| BackgroundCardiovascular disease(CVD)is still a critical public health problem threatening human health worldwide,and atherosclerosis(AS)is the main cause of CVD.Notably,increasing evidence has demonstrated that the development of AS is inseparable from cellular senescence,and cellular senescence is involved in different stages of AS.Endothelial dysfunction mediated by endothelial cells(ECs)senescence occured in the initial stage of AS,and it triggered an imbalance of vascular homeostasis,which accelerated the process of AS by vasoconstriction,leukocyte adhesion and platelet activation.Therefore,exploring the pathological mechanisms underlying EC senescence is of great significance for the prevention and treatment of aging-related AS and ASCVD.Cellular senescence is mainly divided into replicative senescence and stress-induced premature senescence(also known as stress-induced senescence),both of which have similar consequences.Angiotensin II(Ang?)is the main effector of the renin-angiotensin-aldosterone system and is associated with stress-induced senescence of vascular ECs.Studies have shown that Ang? plays a key role in changes in extracellular matrix composition,activation of inflammatory responses and generation of oxidative stress,while its signaling cascade increases with age,and inhibition of Ang? activity could reduce morbidity and mortality of ASCVD.Signal transducer and activator of transcription 3(STAT3)is a cytoplasmic signal transducer and transcription activator.Upon phosphorylation,it is activated and transferred to the nucleus where it binds to the DNA to exert its biological effects.Studies have shown that STAT3 is a key regulator of cellular senescence and growth arrest under certain conditions.In addition to inducing cellular senescence via P53/P21 signaling pathway activation,it is a major component of the oxidative stress signaling pathway.Of note,inhibition of STAT3 activation downregulates the expression of genes associated with senescence and SASP,and its activator interleukin-6 is a key factor in inducing cell cycle arrest and SASP.Therefore,STAT3 signaling pathway plays an important role in cellular senescence.However,the regulatory mechanism of STAT3 in ECs senescence remain unclear.Studies have reported that while STAT3 is activated by upstream growth factors and cytokine receptors,it can also be directly or indirectly regulated by non-coding RNA(nc RNAs).Micro RNAs(miRNAs)are a class of small nc RNAs consisting of approximately 18–25 nucleotides.They primarily interact with the 3'-untranslated region(UTR)of a target m RNA through complementary base pairing.Consequently,the target m RNA is degraded or its protein translation is inhibited.Several studies have demonstrated that miRNAs regulate vascular cell senescence.For example,the expression of miR-21,miR-216,miR-217,miR-181 b,miR-31 b and miR-34 a was increased in the replicative senescence of human umbilical vein ECs,while the expression of members of the miR-17-92 cluster was decreased.Meanwhile,serum miR-106 b was also decreased gradually in the process of aging.It was found that miR-106b-5p was down-regulated during the JQ1-induced senescence of gastric cancer cells(MKN28),and overexpression of miR-106b-5p could inhibit the expression of P21 to block the stress-induced senescence,which suggested that miR-106b-5p was related to cellular stress-induced senescence.We found that miR-106b-5p has a binding site with STAT3 by searching the database,but it is not clear whether miR-106b-5p targets STAT3 to participate in the regulation of ECs senescence.It has been shown that circular RNAs(circRNAs)could jointly participate in the regulation of miRNA target genes by adsorbing miRNA.At the same time,circRNAs have the characteristics of strong stability,good conservation between species and high expression abundance,avoiding the disadvantages of low stability and poor conservation of miRNAs,and improve the transformation value to clinical disease diagnosis and treatment targets.CircRNAs are another special class of nc RNAs,which can not only neutralize endogenous miRNAs,but also bind to proteins,regulate the expression of parental genes or translate into functional proteins and perform other multi-level regulation of gene expression.Studies have shown that circRNAs are involved in the regulation of cellular senescence.Among them,circ?AFF1 is transcribed from a gene located at the human autosomal 4q21.3 locus,and its expression in the peripheral blood of the elderly was significantly higher than the young;and it was also elevated in the replicative senescence of three different types of cells,suggesting that circ?AFF1 may be involved in cellular senescence.At the same time,the regulatory mechanism of circRNAs involved in cellular senescence by adsorbing miRNAs has been confirmed.For example,the expression of circ PVT1 was reduced in WI-38 replicative senescence,and silencing circ PVT1 could significantly reduce the expression level of the proliferation protein encoded by let-7 target m RNA,resulting in cell cycle arrest to induce cellular senescence.In addition,circ PVT1 also targeted miR-199a-5p and attenuated its negative regulation of SIRT1 expression to delay cellular senescence.We found that circ?AFF1 has a binding site with miR-106b-5p by searching the databases.At the same time,studies have shown that circ?AFF1 could attenuate its inhibitory effect on SAV1 by adsorbing miR-516 b to increase the phosphorylation level of Yes-related proteins,thereby inhibiting the proliferation and migration of vascular ECs.Therefore,we speculated that circ?AFF1may affect ECs senescence by regulating the expression of STAT3 by adsorbing miR-106b-5p.ObjectiveIn this experiment,human coronary artery ECs(HCAECs)were used as the research object to investigate the effect of circ?AFF1 on Ang?-induced stress senescence in HCAECs,trying to elucidate that circ?AFF1 affected the expression of STAT3 by adsorbing miR-106b-5p,and then participated in the regulation mechanism of HCAECs senescence.This provides a theoretical basis for finding targets to intervene EC senescence and a new idea for the early prevention and treatment of aging-related ASCVD.Methods1.We constructed the stress-induced senescence model of HCAECs by Ang?.First,HCAECs were stimulated with different concentrations of Ang?(0 mol/L,10-8mol/L,10-7 mol/L,10-6 mol/L,10-5 mol/L,10-4 mol/L)for 48 hours.We detected intracellular reactive oxygen species(ROS)level by DCFH-DA probe,the expression of senescence-related proteins(P53,P21,P16,?-galactosidase)by Western Blot(WB),and SA-?-gal positive cells by senescence-associated ?-galactosidase(SA-?-gal)staining to select the optimal concentration of Ang? to induce cell senescence.Secondly,HCAECs were stimulated with the optimal concentration of Ang? for different times(0 h,12 h,24 h,48 h and 72 h).We detected intracellular ROS level,the expression of senescence-related proteins and SA-?-gal positive cells at different time points,and the best time of Ang? induced cell senescence was selected.Furthermore,we detected cell cycle distribution by flow cytometry,nitric oxide(NO)content in cell culture supernatant by NO detection kit,endothelial nitric oxide synthase(e NOS)by enzyme-linked immunosorbent assay(ELISA)kit and intracellular SOD activity by superoxide dismutase(SOD)activity detection kit to detect the changes of cell cycle,NO,e NOS and SOD during Ang?-induced stress senescence in HCAECs.2.To explore the regulatory mechanism of STAT3 involved in stress-induced senescence in HCAECs.First,the expression of STAT3 in Ang?-induced HCAECs senescence were detected by real-time quantitative polymerase chain reaction(q RT-PCR)and WB,and it was confirmed that STAT3 was related to stress-induced senescence in HCAECs.Secondly,si-STAT3 and pc DNA3.1-STAT3 were transfected respectively,and the transfection efficiency of silencing and overexpressing STAT3 was detected by q RT-PCR and WB.Then,we analyzed the expression of senescence-related proteins,SA-?-gal positive cells,cell cycle distribution,NO content in cell culture supernatant,intracellular e NOS expression and SOD activity in each group(Control,Ang?,si-NC,si-NC+Ang?,si-STAT3,si-STAT3+Ang?,pc DNA3.1,pc DNA3.1 + Ang?,pc DNA3.1-STAT3,pc DNA3.1-STAT3+Ang?)to explore the effect of STAT3 on Ang?-induced stress senescence in HCAECs.3.To explore whether miR-106b-5p participated in the regulatory mechanism of stress-induced senescence in HCAECs.First,the expression of miR-106b-5p in HCAECs induced by Ang? were detected by q RT-PCR to clarify whether miR-106b-5p was related to HCAECs senescence.we analyzed the expression of senescence-related proteins,SA-?-gal positive cells,cell cycle distribution,NO content in cell culture supernatant,intracellular e NOS expression and SOD activity in each group(NC,NC+Ang?,miR-106b-5p mimics,miR-106b-5p mimics +Ang?,in-NC,in-NC +Ang?,miR-106b-5p inhibitor,miR-106b-5p inhibitor + Ang?)to verify that miR-106b-5p was involved in the regulation of HCAECs senescence.Secondly,the binding of miR-106b-5p to STAT3-3'UTR was verified by double luciferase assay.Then,the expression of STAT3 m RNA and protein were detected by q RT-PCR and WB in HCAECs with silencing and overexpressing miR-106b-5p to analyze the regulatory relationship of miR-106b-5p on STAT3.Finally,a reverse experiment was performed through the co-transfection of miR-106b-5p and STAT3,and we analyzed the expression of senescence-related proteins,SA-?-gal positive cells,cell cycle distribution,NO content in cell culture supernatant,intracellular e NOS expression and SOD activity in each group(Control,Ang?,NC + Ang?,miR-106b-5p mimics + Ang?,NC + pc DNA 3.1-STAT3 + Ang?,miR-106b-5p mimics + pc DNA 3.1-STAT3 + Ang?).Furthermore,miR-106b-5p was involved in the regulation of HCAECs senescence by targeting STAT3.4.To explore whether circ?AFF1was involved in the regulatory mechanism of stress-induced HCAECs senescence through miR-106b-5p/STAT3.First,the expression of circ?AFF1 in HCAECs by Ang? was detected by q RT-PCR to determine whether circ?AFF1 was related to Ang?-induced HCAECs senescence.At the same time,we detected the effects of the exonuclease RNase R-mediated digestion and actinomycin D-induced blockade of gene transcription on the expression of circ?AFF1 and linear AFF1 to identified circ?AFF1 as a real circRNA.Secondly,si-circ?AFF1 and p Lenti-CMV-circ?AFF1 were transfected respectively,and the transfection efficiency of silencing and overexpressing circ?AFF was detected by q RT-PCR.We analyzed the expression of senescence-related proteins,SA-?-gal positive cells,cell cycle distribution,NO content in cell culture supernatant,intracellular e NOS expression and SOD activity in each group(Control,Ang?,circ-NC,circ-NC+Ang?,si-circ?AFF1,si-circ?AFF1+Ang?,p Lenti-CMV,p Lenti-CMV+Ang?,p Lenti-CMV-circ?AFF1,p Lenti-CMV-circ?AFF1 +Ang?)to clarify the role of circ?AFF1 in HCAECs senescence.Subsequently,the binding of circ?AFF1 to miR-106b-5p was detected by RNA immunoprecipitation(RIP)and dual luciferase assay;FISH experiment was used to observe the co-localization of circ?AFF1 and miR-106b-5p in HCAECs.We detected the expression of miR-106b-5p and STAT3 in HCAECs with silencing and overexpressing circ?AFF1 by q RT-PCR to clarify the regulation of circ?AFF1 on miR-106b-5p and STAT3.Finally,a reverse experiment was performed through co-transfection of miR-106b-5p and circ?AFF1,and we analyzed the expression of senescence-related proteins,SA-?-gal positive cells,cell cycle distribution,NO content in cell culture supernatant,intracellular e NOS expression and SOD activity in each group(Control,Ang?,circ-NC+Ang?,si-circ?AFF1+Ang?,circ-NC+miR-106b-5p inhibitor +Ang?,si-circ?AFF1+miR-106b-5p inhibitor +Ang?).At the same time,the expression of STAT3 in each group was also detected by WB,which further verified that circ?AFF1targeted miR-106b-5p to affect the expression of STAT3,and then regulated the mechanism of Ang?-induced senescence in HCAECs.Results1.After HCAECs being stimulated by different concentrations of Ang? for 48 h,the levels of intracellular ROS,the expression of senescence-related proteins(P53,P21,P16,?-gal),and the rate of SA-?-gal positive cells increased with the increase of Ang?-stimulated concentration.The senescence-related phenotype changes of HCAECs were the most significant when the concentration of Ang? was 10-5 mol/L.The level of intracellular ROS,the expression of senescence-related proteins and the rate of SA-?-gal positive cells increased with the prolongation of Ang? stimulation time,and the senescence-related phenotypes changed most significantly in HCAECs after Ang? stimulation for 48 h.In addition,the percentage of cells in G0/G1 phase of the cell cycle increased,while NO concentration in cell culture supernatant,the level of intracellular e NOS and SOD activity decreased with the prolongation of Ang? stimulation time.2.The expression of STAT3 m RNA and protein increased with a time-dependent manner in Ang?-induced HCAECs senescence,suggesting that STAT3 may be related to Ang?-induced stress senescence in HCAECs.Regardless of the presence or absence of Ang? stimulation,the expression of senescence-related proteins,SA-?-gal positive cells and the percentage of cells in G0/G1 phase was reduced,while NO concentration in cell culture supernatant,intracellular e NOS content and SOD activity was increased in si-STAT3 group compared with the si-NC group,suggesting that si-STAT3 could inhibit HCAECs senescence.However,the results in the overexpression group of STAT3 were the opposite.3.The expression of miR-106b-5p decreased in Ang?-induced HCAECs senescence,suggesting that miR-106b-5p may be related to Ang?-induced stress senescence in HCAECs.Regardless of the presence or absence of Ang? stimulation,the expression of senescence-related proteins,SA-?-gal positive cells and the percentage of cells in G0/G1 phase was reduced,while NO concentration in cell culture supernatant,intracellular e NOS content and SOD activity was increased in miR-106b-5p mimics group compared with the NC group.However,the opposite results were found in the miR-106b-5p inhibitor group.Dual-luciferase assay showed that the fluorescence activity of co-transfected miR-106b-5p mimics and WT-STAT3-3'UTR group was significantly decreased compared with NC group.At the same time,compared with the Control group,STAT3 m RNA and protein expression levels were significantly decreased in miR-106b-5p mimics group,and increased in miR-106b-5p inhibitor group,confirming that miR-106b-5p has a negative effect on STAT3.In the recovery experiment of co-transfected miR-106b-5p and STAT3,overexpression of STAT3 could partially reverse the inhibitory effect of miR-106b-5p on Ang?-induced senescence in HCAECs,suggesting that miR-106b-5p regulated HCAECs senescence through STAT3.4.The expression of circ?AFF1 was increased with a time-dependent manner in Ang?-induced HCAECs senescence.The reverse primer of circ?AFF1 amplified the band only in c DNA,while the forward primer could exist in both c DNA and g DNA;and the tolerance of circ?AFF1 to the exonuclease RNase R-mediated digestion and actinomycin D-induced blockade of gene transcription was significantly higher than that of linear AFF1,proving that circ?AFF1 was a real circRNA.Regardless of the presence or absence of Ang? stimulation,the expression of senescence-related proteins,SA-?-gal positive cells and the percentage of cells in G0/G1 phase was reduced,while NO content in cell culture supernatant,intracellular e NOS expression and SOD activity was increased in si-circ?AFF1 group compared with the circ-NC group.However,the opposite results were found in the overexpression of circ?AFF1group.Dual luciferase assay showed that the luciferase activity of co-transfected miR-106b-5p mimics and WT-circ?AFF1 group was significantly decreased compared with the NC group;the RIP experiment showed that the abundance of circ?AFF1 and miR-106b-5p in the IP group treated with anti-Ago2 antibody was significantly higher than Ig G,confirming that circ?AFF1 specifically binded to miR-106b-5p.circ?AFF1 and miR-106b-5p predominated in the cytoplasm of HCAECs.The expression of miR-106b-5p in the si-circ?AFF1 group was increased,while the expression of STAT3 was decreased compared with the circ-NC group.However,the opposite results were found in the overexpressing circ?AFF1 group,suggesting that circ?AFF1 has a regulatory effect on miR-106b-5p and STAT3.In the recovery experiment of co-transfected miR-106b-5p and circ?AFF1,silencing miR-106b-5p could partially reverse the inhibitory effect of si-circ?AFF1on Ang?-induced senescence in HCAECs,and also reversed the effect of circ?AFF1 on STAT3,suggesting that circ?AFF1 targeting miR-106b-5p/STAT3 was involved in the regulation of stress senescence in HCAECs.Conclusions1.Ang? can induce stress senescence in HCAECs accompanied by activation of STAT3.knocking down STAT3 could inhibit Ang?-induced stress senescence in HCAECs.2.Mi R-106b-5p combines with STAT3 to inhibit its expression,and STAT3 could partially reverse the inhibitory effect of miR-106b-5p on Ang?-induced stress senescence in HCAECs,indicating that miR-106b-5p targetes STAT3 to inhibit Ang?-induced stress senescence in HCAECs.3.Circ?AFF1 binds to miR-106b-5p and inhibits the inhibitory effect of miR-106b-5p on STAT3,while knocking down miR-106b-5p could partially reverse the inhibitory effect of knocking down circ?AFF1 on Ang?-induced stress senescence in HCAECs,indicating that circ?AFF1 targets miR-106b-5p/STAT3 to promote Ang?-induced stress senescence in HCAECs.Innovation and significanceThis experiment verified the relationship between circ?AFF1 and Ang?-induced stress senescence in HCAECs,and further elucidated the mechanism of circ?AFF1targeting miR-106b-5p/STAT3 to promote Ang?-induced stress senescence in HCAECs.From the perspective of circRNA,miRNA and gene regulation,this experiment explored the regulatory mechanism of circ?AFF1 on Ang?-induced senescence in HCAECs,which revealed new targets of circRNA intervention on HCAECs senescence and provided new scientific clues for the prevention and treatment of aging-related ASCVD. |
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