et al. 2014b; Robinson and Baker 2012). Thus, expression of miR-125b-5p and miR-143-3p could be related to disease progression or severity, and could perhaps predict clinical outcome.
It is well known that in humans, conserved miRNA preferentially targets many sets of mRNA, which play vital roles in multiple biological pathways (Cortez et al. 2011). However, the contribution of gene networks targeted by the deregulated miRNAs in aneurysm biology is not known. Although few studies explicitly link miRNA to its downstream target gene, no study has experimentally validated the gene targets of miRNAs in aneurysm tissue. To address this, we used the interaction of the experimentally verified miRNA with gene information module to find the genes targeted by the miRNAs, and found that these miRNAs targeted the expression of 3822 unique genes in aneurysm tissue. KEGG pathway analysis indicated that the target genes of the dysregulated tissue miRNA were exclusively associated with several signaling pathways, including, TGF-β, MAPK, and NF-kB signaling pathway underlying its importance in aneurysm biology.
In recent years, the TGF-β signaling pathway has gained special attention since TGF-β is a multifaceted cytokine that regulates a diverse range of cellular activities (Morikawa et al. 2016; Moustakas et al. 2002). Several studies report that numerous miRNAs can induce TGF-β, and it is a chief domain which activates key components of the downstream cell signaling cascade. Various reports suggest that TGF-β is essential for maintaining vascular integrity and function. In rats, middle cerebral artery occlusion resulted in elevated TGF-β expression (Vincze et al. 2010). Experimental evidence in mice reported that TGF-β signaling amplifies with an increase in age and signals to innate immune cells and astrocytes after stroke (Doyle et al. 2010). The relationship between TGF-β and IA is still poorly understood. Mutation in the genes encoding ENG/endoglin and TGFBR3/ betaglycan transmembrane proteins that modulate TGF-β predispose to aneurysm pathogenesis (Santiago-Sim et al. 2009). In our present study, we found that expression of miR-26b and miR-199a markedly decreased and the expression of TGF-β1 and TGF-β2 in aneurysm tissue increased compared to controls. In addition, 3'UTR of SMAD2 and SMAD4 mediator of TGF-β signal transduction was targeted by miR-497 and miR-26b. Expression of miR-497 and miR-26b was downregulated and SMAD2 and SMAD4 levels were over expressed in aneurysm tissue. These findings suggest that abnormally expressed miR-26b, miR-199a and miR-497 may play a role in aneurysm biology through TGF-β signaling mediated by SMAD2 and SMAD4 transcription factors.
Mitogen-activated protein kinases (MAPKs) are serine/ threonine protein kinases, and the two MAPKs: MAPK1/ERK2 and MAPK3/ERK1, play central roles in the MAPK/ERK signaling. Typically, they control various transcription factors that govern the transcription of an array of genes involved in endothelial cell proliferation, cytoskeletal, and vascular remodeling by phosphorylating protein kinases (Bogatcheva et al. 2003; Plotnikov et al. 2011). Also, MAPK cascade is activated by a wide range of extracellular stimuli such as growth factors, cytokines, and also in response to cellular stress (Yoon and Seger 2006). In experimental animals, rapid activation of ERK was seen in balloon-injured arteries, hypertensive vascular tissue (Kim et al. 1998). In addition, ERK was activated in rat aortic vascular smooth muscle cells in vitro in response to both cyclic strain and shear stress (Hu et al. 1998). Interestingly, ERK activation in cultured endothelial cells was higher in response to shear stress compared to cyclic strain (Azuma et al. 2000). Furthermore, the involvement of MAPKs in the regulation of miRNAs expression has also been reported (Hong et al. 2013). However, the role of miRNA and MAPKs in the pathobiology of IAs is unclear. In this study, we examined the expression of miR-365 and miR-497, targeting MAPK1 and MAPK3, respectively, in aneurysm tissues. Our study revealed that the expression of miR-365a and miR-497 was decreased, and MAPK1 and MAPK3 increased in aneurysm tissues compared to controls suggesting a role for miRNA in regulating the stress-activated kinase, ERK, in the aneurysm wall. Our results are supported by a study by Maddahi et al., which showed that the ERK1/2 pathway was activated in IA tissues post-SAH (Maddahi et al. 2012).
We have systematically analyzed the biological functions and downstream signaling pathways associated with target genes of dysregulated tissue miRNAs (Fig. 9). These data suggest that miRNA could play a significant role in the dysfunction and remodeling of vascular endothelial and smooth muscle cells by influencing inflammatory immune processes via the downstream regulation of genes, which could, in turn, contribute to the pathophysiology of aneurysm rupture and aSAH.
Our study has few limitations. Since we included a relatively small number of patients in the validation cohort, we failed to define significant association between miRNA expression and clinical outcome. Further, target mRNA of tissue miRNAs was validated based on functional analysis and we failed to validate these putative miRNA targets in-vivo. An additional limitation is that miRNA microarray profiling can only measure miRNAs that are included in the array and cannot discover new miRNAs.
To conclude, this study identified several differentially expressed miRNAs in ruptured intracranial aneurysmal tissue through microarray analysis. Bioinformatic analysis showed that miR-26b, miR-199a, miR-497 and miR-365 which were significantly decreased, modulates genes involved in TGF-β and MAPK signaling, which could potentially influence inflammatory processes, extracellular matrix and vascular smooth muscle cell degradation and apoptosis, and ultimately cause vessel wall degradation and rupture. Further functional validations of the target genes of the dysregulated miRNA are necessary for deciphering their exact role in the progression and rupture of IAs.