| Background and PurposeAbdominal aortic aneurysm(AAA)is a permanent and irreversible expansion of the local abdominal aorta,and it can lead to the fatal outcome of aortic rupture.At present,the only feasible treatment for AAA is to use surgery to prevent the larger aneurysm(over 5.5cm)rupture-caused death of the patient.However,there are up to 90%of the patients whose AAA aneurysm diameter is below that surgery standard(below 5.5 cm),and there is no confirmed drug or other strategy for this cohort,a dilemma mainly caused by limited understanding of AAA pathogenesis.It has been recognized that AAA is essentially a vascular inflammatory disorder,the occurrence and development of AAA is driven and maintained by extensive vascular inflammatory reaction.AAA formation is a complicated process involving nearly all blood cell types,with extensively spread and significantly upregulated vascular pro-inflammatory cytokines(such as interleukin-6,IL-6)and chemokine expression.Accordingly,interfering aortic inflammatory reaction has strong theoretical therapeutic potential.However,the complicated networks regarding the inflammation regulation in AAA is far from being fully elucidated,which greatly hinders the understanding and control of AAA.Long non-coding RNA(lncRNA)is a kind of important intracellular regulatory factors.Under a variety of stimulating conditions,it exerts powerful regulation on gene expression and cell differentiation through a variety of molecular mechanisms such as coding gene transcription,epigenetic modifications,and post-translational modifications.LncRNA H19 is a highly conserved imprinted gene cluster that is highly expressed in fetuses and is expressed only in skeletal muscle and myocardium in adulthood.H19 can be reexpressed in multiple cell types in different tissues a variety of disorders.Recent experiments have suggested that the reexpression of H19 is closely related to inflammatory diseases.Such as in the bile duct cancer cells,oxidative stress can induce the reexpression of H19,and reexpressed H19 promotes the up-regulation of inflammatory mediators such as interleukin-6,thereby promoting the migration and invasion of cancer cells.H19 overexpression can activate inflammatory pathways and accelerate atherosclerosis.Inhibition of H19 alleviates the fat generation and inflammatory response induced by oxidized low-density lipoprotein.Epidemiological data suggest that H19 is related to the risk and severity of coronary heart disease in Chinese population.In a mouse model of hyperhomocysteinemia,a non-homeostasis with strong correlation with AAA,H19 was reexpressed in the aortic smooth muscle cell layer.The above evidences suggest that H19 may influence the formation and development of AAA by regulating vasculr inflammation.Therefore,this study aims to explore whether lncRNA H19 can influence the formation and development of AAA by enhancing the inflammatory response.First,we examined the H19 expression in the human AAA specimens and the mouse AAA models.Second,by successfully establishing two recognized AAA models,we overexpressed and knockdowned the H19 content in the aorta and observed its correspondent effects on the development of AAA.In addition,we explored the molecular mechanism conducting H19 promoting the development of AAA by enhancing vascular inflammation.Methods1.Laboratory animals:Healthy adult male C57BJ/6C mice and male ApoE-/-mice.2.Ang Ⅱ-induced AAA model:The experimental group of ApoE-/-mice were infused with angiotensin-2(angiontensin-Ⅱ,Ang Ⅱ,1.44 mg/kg/d)by micropump and ApoE-/-mice in control group was pumped the same amount of normal saline,mouse AAA model was obtained after 28 days of constituent pumping.3.AAA model was induced by calcium chloride.The abdominal aorta was exposed by laparotomy,and a gauze covered with 0.5 mol/1 calcium chloride was applied to the outer surface of the abdominal aorta of C57BL/6J for 15 minutes.The control group was replaced with saline gauze(0.9%).AAA model can be obtained after 6 weeks.4.Determination of AAA:After execution,the mouse abdominal aorta was fixed.To observe whether AAA occurred,the maximum diameter of abdominal aorta was measured,and the AAA was assessed by Ⅰ-Ⅳ types according to severity.5.Elastin staining and elastin degradation evaluation:Van Gieson staining of elastin was carried out with a score of 1 to 4 according to the degree of degradation of elastic fibers.6.Immunohistochemical analysis:Under corresponding conditions,the abdominal aortic wall of IL-6,monocyte chemotactic protein 1(monocyte chemoattractant protein 1,MCP-1),and macrophage antigen-2(macrophage antigen-2,MAC-2)were stained and quantified.7.Western Blotting:Detection of protein expression levels in the abdominal aorta of H19,MCP-1,MAC-2,and IL-6.8.LncRNA in situ hybridization:Detection of H19 distribution and content in mouse specimens.9.PCR:Detection of RNA expression levels of H19,MCP-1,MAC-2 and IL-6 in the abdominal aortic wall.10.Adenovirus vector-mediated H19 knockdown and overexpression:Using adenovirus as the vector,we realized the overexpression as well as knockdown of H19 in the whole body(including the abdominal aorta).11.Luciferase experiment:In vitro,the mutant IL-6 3 ’UTR,mutant H19 3’ UTR,and the wild-type IL-6 3 ’UTR,wild-type H19 3’ UTR were transfected to the smooth muscle cells containing the functional or non-functional analogue of the let-7a.The activity of luciferase reflecting the binding affinity was detected to determine if there exists binding site between intracellular let-7a and IL-6,let-7a and H19.12.Let-7a loss-and gain-of-fucntion experiments:The expression of IL-6 fluorescent protein levels in different groups was detected by adding a let-7 a antagonist and analogue in the cell solution.13.Statistical analysis:Statistical analysis was performed using SPSS 16.0 software.All data were presented as mean ± SD or percentage,and P<0.05 was considered statistically significant.Student’s t tests was used when statistical comparison of two groups data.Differences among groups were determined using one-way ANOVA.Results1.The expression of H19 is upregulated in Ang II-and calcium chloride-induced AAA:PCR,Western Blot,in situ hybridization technique proved that the H19 content was increased significantly in two kinds of successfully built AAA;H19 is mainly expressed in SMCs from the abdominal aortic wall.2.Detection of the efficiency of virus-mediated intervention of H19 in abdominal aorta:The results showed that the adenovirus-mediated overexpression or knockdown of H19 had significant effect from the 30th day,which stayed stable in aorta until the 60th days.3.Knockdown of abdominal aortic H19 reduced AAA induced by Ang Ⅱinfusion and the corresponding inflammatory reaction:Knockdown of H19 significantly reduced the incidence of AAA,and reduced related pathological changes of aortic RNA and protein upregulation of IL-6,MCP-1 and MAC-2.4.Overexpression of abdominal aorta H19 increased AAA induced by Ang Ⅱinfusion and the corresponding inflammatory reaction:Overexpression of H19 significantly increased the incidence of AAA,and promted related pathological RNA and protein expressions of aortic IL-6,MCP-1 and MAC-2.5.H19 functions as a competing endogenous RNA for miRNA let-7a to increase the content of IL-6:The luciferase experimental results showed that the there had binding sites between let-7a and H19,and between let-7a and IL-6.In vitro functional experiments showed that the let-7a was the key downstream molecule mediating H19 induced upregulation of IL-6.To sum up,H19 acts as a competing endogenous RNA for miRNA let-7a to release the suppression of let-7a on IL-6 gene transcription,and thus increase IL-6 content.ConclusionLncRNA H19 in the vascular smooth muscle of the abdominal aorta can mediate AAA formation by promoting aortic inflammation.The stimulative role of H19 in AAA is related to the inhibition of microRNA let-7a activity by functioning as a competing endogenous RNA,thereby inducing the upregulation of abdominal aortic interleukin-6. |
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