| Thoracic aortic aneurysm is a very dangerous cardiovascular-surgery disease,which is characterized by strong latent, poor prognosis, complicated postoperative complications and more. In recent years, the incidence of thoracic aortic aneurysms increased year by year. The current study shows that many factors involved in the occurrence of thoracic aortic aneurysms, such as smoking, hypertension, age, family history, atherosclerosis and so on. There may no symptoms could be detected during the early stage of thoracic aortic aneurysm, only when the aorta continued to expand and oppress the surrounding organs or tissues, some non-specific symptoms such as cough,difficulty breathing, dysphagia could be found. The treatment of thoracic aortic aneurysm is limited. To those involve the ascending aorta or aortic arch, the cardiac surgery for aorta replacement could be the only option. On the other hand, the aorta replacement operation is quite difficult procedure because of high technique requirement,the various complications and poor prognosis. Therefore, to explore the effective molecular mechanism involve in the development of thoracic aortic aneurysm, and as a therapeutic target will provide a new basis for the treatment of thoracic aortic aneurysm.Aortic wall is divided into 3 layer, inner membrane, medial membrane, outer membrane. The medial membrane is considered as the most important part of the aortic wall. The medial membrane is composed of vascular smooth muscle cells (VSMCs),collagen, elastic fibers and a variety of extracellular matrix components. Any pathological changes from the above components may lead to decreased vascular wall systolic function and strength. Under the high-speed high blood pressure inside the aorta,vascular wall could expand gradually and eventually leading to the occurrence of aortic aneurysm. More current research has declared the possible molecular mechanisms; 1,VSMCs transform from the contractile phenotype to the secretion, thus affecting its original contractile function and vascular wall strength; 2, the composition of the vessel wall, such as collagen and elastic fibers,was destructed,leading to increasing blood vessel wall fragility, decreased flexibility; 3, external factors stimulation, VSMCs or secretion of inflammatory cells and activation of matrix metalloproteinase 2 and 9,destroy collagen, resulting in destruction of the vessel wall components; 4, TGFβpathway abnormality in recent years is considered to be an important factor in the occurrence of aortic aneurysm. Any factors involved in or affecting the above process may lead to the occurrence and development of aortic aneurysms.miRNA is a class of endogenous non-coding RNA having regulatory functions found in eukaryotes. It’s about 20-25 nucleotides in length, and its main mechanism of action include: binding to the mRNA-related bases, inhibiting mRNA after transcription and translation; by way of inhibition of mRNA CAP or Poly (a) dependent translation; affect mRNA polyadenylation off. MiRNA precursors under the influence exprotin-5 transport the nuclear complex,has become a mature miRNA dicer cut,and further formation of RNA-induced silencing complex (RISC) in the cytoplasm, the mRNA the 3’-UTR region combine to play a role when the miRNA is fully complementary with related targets, and even can cause degradation of the mRNA. Currently some studies indicate, miRNA affect gene promoter CpG island methylation of target gene regulation directly at the transcriptional level. Numerous studies show that miRNA may be involved in the development of aortic aneurysm. Kim and other studies have shown that miR-712 capable of targeting and inhibiting TIMP3 RECK expression, thus breaking the matrix metalloproteinases and their inhibitors balance in vitro and in vivo experiments, to promote the expansion of aortic aneurysm model mouse vascular wall. Cheng and Cordes other studies have shown inhibition of miR-143/145 has been significantly SMC in a-SMA, Calponin and SM-MHC expression, and thus involved in the development of aortic aneurysm. Some studies have shown that overexpression of SMC miR-21 can promote SMC proliferation and inhibit apoptosis. Maegdefessel are also through animal experiments show that in vivo overexpression of miR-21 can inhibit the expansion of aortic aneurysm. In summary,a variety of miRNA through many different pathways involved in the occurrence of aortic aneurysms, but exploring a new and very effective as an intervention miRNA targets aortic aneurysm is still very necessary.In this research, we intend to solve the following problems :( 1) by miRNA microarray expression differences in the aortic vessel wall miRNA, and verified in the specimen; (2)the in vitro experimental observation Effect of miRNA on VSMCs biological activity; (3)by constructing the aorta through specific expression in transgenic mice, observing the impact of the miRNA of aortic aneurysms occur in vivo; (4) to explore the miRNA new downstream target genes, and verify its functionality. The following will be discussed on the four parts to discuss.Part Ⅰ The differentially expressed miRNA in the vascular wall from thoracic aortic aneurysmObjective Screening the differentially expressed miRNA in the vascular wall from thoracic aortic aneurysmMethod By bioinformatic analysis to identify more than an important role in the TGFβ signaling pathway of miRNA as an alternative to be detected miRNA. Aortic dilatation was 1.5 times aortic aneurysm diagnostic criteria collected in the vessel wall,Changhai Hospital cardiovascular surgical specimens of patients with thoracic aortic aneurysm, which exclude hereditary disease or an autoimmune disease caused by aneurysms, syphilitic arterial Tumor, traumatic aneurysm and so on. Take aortic graft or aortic valve replacement and other patients with aortic wall as a normal control group. In specimens obtained in the above-described alternative miRNA detection by fluorescence quantitative PCR, selected as the object of the research the most significant difference in the expression of miRNA. By in situ hybridization to detect the miRNA localization in the specimen.Result RT-PCR results suggest that in the TGFβ pathway of miRNA, miR-494 expression was significantly decreased, miR-494 in situ hybridization prompted mainly located in the middle of the vessel wall in the vascular smooth muscle cells in the vessel wall in patients with aortic aneurysms.Conclusion miR-494 expression level is lower than normal in patients with aortic aneurysms the vessel wall, and are mainly located in the middle of the vessel wall.Part Ⅱ Impact of miR-494 on the biological function of vascular smooth muscle cellsObjective To investigate the effect of miR-494 on the biological function of aortic vascular smooth muscle cells.Methods Cultured primary vascular smooth muscle cells (h-VSMCs) were cultured in 6-well plates until cell density and phenotype were stable. The cell density was 70%and treated with TGFβ(10 ng / ml) H-VSMCs were detected at different time points. The miR-494 expression was observed and the changes of phenotypic transformation related genes were detected at time. (SM22, MYH11) were detected by lipo2000 transfection with miR-494-mimic. The expression of h-VSMCs was detected by Cck-8. The proliferation of h-VSMCs was detected by Cck-8. The migration of h-VSMCs was detected by scratch test. The secretion of h-VSMCs was detected by ELISA.Results TGFβ could significantly increase the level of miR-494 in h-VSMCs, and gradually increased with time point. TGFβ could promote the expression of contractile-related genes and began to decrease at 72 h after stimulation. The expression of miR-494 in h-VSMCs was inhibited by transfection of miR-494-mimic at 48 h after TGFβ treatment. The expression of miR-494-inhibitor was down-regulated after 72 h of TGFβ treatment H-VSMCs in miR-494 levels, can promote the expression of contractile-related proteins. Overexpression of miR-494 in h-VSMCs significantly inhibited the expression of contractile-associated proteins and promoted the proliferation and migration of h-VSMCs. The expression of Collagen could significantly inhibit the expression of Elastin and inhibit the secretion of MMP2.Conclusion miR-494 can induce the expression of miR-494. When miR-494 reaches a certain level, it can inhibit the effect of TGFβ by negative feedback. MiR-494 can induce the transformation of h-VSMCs from synthetic to contractile type. MiR-494 can significantly inhibit MMP2 secretion.Part Ⅲ The mechanisms of miR-494 impact the biological function of vascular smooth muscle cellsObjective To explore and determine the function of miR-494 target gene and target gene, and to explore the mechanism of miR-494 inhibiting MMP2 secretion.Methods The miR-494 potential target gene was further investigated by GO analysis and the miR-494 target gene was further searched by GO analysis. The luciferase reporter gene was constructed and the direct target gene of miR-494 was determined by luciferase reporter assay. The expression level of target gene after miR-494 expression in h-VSMCs was detected by Western Blot, and the target gene and its function were further verified by rescue test (Rescue test). Western blot analysis showed that miR-494 could inhibit the expression of Syndecan-1 (SDC-1) and inhibit the level of MMP2, and the effect of SDC-1 on miR-494 inhibition of MMP2 expression was clarified by rescue experiment.Results The transcription factor nuclear factor of activated T-cells 5 (NFAT5) was the direct target gene of miR-494, and the luciferase reporter gene was further screened by multiple data screening and GO analysis. The experiment confirmed that NFAT5 binds directly to miR-494. Further overexpression of miR-494 in h-VSMCs significantly inhibited the expression of NFAT5. Salvage test demonstrated that miR-494 could promote the phenotype transformation, proliferation and migration of h-VSMCs by inhibiting NFAT5, and overexpression of NFAT5 could reverse this process.Overexpression of miR-494 can significantly inhibit the expression of SDC-1 and MMP2 in h-VSMCs, while increasing the level of SDC-1 while overexpressing miR-494 can alleviate the inhibitory effect of miR-494 on MMP2.Conclusion miR-494 can promote the transformation,proliferation and migration of h-VSMCs by direct inhibition of NFAT5. On the other hand, miR-494 inhibits the synthesis of MMP2 by SDC-1.Part Ⅳ The mechanisms of miR-494 relieve the development of aortic aneurysmsObjective To explore the effect of miR-494 on the development of aortic aneurysm by in vivo experiment.Methods The mouse model of aortic aneurysm was established by using Apoe - / -mice combined with Angiotensin II (Ang II). Subcutaneous injection of miR - 494 inhibitor miR - 494 - Antagomir was performed by mouse aortic ultrasonography Aortic aneurysm formation. The pathological changes of the vessel wall were observed by HE staining and VB staining. Construction of aortic ovarian-specific miR-494 overexpression of transgenic mice, and the use of the mouse to construct aortic aneurysm,through the above method to detect the formation of aortic aneurysm and pathological changes. The mechanism of miR-494 on the development and progression of aortic aneurysm was explored by ELISA, immunohistochemical staining and in situ gelatin zymography.Results Apoe-/- mice combined with Ang II could effectively construct aortic aneurysm model. In the miR-494-Antagomir injection group, the diameter of the aorta was significantly larger than that of the model group. VB staining indicated that the miR-494-Antagomir injection group Elastic fibers and collagen damage is also more serious. In aortic aneurysm model constructed by miR-494 transgenic mice, aortic aneurysm dilatation was significantly improved. VB staining suggested that the damage of vascular fibers and collagen in vascular wall of transgenic mice was improved compared with that of wild type mice. The results of ELISA showed that the level of MMP2 in plasma of miR-494 overexpressing mice was significantly lower than that of wild-type mice. Immunohistochemistry showed that miR-494 could significantly inhibit the expression of MMP2 in mouse vascular wall. The gelatin zymogram demonstrated that miR-494 was able to inhibit the activity of MMPs in the vessel wall.Conclusion In vivo experiments show that overexpression of miR-494 has a protective effect on aortic aneurysm model. This protective effect is mainly through miR-494 inhibition of MMPs synthesis, secretion and activity to achieve. |
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