The Mechanism Of Long Non-coding RNA NR₀24430 In The Aneurysm Development Of Marfan Syndrome

Marfan syndrome （MFS） is an autosomal dominant connective tissue disorder caused by fbnl mutation, the rupture of aortic aneurysm is the main causes of mortality in these patients. Current therapies such as surgery and medicine are not ideal, a more effective method should be found based on better understanding of the pathogenesis. Smooth muscle cells and elastic fibers are basic units to maintain the function of aorta, the dysfunction and apoptosis of the smooth muscle cells, degradation of the elastic fibers are the main causes of aneurysm development of Marfan patients. The reasons of behavior disorder of smooth muscle cells and the reasons of extracellular degradation become the main research orientations. Long noncoding RNA （lncRNA） are a kind of non-protein coding RNA longer than 200nt, recent studies revealed that they paly vital roles in various biological processes such as epigenetic regulation, transcription regulation and post-transcription regulation, moreover, some researches showed that some kinds of lncRNA play vital role in regulating the behavior of smooth muscle cells, however, there are on reports about the roles of certain lncRNA in the aneurysm development of Marfan syndrome. Given its various regulatory roles in the cardiovascular development and smooth muscle cells, we proposed that some kinds of lncRNA play vital roles in the aneurysm development of Marfan syndrome. In this study, we use human aortic tissues as the research material, and microarrays were used to screen the differentially expressed mRNA and lncRNA, furthermore, bioinformatic analysis were used to find the lncRNA which are closely related to the aneurysm development of Marfan syndrome, focusing on its roles in regulating the behavior of smooth muscle cells and the remodeling of the extracellular matrix, trying to find key signaling pathway in the aneurysm development to determine a new, specific, and sensitive therapeutic target, which then provides substantial rationale for accurately treatment for Marfan syndrome and the improvement of clinical situation.Part I:Expression profiles of LncRNA and mRNA in Marfan patients and related bioinformatic analysisWe performed microarray analysis in human aortic root tissues derived from the patients who underwent the coronary artery bypass surgery （no pathological changes in aortic root, control group, n=5） and Bentall surgery （Marfan patients, experimental group, n=5）, TRIZOL method was used to extracted the total RNA, and we use limma method to screen the differentially expressed mRNA and lncRNA, GO analysis was performed to find the significantly affected functions; pathway analysis was performed to find out the significantly affected pathways; GO trees were constructed to better understand the relationships of all the significantly affected functions; Gene-Act-Net was constructed to find out the core regulatory genes; Co-expression-Network was constructed to find out the core regulatory lncRNA. qRT-PCR was performed to validate the expression of related mRNA and lncRNA.The microarray results showed that there are 602 differentially expressed mRNA and 376 differentially expressed lncRNA （Fold change>1.5, P<0.05） in the aortic tissues derived from MFS patients, GO analysis revealed that the differentially expressed mRNA are mainly about inflammatory response and the extracellular remodeling; pathway analysis showed that PPAR and some metabolism related pathways significantly down-regulated, while ECM-receptor interaction, protein digestion and absorption, PI3K-AKT and TGF- βsignificantly up-regulated. mRNA-lncRNA co-expression-network analysis indicate that a significantly down-regulated lncRNA named NR₀24430 is closely related to the aneurysm development of MFS, and the following qRT-PCR assay validated its expression level in aortic tissues.Part II:The role of NR 024430 in the aneurysm development of MFS and related mechanismsPrimary human aortic smooth muscle cells （HASMCs） were used to detect the function of NR₀24430 and related mechanisms. RNA-FISH was used to localize the NR₀24430 in the HASMCs; ASOs were used to knockdown NR₀24430 in the HASMCs, after that, RNA-seq and bioinformatic methods were used to find out the downstream molecular and biological function, furthermore, qRT-PCR assays were performed to validate the expression level of NR₀24430 downstream molecular. In vitro transcription assay combined with the proteomic technology were used to detect the NR₀24430 binding proteins. Human recombinant TGF-β1 was used to stimulate HASMCs in order to verify the relationship between non-canonical TGF-β 1 signaling and the NR₀24430 signaling pathway.The RNA-FISH results indicated that NR₀24430 are mainly expressed in the nucleus, RNA-seq results showed that after knockdown NR₀24430, the apoptotic rate of HASMCs increased significantly, as well as inflammatory cytokines and MMP-1, MMP-9, and these changes are key biological processes in the aneurysm development of MFS. Further research revealed that the NR₀24430 signaling pathway is independent of non-canonical signaling pathway, and both of them play important roles in the aneurysm development of MFS. The proteome results showed that most of the NR₀24430 binding proteins are transcriptional factors, we proposed that the function of NR₀24430 are closely related to the regulation of these transcriptional factors.Part â…¢:Changes of smooth muscle cells in aortic tissues of MFSA kind of MFS mouse model （FBN1 C1O39G/+） was used in this part, HE combined with immunofluorescent staining to detect the morphological change of smooth muscle cells in MFS. TUNEL assay was used to detect the apoptotic smooth muscle cells in mouse aortic tissues; qRT-PCR assays were used to detect the expression level of calcium related genes; Fluo-4 was used to label the cytoplasmic calcium ion, and combined with confocal microscopy, the calcium store was detected in the smooth muscle cells.The results showed that the abnormal arrangement of smooth muscle cells in MFS aortic tissues, smooth muscles aggregates abnormally. The apoptotic rate of smooth muscle cells are higher in MFS group, the mRNA of ryr2 in MFS group are significantly down-regulated, furthermore, the calcium store in primary smooth muscle cells isolated from MFS mouse is higher than that in the control group.Conclusions1. NR₀24430 are significantly down-regulated in aortic tissues derived from MFS patients.2. NR₀24430 are mainly expressed in the nucleus.3. NR₀24430 are involved in aneurysm development of MFS.4. NR₀24430 knockdown can induce apoptosis of smooth muscle cells.5. NR₀24430 knockdown can promote smooth muscle cells secrete inflammatory cytokines.6. NR₀24430 knockdown can promote smooth muscle cells secrete MMP-1 and MMP-9.7. TGF- β1 can promote smooth muscle cells secrete MMP-2.8. The NR₀24430 related signaling pathway is independent of noncanonical TGF-β1 signaling pathway, and both of them play important roles in the aneurysm development of MFS.9. The behavior disorder of smooth muscle cells in MFS （increased apoptotic rate, abnormal arrangement, increased calcium store）.