MicroRNA/mRNA profiling and regulatory network of intracranial aneurysm

Yugang Jiang 1 Mingming Zhang 1 Hua He 0 Jia Chen 0 Hua Zeng 0 Jia Li 0 Ranhui Duan 0 0 State Key Laboratory of Medical Genetics, Central South University , Changsha, Hunan Province , China 1 Department of Neurosurgery, Second Xiang-ya Hospital of Central South University , Changsha, Hunan Province , China Background: Intracranial aneurysm (IA) is one of the most lethal forms of cerebrovascular diseases characterized by endothelial dysfunction, vascular smooth muscle cell phenotypic modulation, inflammation and consequently loss of vessel cells and extracellular matrix degradation. Besides environmental factors, genetics seem to be a very important factor in the genesis of this disease. Previous mRNA expression studies revealed a large number of differentially expressed genes between IA and control tissue. However, microRNAs (miRNA), small non-coding RNAs which are post-transcriptional regulators of gene expression, have been barely studied. Studying miRNAs could provide a hypothetical mechanism underlying rupture of IA. Methods: A microarray study was carried out to determine difference in microRNAs and mRNA between patients' IA tissues and controls. Quantitative RT-PCR assay compared the expression level between two groups (14 IA domes vs. 14 controls) were used for validation. Validated miRNAs were analyzed using Ingenuity Pathway Analysis (IPA) to identify the networks and pathways. Results: 18 miRNAs were confirmed by qPCR to be robustly down-regulated in 14 ruptured IA patients including hsa-mir-133b, hsa-mir-133a, hsa-mir-1, hsa-mir-143-3p, hsa-mir-145-3p, hsa-mir-145-5p, hsa-mir-455-5p, hsa-mir-143-5p, hsa-mir-23b-3p etc., of which 11 miRNAs are clusters: hsa-mir-1/has-mir-133a, hsa-mir-143/hsa-mir-145, hsa-mir-23b/ hsa-mir-24-1, and hsa-mir-29b-2/hsa-mir-29c. 12 predicted functions were generated using IPA which showed significant associations with migration of phagocytes, proliferation of mononuclear leukocytes, cell movement of mononuclear leukocytes, cell movement of smooth muscle cells etc. Conclusion: These data support common disease mechanisms that may be under miRNA control and provide exciting directions for further investigations aimed at elucidating the miRNA mechanisms and targets that may yield new therapies for IA. - Background As one of the most devastating neurological conditions known to date, intracranial aneurysm (IA) has a high mortality rate and undesirable prognosis with spontaneous cerebral hemorrhage, cerebral vasospasm, and oculomotor nerve palsy as the main clinical feature. IA is common result of vascular abnormalities in the brain, with a prevalence of 3.2% in the general population, and an overall risk of rupture around 1.2% in western populations and 2.3% in Japanese series [1]. A significant proportion of aneurysmal patients are around the age of 4060 [2,3]. Cigarette smoking, excessive alcohol consumption, hypertension and female gender are significant risk factors for IA formation and growth, and family history of IA has also been suggested to be evidence for genetic causality of cerebral aneurysms. Dysfunction of vessel cells, degeneration of vessel wall and activation of immune system were identified to be the intrinsic factors of IA development [3-6]. Its unpredictable nature and the catastrophic consequences of IA rupture remain a challenge for clinicians. Comprehensive understanding of IA pathobiology is crucial for reasonable management of IA carriers. Due to the fact that animal models of IA are imperfect and human aneurysmal tissues are difficult to obtain, the molecular mechanisms of IA remain poorly understood. Most studies focus on mRNA expression in aneurysmal and healthy tissue to identify the alteration of gene expression within the vessel wall, which has implied some mechanisms underlying the development of IA. For example, in 2008 Krischek et al. found differentially expressed genes, which indicated that antigen processing was the most significantly associated; another study in 2009 by Shi et al. indicated that misregulated genes were mostly correlated with focal adhesion, ECM-receptor and cell communication etc. Because the large amounts of data created with each study, make a comparison or interpretation of results difficult, Roder et al. (2012) performed a meta-analysis which found seven genes showing altered expression in more than three studies: BCL2, COL1A2, COL3A1, COL 5A2, CXCL12, TIMP4, TNC [7-13]. Functional studies on these genes showed that COL1A2, COL3A1, COL5A2, TIMP4, and TNC could modulate processes in the formation of the extracellular matrix (ECM), which have been described in association with IAs [10,14]. miRNA may be another layer of control in gene expression which modulates pathways and mechanisms of IA, however, expression of miRNA in IA is rarely studied. A novel direction for IA research is the modulation of miRNA, endogenous approximately 23 nt non-coding RNAs. By binding to the 3 UTR of complementary protein-coding mRNAs, miRNA primarily acts in the posttranscriptional repression of gene expression in animals and plants. miRNAs are incorporated into the RNA induced silencing complex (RISC) and then inhibit gene expression by either mRNA degradation or inhibiting translation which can thereby regulate up to 75% of the human genome which belong to many biological pathways including immune response and apoptosis [15-19]. Dysregulation of miRNAs have been found to have relevance to tumorigenesis, neurological, cardiovascular and developmental and other diseases [20]. Recent studies have demonstrated that miRNAs play roles in vascular remodelling and atherosclerosis [21,22]. miRNA may be another layer of control in gene expression which modulates pathways and mechanisms of IA, however, expression of miRNA in IA is rarely studied. The role of miRNA in the molecular mechanism of IA has been of particular interest. Our study focused on investigating how the differential expression patterns of regulatory microRNAs in IA act as a potential regulator in its pathological mechanism. We generated a microRNA array followed by confirmation of miRNAs individually with qRT-PCR. We identified 18 miRNAs in 14 patients which were significantly down-regulated between IA and control tissue, 11 of these miRNAs in the cluster including hsa-mir-1/has-mir-133a, hsa-mir-143/hsa-mir-145, hsamir-23b/hsa-mir-24-1, hsa-mir-29b-2/hsa-mir-29c. Functional analysis indicates these miRNAs are involved with dysfunction and remolding of vascular endothelial cells, vascular smooth muscle cell and involvement of inflammatory/immune processes. Methods Patients and tissue samples Full-thickness vessel wall samples from 14 ruptured IA domes were prospectively collected from patients (10 female, 4 male, age: 52.7 8.5 ) undergoing microsurgical clipping. 14 middle meningeal artery (MMA) segments with matched sex and age were obtained during standard neurosurgical procedures (traumatic hematoma, tumor resection, IA clip (...truncated)