| Background:Abdominal aortic aneurysm(AAA)refers to abdominal aortic morphological changes and progressive dilatation,aortic diameter greater than 3.0 cm or 1.5 times the expected normal diameter of the disease.Abdominal aortic aneurysms mainly occur in men over 65 years of age.The prevalence fluctuates between 1.3%and 12.5%,which can cause more than 175,000 deaths worldwide.It has become a major public health problem.Patients usually do not show specific symptoms or signs before rupture of aneurysms in the early stage of disease.Once rupture of aneurysms occurs,the mortality rate will be as high as 90%.With the increase of the diameter of aneurysms,the probability of rupture increases significantly,so it is particularly important to make early diagnosis and treatment before rupture.At present,the effective treatment of abdominal aortic aneurysm is still based on surgery,and no specific drug has been found for the treatment of abdominal aortic aneurysm.Clinical medication can only slow down the course of disease by controlling blood pressure,heart rate,blood lipid and other indicators.However,the traditional surgical treatment has the disadvantages of large trauma,easy to cause complications and high mortality.Therefore,it is controversial whether the abdominal aortic aneurysm with diameter less than 5.0 cm needs surgery.To study the molecular mechanism of abdominal aortic aneurysm is of great significance for the early diagnosis and treatment of abdominal aortic aneurysm.Extracellular matrix(ECM)is mainly composed of collagen,proteoglycan,elastin and glycoprotein,which form a complex network structure through interconnection and interweaving,and participate in maintaining the tension strength and elasticity of the arterial wall.Under normal physiological conditions,the synthesis and degradation of extracellular matrix can always maintain a dynamic balance.Abnormal synthesis or degradation of extracellular matrix may lead to various functional disorders and diseases.The key pathogenesis of abdominal aortic aneurysm is ECM destruction due to the high activity of protein hydrolysis.At the same time,vascular smooth muscle cells(VSMCs),the main component of arterial wall media,usually undergo apoptosis when AAA occurs.In addition,inflammatory changes such as angiogenesis,mononuclear cell phagocytosis and adventitia transition from innate immunity to adaptive immunity were observed during the occurrence of abdominal aortic aneurysm.In a word,the occurrence and progress of abdominal aortic aneurysms involve many changes in cell and molecule functions and quantities,but the specific molecular mechanism has not yet been elucidated.Therefore,it is necessary to carry out in-depth discussion in order to facilitate the discovery of effective treatment strategies and the development of novel targeted drugs.MicroRNAs(miRNAs,miRs)are small endogenous non-coding RNAs with 18 to 25 nucleotides in length.Recent discoveries of microRNAs associated with different atherosclerosis have led to new insights into the diagnosis and treatment of cardiovascular diseases.By binding with 3'-UTR of target gene,the degradation or translation of target gene is inhibited,and the gene expression is inhibited at the post-transcriptional level.More and more studies have shown that microRNA is closely related to angiogenesis by regulating angiogenesis-related cellular biological functions and angiogenesis factors,which are related to aneurysm,aortic dissection,atherosclerosis,thrombosis,stroke and other cardiovascular and cerebrovascular diseases.Let-7 is an ancient family of microRNAs,which plays an important role in many physiological and pathological processes,such as ontogenesis,cell proliferation,differentiation,stem cell maintenance,aging and tumorigenesis.In recent years,it has been pointed out that let-7 plays an important role in regulating cardiovascular diseases,especially in abdominal aortic aneurysm patients.Abnormal expression of let-7 is found in plasma and tissues,which means that let-7 may be involved in the regulation of the pathogenesis of this disease,although the specific molecular mechanism is still unknown.let-7 was conventionally considered as an anti-oncogene,which can promote the apoptosis and senescence of tumor cells.Unlikely,it may play an opposite role in vascular-related diseases.It has been reported that let-7 overexpression can promote the proliferation,migration and angiogenesis of vascular endothelial cells.On the other hand,let-7 extensively inhibits the activities of ECM hydrolases such as collagenase and MMPs,thus inhibiting the metastasis of tumors.Therefore,we speculate that some members of let-7 family may regulate the pathogenesis and progression of abdominal aortic aneurysms by regulating the abnormal proliferation of synthetic VSMC and promoting ECM decomposition.Methods:1.Sample collection and detection of let-7 and matrix protease expression in plasma of patients with abdominal aortic aneurysmA total of 21 patients with lower abdominal aortic aneurysm(age 67.2±5.53 years)and 25 healthy controls in Jining No 1 People' hospital from Mar 2016 to Sep 2018 were enrolled in this study.There was no statistical difference in age and sex ratio among all patients.All patients were diagnosed by CTA examination.The general clinical characteristics of patients and healthy controls included age,gender,BMI,history of disease and medication history.The difference between the two groups was used to evaluate the risk factors of abdominal aortic aneurysm.The abdominal aortic aneurysm tissues from patients and normal abdominal aorta tissues were obtained.The pathological changes of abdominal aortic aneurysm were observed by HE staining,while the fibrous structure in the aortic tissue was observed by EVG staining.2.Expression of let-7 family and inflammatory-related MMPs in plasma of patients with abdominal aortic aneurysmPeripheral blood was collected and plasma samples were obtained by centrifugation.The plasma concentrations of MMP-2,MMP-9 and MMP-13 were measured by enzyme-linked immunosorbent assay(ELISA).Quantitative PCR was used to detect the expression of let-7 family members(including let-7a,let-7b,let-7c,let-7d,let-7e,let-7f,let-7g,let-7i and microRNA-98)in plasma.According to the median of let-7i,the patients were divided into two groups with high and low expression.The clinical characteristics of the two groups were compared,and the correlation between let-7i expression and tumor size and other clinical characteristics was analyzed.3.Induction of abdominal aortic aneurysm mice model and detection of let-7i expression in miceApoE-/-mice were subcutaneously injected with Ang II for 4 weeks to induce abdominal aortic aneurysm animal models.The sham-operated mice were continuously pumped into PBS buffer.The maximum diameter of abdominal aorta was examined by ultrasonography after 4 weeks.Abdominal aorta tissues were isolated from mice,and the abnormal expression of let-7i in abdominal aorta tissues of model mice was detected by quantitative PCR.4.The effect of let-7i on the formation of abdominal aortic aneurysm and the expression of MMPs in the tissues of miceTo study whether let-7i down-regulation is the initiating factor of abdominal aortic aneurysm,we used adeno-associated virus-coated overexpression vector(AAV-let-7i mimic)or control plasmid(AAV-mimic NC)to intervene abdominal aortic aneurysm model mice.One day before the osmotic pump was implanted,the virus vector was injected,once every other day until the end of the animal modeling cycle,and the mice were sacrificed on the twenty-eighth day.The formation rate of abdominal aortic aneurysm and the maximum diameter of abdominal aorta in mice of each group were compared,and the effect of let-7i overexpression on the formation and progression of abdominal aortic aneurysm was judged.Finally,Western blot was used to detect the expression levels of MMP-2,MMP-9 and MMP-13 in tissue samples.5.let-7i expression in vascular smooth muscle cellsVSMC cells can be transformed from differentiated(contractile)to dedifferentiated(synthetic)when stimulated by the outside world,in order to synthesize a large number of extracellular matrix and repair damaged blood vessels.VSMC cells were dedifferentiated by 10 ng/mul PDGF to simulate the inflammatory environment of abdominal aortic aneurysm in vivo.Quantitative PCR was used to detect the expression of let-7i in different time periods.6.Potential relationship between let-7 family members and MMPsTargetscan and RegRNA were used to analyze their possible sites of action.The predicted 3'-UTR binding sequence of MMP-9 gene was cloned downstream of psiCHECK2 vector luciferase.HEK293T cells were transfected with let-7 family members'expression vectors to detect luciferase activity.Further,let-7i expression vectors with decreased plasma content were co-transfected with wild-type and mutant MMP-9 fluorescent vectors into HEK293T cells,and the relationship between the two genes was determined by double luciferase reporter gene assay.7.Regulation of let-7i on MMPsLet-7i mimic and antisense let-7i were transfected into VSMC to up-regulate and down-regulate the expression of let-7i,respectively.Quantitative PCR was used to detect the expression of let-7i in cells and evaluate the transfection efficiency.Enzyme-linked immunosorbent assay(ELISA)was used to determine the effect of up-regulation and down-regulation of let-7i on MMP-9 secretion.Western blot was used to detect the expression of MMP-2 and MMP-9 in VSMC cellsIn addition,let-7i mimic and antisense let-7i were transfected into human umbilical vein endothelial cells(HUVEC)to interfere with let-7i.The expression of MMP-9 after transfection was detected by ELISA8.Effect of let-7i on proliferation,migration,and apoptosis of dedifferentiated VSMCsSynchronized VSMC cells were induced to dedifferentiate by PDGF(10 ng/ml).After 48 hours,quantitative PCR was used to detect the expression of let-7i in differentiated and dedifferentiated VSMC cells.Let-7i was overexpressed or silenced in VSMCs before 48-hour treatment with PDGF,the effects of let-7i on the viability and migration ability of dedifferentiated VSMC cells were determined by CCK-8 and cell migration experiments.After 48 hours of PDGF treatment,cell apoptosis was analyzed by flow cytometry after transfection of let-7i mimic and anti-let-7i.Western blot was used to detect the expression of Caspase-3 and caspase-9 in the precursor and shear form of apoptosis-related factors in the lysate of VSMC cells.Results:1.Statistical data and pathological changes of patients with abdominal aortic aneurysmHypertension and smoking were risk factors for abdominal aortic aneurysm(P<0.05).The results of HE staining showed that the intima,media and adventitia of normal tissues had clear layers and complete structures,while the abdominal aortic aneurysm wall had disordered structure,tissue was seriously damaged,infiltrated by a large number of inflammatory cells,and the number of smooth muscle cells in the media decreased significantly,and fibroblasts increased significantly.EVG staining showed that the elastic fibers in the normal abdominal aortic wall tissues were intact and wavy,while in the abdominal aortic aneurysm tissues,the elastic fibers were broken and severely damaged,while the remaining elastic fibers were linear and not corrugated.2.The expression of let-7 family members and MMPs in plasma of patients with abdominal aortic aneurysmThe results of enzyme-linked immunosorbent assay(ELISA)showed that the levels of MMP-2,MMP-9 and MMP-13 in plasma of patients were higher than those of control group(P<0.05 or P<0.01).At the same time,the levels of let-7a,let-7b,let-7c,and miR-98 in abdominal aortic aneurysm patients were significantly higher than those in healthy controls(P<0.05 or P<0.01),and let-7i expression was most significantly down-regulated.These data suggest that some members of the let-7 family may play a key role in the occurrence or progression of abdominal aortic aneurysms.3.The expression of let-7i in plasma is correlated with the severity of diseaseThere was no significant correlation between the expression level of let-7i and age,sex,BMI index,disease history and drug history(P>0.05).The patients with low expression of let-7i had a larger diameter than those in the high let-7i expression group,and the difference between the two groups was statistically significant(8.07±0.93 vs 6.32±1.02,P<0.05).Therefore,let-7i may play a regulatory role in the occurrence and progression of abdominal aortic aneurysms4.let-7i is down-regulated in abdominal aortic aneurysm miceUltrasound results confirmed that after 4 weeks of continuous injection of Ang ?,the largest diameter of the abdominal aorta in the model group was 1.423±0.109 mm,while the diameter of the aorta in the sham operated group was 0.613±0.012 mm,and the difference between the two groups was significant(P<0.05),indicating that the animal model of abdominal aortic aneurysm was successfully established.In addition,the blood pressure of mice increased from 102.0±7.4 mmHg to 146.7±9.3 mmHg before induction,and there was a significant difference between the model group and the sham-operated group(P<0.05).There was no significant change in heart rate and body weight between the model group and the sham-operated group(P>0.05).5.let-7i controls the formation of abdominal aortic aneurysm in miceThe expression of let-7i in the abdominal aortic wall of AAV-let-7i mimic mice was significantly increased,but the expression of let-7i in the abdominal aortic wall of AAV-mimic NC mice was not significantly changed compared with that of non-injected mice.The rate of abdominal aortic aneurysm formation in mice treated with let-7i mimic overexpression vector was significantly lower than that in mice without intervention,but the introduction of control vector could not cause this change.By measuring the maximum diameter of the abdominal aorta,we found that although the maximum diameter of the abdominal aorta increased significantly after 4 weeks of Angll induction,the maximum diameter of the abdominal aorta decreased significantly when injected with the AVV carrying let-7i mimic,which was significantly different from that of the negative control group.In conclusion,up-regulation of let-7i in abdominal aortic wall can inhibit the formation of abdominal aortic aneurysm induced by Ang ? in mice.6.let-7i overexpression down-regulates the expression of MMPs in tissues.The expression levels of MMP-2,MMP-9 and MMP-13 proteins in abdominal aortic aneurysm model mice were significantly higher than those in normal mice,but when let-7i was overexpressed,these proteins decreased significantly.It is suggested that let-7i can inhibit the expression of MMPs in mice,which may be one of the main reasons for its inhibition of abdominal aortic aneurysm formation.7.Reduced expression of let-7i in synthesized vascular smooth muscle cellsQuantitative PCR results showed that the expression of let-7i in dedifferentiated VSMC decreased after 48 hours of PDGF induction compared with untreated cells(P<0.05),but the level of let-7i was restored again when the stimulation was removed,which was similar to the results obtained in plasma of patients.Therefore,we used this cell model to study the function mechanism of let-7i in vitro.8.let-7i directly targets to 3'UTR of MMP-9 geneThere are conservative binding sites between 3'-UTR of MMP-9 gene and members of let-7 family.Double luciferase reporter gene assay showed that let-7a,let-7b,let-7f and let-7i could inhibit luciferase activity in wild-type MMP-9 transfection group compared with empty vector control(p<0.01).Moreover,we found that let-7i could significantly inhibit the fluorescence intensity of cells,while the fluorescence intensity of mutant MMP-9 group did not change significantly,suggesting that let-7i may have a direct interaction with the MMP-9 coding gene.9.let-7i down-regulates the expression of MMPs in VSMC cellsThe expression of let-7i in VSMC cells transfected with let-7i mimic was significantly increased(P<0.001),while the expression of let-7i in antisense let-7i transfected VSMC cells was significantly decreased(P<0.01).The results showed that the transfection efficiency was high and could be used in subsequent functional experiments.Enzyme-linked immunosorbent assay(ELISA)showed that over-expression of let-7i significantly decreased the expression of MMP-9 in cell culture supernatant(p<0.01),while inhibition of let-7i significantly increased the expression of MMP-9(P<0.001).Overexpression of let-7i also resulted in the decrease of the expression of MMP-2 and MMP-13 in VSMC cells.On the contrary,inhibition of let-7i could increase the expression of MMP-2 and MMP-13.It is suggested that let-7i may play a role in VSMC cells by directly binding to and interacting with MMP-9 matrix proteinase,which can decrease the expression of MMP-9 and other proteinases,thus protecting the extracellular matrix from degradation.The expression of MMP-9 in HUVEC cells transfected with let-7i mimic was inhibited(P<0.01),while the expression of MMP-9 in HUVEC cells transfected with antisense let-7i was significantly increased(p<0.001).10.let-7i inhibits the proliferation and migration of dedifferentiated vascular smooth muscle cellsQuantitative PCR results showed that after 48 hours of PDGF induction,let-7i expression in dedifferentiated VSMC was lower than that in untreated(i.e.differentiated)cells,but let-7i level was restored again when stimulation was removed.CCK-8 results showed that after 48 hours of PDGF stimulation,let-7i overexpression significantly inhibited the viability of VSMC cells compared with the negative control cells(P<0.01),while after interference with let-7i expression,the viability of VSMC cells increased significantly compared with the negative control cells(P<0.05).After let-7i overexpression,the number of migrating VSMC cells decreased significantly compared with the negative control group,while the number of migrating cells increased significantly after let-7i interference,which indicates that let-7i can reduce the migration ability of vascular smooth muscle cells,which may be achieved by inhibiting the degradation of ECM.11.let-7i induces apoptosis of vascular smooth muscle cellsFlow cytometry was used to analyze the apoptosis of cells transfected with let-7i mimic and anti-let-7i.After 48 hours of PDGF treatment,the apoptotic rate of VSMC cells overexpressed by let-7i was significantly higher than that of the control group,while anti-let-7i transfection could reduce the apoptotic rate of cells(p<0.01).Overexpression of let-7i can promote the expression of active forms Caspase-3 and Caspase-9 and increase the ratio of them to precursor proteins(P<0.001),while the effect of interference with let-7i expression on cells is contrary to its effect on cells.These data suggest that let-7i significantly induces apoptosis in dedifferentiated VSMC cells.Conclusion:In conclusion,our results suggest that let-7i can reduce ECM degradation and inhibit PDGF-induced dedifferentiation,proliferation and migration of vascular smooth muscle cells by interacting with MMP-9 and inhibiting its expression.On the other hand,let-7i can induce apoptosis of dedifferentiated VSMC cells.These results suggest that microRNA let-7i may be an important regulator in the pathogenesis of abdominal aortic aneurysms,as a new biomarker for diagnosis and treatment. |
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