| BackgroundAbdominal aortic aneurysm(AAA)refers to an aneurysm-like enlargement and expansion of abdominal aorta.The incidence of intracranial aneurysms surpasses other aneurysms.The clinical outcome of this disease is an acute rupture of the tumor,which often results in a poor prognosis with a high mortality rate of about 80%.With the gradual development of our country to the aging,abdominal aortic aneurysm has become a common disease threatening the health of the elderly.At present,abdominal aortic aneurysms can only be treated by surgery,and no specific drugs that can be used for clinical treatment have been found.Therefore,in-depth study of the pathogenesis of abdominal aortic aneurysm,to find the appropriate drug targets for the prevention and treatment of abdominal aortic aneurysm is of great significance.At present,atherosclerosis is currently considered a direct result of the occurrence of abdominal aortic aneurysm,the main reason is due to the lack of nourishing the aorta of the renal veins,mainly rely on the dispersion of the nourishing artery supply nutrition.The formation of atherosclerotic plaque and its mural thrombus directly cut off the nourishment of the kidneys under the blood.In the meantime,the formation of atherosclerotic plaque reduces the stimulation of vascular smooth muscle cells(ASMCs)by bloodstream pulses,and greatly reducesthe ability tosynthesize the extracellular matrix(ECM)of the aortic wall,whilst promoting arterial intimal and neutrophilic necrosis and leading to destruction of vascular cell matrix,elastin synthesis decreases,eventually making the blood vessel elasticity decreased,weakness of the wall,under shear stress,making the abdomen Aortic aneurysm expansion.The previous study revealed that during the development of abdominal aortic aneurysm,the apoptosis of aortic smooth muscle cells and the imbalance of extracellular matrix played a major role in the formation of abdominal aortic aneurysms.The activity of protease increased and the expression of elastin and collagen Protein loss is the main reason for the imbalance of extracellular matrix metabolism.The main components of ECM are collagen,elastin,proteoglycan and glycoprotein,which are macromolecular substances secreted by cells into the extracellular matrix,forming a complex network structure,supporting and connecting the tissue structure,and regulating the occurrence of the tissue And cell physiology.Abdominal aorta,the extracellular matrix is mainly involved in maintaining the normal tensile strength and elasticity of the arterial wall plays an important role in maintaining its normal structure and physiological function.Usually,the extracellular matrix is always in the dynamic balance of synthesis and degradation,and the synthesis or degradation of it may lead to the development of abdominal aortic aneurysm.ASMC is a major component of the aortic tunica media that interacts with ECM to stabilize the mechanical strength of the vessel wall.In the development of abdominal aortic aneurysm,the role of ASMC includes the synthesis of matrix,the secretion of proteases,recruitment of inflammatory cells.Inflammation as an important factor to promote the enlargement of abdominal aortic aneurysm is widely accepted in recent years.Inflammatory cells in the adventitia and tunica media,as well as their secreted cytokines and proteolytic enzymes,stimulate sustained proteolysis,thinning the aortic wall and promoting aneurysmal expansion.Preventing the hydrolysis process can be seen as an effective means of treatment of aneurysm expansion.Cyclooxygenase 2 is widely expressed in the aneurysmal wall and increases the synthesis of prostaglandin E2(PGE2),which may further reduce collagen synthesis.miRNAs are a class of small,non-coding 20-24 nt RNAs.The miRNA can bind to the 3’UTR or CDS region of the target gene by means of partial pairing of base pairs to inhibit the expression of the target gene at post-transcriptional level.MiRNAs are widely involved in the regulation of various physiological processes,including organ development,cell differentiation,immune responses and more.In recent years,studies have shown that small RNAs are also involved in the regulation of the formation and development of abdominal aortic aneurysms.The miR-29 family is an important regulator of the extracellular matrix microenvironment that inhibits the expression of extracellular matrix proteins such as collagen,fibrillin and elastin at the transcriptional level.In addition,miR-29 is a pro-fibrotic factor that may serve as a potential therapeutic target in fibrosis-related diseases.The mechanism of a pro-fibrotic response is usually considered pathological,and its occurrence is accompanied by significant damage oforgans and systems.For example,miR-29b downregulation has been identified that is closely linked to controlling myocardial fibrosis after myocardial infarction and ischemia as well as liver and kidney fibrosis.MiR-29b was found to be suppressed in abdominal aortic aneurysms,suggesting that it is likely to be involved in the regulation of aneurysm development.In the process of crude fibrosis induced by abdominal aortic aneurysm expansion,the increase of the expression of collagen and other matrix proteins is directly controlled by the decrease of miR-29b level.Thus,further reduction of the expression of miR-29b may have a potential role in preventing the continued expansion and eventual rupture of the aorta.Targeting to reduce the expression of miR-29b to induce increased pro-fibrogenic response is to prevent the expansion of abdominal aortic aneurysm is an effective means.Prostaglandin E2(PGE2)not only participates in the regulation of many important biological processes,but also plays a beneficial role in the inhibition of fibrosis.Non-steroidal anti-inflammatory drugs can reduce PGE2 secretion.In the meantime,compared with 15 patients taking NSAIDs and 63 controls,it was found that the administration of drugs significantly slowed the expansion of abdominal aortic aneurysms.This shows that PGE2 in the expansion of abdominal aortic aneurysm plays an important regulatory role.ObjectivesThis study aims to investigate whether PGE2 can regulate the expression of miR-29b in ASMCs,therefore regulating the secretion of ECM components so as to reveal the specific role of NSAIDs in preventing the expansion of abdominal aortic aneurysm mechanism.Further hope that through the development of this topic for us to miR-29b or PGE2 synthesis-related genes as the target of treatment of abdominal aortic aneurysm lay a good theoretical basis.Methods1.To assess the effect of PGE2 on miR-29b:The smooth muscle cells cultured in vitro were treated with PGE2 and indomethacin,a non-steroidal anti-inflammatory drug,respectively.The expression of miR-29b in cells was detected by quantitative PCR and compared to unused patients,the expression of miR-29b in ASMCs of abdominal aortic aneurysm.2.Using bioinformatics to predict the target site of miR-29b,and whether dual-luciferase reporter gene miR-29b can regulate the promoter of COL1A1 gene predicted to bind the most with its binding site.Western blot was used to detect whether the latter is regulated by miR-29b in arterial smooth muscle cells.3.To assess the effect of PGE2 and indomethacin on extracellular matrix components of ASMC,including COL1A1,COL3A1,elastin and fibronectin;4.Silencing miR-29b expression in cells by transfecting miR-29b inhibitor.To determine whether PGE2 affect the extracellular matrix by regulating miR-29b.5.Clinical level of validation:Clinically taking indometacin in patients with abdominal aortic aneurysm,ASMC cells in their tissue to detect the secretion of soluble collagen content and compared with patients not taking drugs to evaluate prostaglandin inhibition in vivo effect.Results1.PGE2 promotes the expression of miR-29bThe study found that a large number of arterial smooth muscle cell apoptosis can be observed in patients with abdominal aortic aneurysm.And this phenomenon and the occurrence of inflammation are inextricably linked.In our earlier study,we found that miR-29b is increased in arterial smooth muscle cells stimulated by prostaglandin PGE2.We treated normal artery smooth muscle cells with 500 ng/mL PGE2 and found that miR-29b expression was significantly increased in cells compared to untreated cells(2.162 ± 0.117 vs 1.004 ± 0.010,P<0.001).The results are strictly consistent.Thus,prostaglandin E2 has a strong role in promoting the synthesis of miR-29b in arterial smooth muscle cells.We then treated the cells with 10 mmol/l indometacin and found that the expression of miR-29b was significantly down-regulated in the presence of indomethacin and even down-regulated compared to the untreated control(0.665 ± 0.015 vs 1.004 ± 0.010 vs.untreated P<0.01).Further,we validated this experimental result in arterial smooth muscle cells of abdominal aortic aneurysm origin.We divided the patients into experimental group taking indometacin enteric-coated tablets and control group without taking drugs,and then collected the tissue samples from two groups of aneurysms and isolated the ASMC cells.The quantity of miR-29b was detected by quantitative PCR.The amount of miR-29b in the tissue of patients taking NSAIDs was significantly lower than that in patients without medication,the difference was statistically significant(P<0.05).2.MiR-29b can directly regulate the COL1A1 geneThe data from TargetScan showed that COL1A1 is a potential miR-29b downstream target gene and that miR-29b interacts with at least three sites on the 3’-untranslationregion(UTR)of COL1A1 We directly transfected the 3’-UTR binding site sequence of miR1A1 and miR-29b into cells,and observed the change of fluorescence intensity to determine whether COL1A1 could complement with miR-29b.In the presence of miR-29b mimics,the fluorescence intensity of smooth muscle cells transfected with COL1A1 3’-UTR was significantly lower than that of untransfected cells,down to about half(P<0.01)Cells transfected with COL1A13’-UTR and Scr-miR showed no significant change in fluorescence intensity compared to untransfected cells.This suggests that the change in fluorescence intensity is caused by miR-29b mimics,so we can conclude that the COL1A1 3’-UTR interacts directly with miR-29b.We further assessed the effect of miR-29b on COL1A1 expression in ASMCs by quantitative PCR analysis.The results showed that the expression of COL1A1 in ASMCs transfected with miR-29b mimics was significantly reduced and statistically significant(0.587 ± 0.178 fold,P<0.05,compared with the untransfected cells defined as 1.0-fold expression),while miR-29b inhibitor significantly increased the expression of COL1A1(1.630 ± 0.203 fold,P<0.01).These results indicate that the inhibitory effect of miR-29b on COL1A1 is achieved by direct targeting of its 3’-UTR sequence.Western blot was used to verify the inhibitory effect of miR-29b on COL1A1 at the protein level.The expression of COL1A1 in miR-29b mimics-transfected cells was much lower than that in untransfected cells or miR-src-transfected cells.However,the expression of COL1A1 protein was significantly increased in miR-29b transfected cells,Further indicating that miR-29b has an inhibitory effect on COL1A1 expression.3.Prostaglandin E2 can promote miR-29b-mediated extracellular matrix degradation in arterial smooth muscle cellsAfter PGE2 treatment,COL1A1 and COL3A1 mRNA expression was significantly inhibited(0.690 ± 0.193 and 0.710 ± 0.145 times before treatment,respectively,P<0.05).Likewise,PGE2 also inhibited the expression of elastin and fibronectin FBN1 in arterial smooth muscle cells(0.657 ± 0.105 and 0.643 ± 0.235,respectively,P<0.01,respectively).It is notable that the expression of various extracellular matrix components is increased in response to indometacin in response thereto.We speculated that this may be due to PGE2 can increase the expression of miR-29b,and miR-29b can inhibit extracellular matrix collagen gene expression,and then speculated that the role of PGE2 by regulating miR-29b completed.First,the inhibitory effect of miR-29b inhibitor was tested by RT-PCR and the results showed that the inhibitor could successfully inhibit the expression of miR-29b even in the presence of prostaglandin E2(the expression level was lower than 30%of that before transfection).Although PGE2 can reduce the expression of extracellular matrix,its expression is increased when transfected with miR-29b inhibitor,which is higher than that of untreated cells.From this we can see that the effect of inhibitor on extracellular matrix stronger than prostaglandin,we speculated that PGE2 may be due to an increase in intracellular miR-29b production,so that the matrix gene expression levels,and inhibitors not only can resist this effect,but also can inhibit the background of miR-29b expression in cells.As a result,even if prostaglandin is transfected,the level of miR-29b in cells transfected with the inhibitor is still very low,which further relieves the secretion of extracellular matrix and increases the secretion of collagen,elasticity and fibronectin.We further examined the amount of soluble collagen secreted by ASMCs for validation.In the prostaglandin stimulation,the expression of soluble collagen decreased significantly(P<0.05).When cells were treated with indomethacin,which blocked cellular prostaglandin synthesis,the proportion of collagen increased significantly(P<0.01).PGE2 can regulate the synthesis of extracellular matrix.The effect of prostaglandin E2 was counteracted when miR-29b inhibitor was transfected,and the effect of PGE2 was enhanced when miR-29b mimics was transfected.To further confirm the efficacy of indomethacin in patients and to explore its possible mechanism,we examined soluble collagen secreted by ASMC cells from both groups.The soluble collagen secreted by the cells of NSAID patients was significantly higher than that of the patients without medication,the difference was statistically significant(P<0.05).ConclusionPGE2 promotes the expression of miR-29b,and thus the clinical use of NSAIDs inhibits the synthesis of PGE2 thereby reducing the expression of miR-29b;in arterial smooth muscle cells,miR-29b regulates COL1A1 expression by direct binding to its 3’-UTR binding site;PGE2 can promote arterial smooth muscle cell extracellular matrix degradation,and this effect of PGE2 is accomplished by regulating miR-29b and can be antagonized by the action of indomethacin.Clinical trials further demonstrate the potential use of indomethacin in the clinic:to alter miR-29b levels by modulating PGE2,leading to increased secretion of collagen and other fiber-related proteins in the extracellular matrix,limiting further aneurysm expansion.However,there is no conclusive evidence to confirm this conclusion,and the relevant verification experiments are still in progress. |
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