MicroRNA-199a-5p/sirtuin-1 Signaling Pathway Mediates Vascular Smooth Muscle Cells Senescence In Abdominal Aortic Aneurysms

Objective:The death rate of abdominal aortic aneurysm(AAA)was higher due to aneurysm rupture.A cumulative evdicence has shown that the cellular senescence of vascular smooth muscle cells(VSMCs)plays a critical role in regulating AAA formation.This study aimed to investigate the regulatory role of miR-199a-5p/SIRT1 pathway in VSMCs senescence in AAA.Methods:The Control-VSMCs and AAA-VSMCs were isolated from the abdominal aortic tissues of organ donors and patients with AAA respectively.The protein expressions of p-p53,p21 and SIRT1 were detected by Western blotting.They were stained with HE and SA-β-gal.The proliferation and VSMCs senescence in the abdominal aorta tissue were examined.Cumulative Population Doubling assay,5-Bromodeoxyuridine assay,Ki67 immunofluorescence assay and γH2AX assay were used to detect the proliferative ability and injured degree of VSMCs.Serum AngⅡ concentration was detected by ELISA.The level of reactive oxygen species were detected by fluorescence staining.The senescence model of VSMCs in vitro was established by AngⅡ.Control-VSMCs were divided into Control group,AngⅡ group and NAC group.The ROS level and VSMCs senescence among the three groups were evaluated.Furthermore,Control-VSMCs were divided into Control group,AngⅡ group,DMSO group and Resveratrol group.The express of SIRT1 protein,ROS levels and VSMCs senescence in the four groups were detected.The miRNA sequencing was used to detect the differences of serum miRNA between the AAA and Control groups.Q-PCR was used to verify miRNA in serum,tissues and VSMCs.The target genes of miR-199a-5p were verified by dual luciferase reporter gene assay.VSMCs was co-cultured by miR-199a-5p inhibitor and SIRT1-siRNA.ROS level,protein expression of SIRT1,p-p53,p21 were detected.Results:There were cystic changes in the media of abdominal aorta in patients with AAA,accompanied by inflammatory cell infiltration,which was consistent with the pathological characteristics of AAA.Compared with the control group,the protein expression of p-p53 and p21 in the abdominal aorta tissues of AAA patients was greatly increased accompanied by increased positive rate of SA-β-Gal staining and proportion of α-SMA/p-p53 double-positive cells.Compared with the Control-VSMCs,the cumulative population multiplicative number,BrdU absorbance and Ki67 expression were decreased in AAA-VSMCs.In contast,the expression of yH2AX was increased in AAA-VSMCs,indicating the damage degree of VSMCs increased.Serum AngⅡ levels in AAA patients were higher than those in control group.ROS levels in tissues and VSMCs of the AAA group were higher than those in control group.ROS level,SA-β-Gal staining positive cell rate and p-p53 and p21 protein expression were all higher in AngⅡ group than those in the Control group.After NAC treatment,the above indexes decreased in different degrees.The expression of SIRT1 protein in AAA-tissues and AAA-VSMCs were lower than those in Control-tissue and Control-VSMCs.Compared with Control-VSMCs,the expression level of SIRT1 protein was decreased in AngⅡ group.Compared with AngⅡ group,the expression level of SIRT1 protein was increased in Resveratrol group.The ROS level,SA-β-gal staining positive cell rate and the expression level of p-p53 and p21 protein were lower in Resveratrol group than those in AngⅡ group.The miR-199a-5p expression in serum of the AAA group was higher than that in control group by miRNA sequencing.The same changes were confirmed by Q-PCR in serum,tissues and VSMCs.The expression level of SIRT1 protein in miR-199a-5p mimic VSMCs group was lower than that in miR-Control VSMCs group.Compared with miR-Control VSMCs,the expression level of SIRT1 protein was increased in miR-199a-5p inhibitor group.Luciferase activity of SIRT1 wild-type reporter gene was significantly reduced by miR-199a-5p mimic with dual luciferase reporter assay.Incontast,there was no effect on the luciferase activity of SIRT1 mutant reporter gene.MiR-199a-5p inhibitor accelerated expression of SIRT1 protein,prohibitored ROS production and the expression of p-p53 and p21 protein,delaying VSMCs senescence.MiR-199a-5p inhibitor and SIRT1-siRNA were co-transfected into VSMCs.The results showed that SIRT1-siRNA partially eliminated the effects of miR-199a-5p inhibitor on ROS production,expression of SIRT1 protein and p-p53/p21 protein.Conclusion:It was showed that miR-199a-5p induced ROS production and up-regulated p-p53 expression by targeting SIRT1 regulation,thereby aggravating VSMC senescence.MiR-199a-5p inhibitor accelerated expression of SIRT1 protein,prohibitored ROS production and the protein expression of p-p53 and p21,delaying VSMCs senescence.MiR-199a-5p inhibitor and SIRTl-siRNA were co-transfected into VSMCs.SIRT1-siRNA partially eliminated the effects of miR-199a-5p inhibitor on ROS production,expression of SIRT1 protein and p-p53/p21 protein.