The Mechanism Of CircRNA CDR1as Regulating H₂O₂-induced Pyrotosis Of Human Coronary Endothelial Cells Through MiR-135a/SIRT1 Axis

BackgroundThe cardiovascular disease(CVD)caused by atherosclerosis(AS)is the leading cause of death worldwide,which is also a major public health problem in the world.At present,the mechanism of AS has not been clarified.The prevention and treatment of AS has not been thoroughly solved.Therefore,it can reduce the morbidity and mortality of atherosclerotic CVD by exploring the pathogenesis of AS.studies indicate that oxidative stress and chronic persistent inflammation play a very important role in AS.Long-term oxidative stress and inflammatory stimulation cause vascular endothelial dysfunction and even vascular injury reactions,including autophagy,apoptosis,pyroptosis,necrosis,etc.Different from other types of injury,pyroptosis is a kind of programmed inflammatory cell death,which is closely related to inflammation,while inflammatory response runs through the whole process of AS.It was found that pyroptotic cells were found in atherosclerotic plaques.Moreover,many factors induce pyroptosis of endothelial cell,further leading to AS.These suggest that cell pyroptosis is closely related to AS.Pyroptosis is a programmed cell death characterized by inflammasome formation,gasdermin D(GSDMD)dependent cell perforation,and release of inflammatory cytokines(IL)-1?,IL-18.The NLR Family(Pyrin Domain Containing Protein 3,NLRP3)inflammasome is a classic cell pyroapoptosis pathway that has been studied extensively at present.Studies show that ROS is a common key factor in the activation of NLRP3 inflammasomes by multiple pathological factors,and oxidative stress runs through the whole process of AS.Moreover,Studies have shown that the pyroptosis induced by ox-LDL,high glucose and reactive oxygen species plays an crucial role in the occurrence and development of AS.Therefore,intervention of endothelial cell pyroptosis is of great significance in the prevention and treatment of AS.Inhibition of pyroptosis by pharmacological or genetic intervention has a cardioprotective effect.Although pharmacological studies have found that a variety of drugs such as resveratrol and trimetazidine can be used in anti-pyrotosis therapy,gene intervention therapy may be a better choice due to its stronger targeting.Current studies have found that non-codingRNAs are involved in a variety of pathophysiological processes through the post-transcriptional regulation of genes.Circular ribonucleic acid(circRNA)is a kind of non-codingRNA generated by back splicing of 5?and 3?ends.Because its ring structure is not easy to be degraded by nuclease,it plays a stable regulatory role.Studies have found that circRNAs function in the process of pyroptosis.In humans,cerebellar degeneration associated protein 1 antisense transcripts(CDR1as)is transcribed from a gene located at Xq27.1 with 1485bp.As a well-studied circRNA,CDR1as has been found to regulate a variety of pathological processes including inflammation.One study found CDR1as was involved in the regulation of inflammatory factors such as IL-6,IL-8,IL10 and TNF-?in the inflammatory model induced by IL-1?.As one way of inflammatory death,pyroptosis is closely related to inflammatory response.Therefore,we considered that CDR1as might be involved in the pyroptosis of endothelial cells.MicroRNA is a single strandRNA with a length of about 22nt,which regulates the expression of target genes at the post-transcriptional level and plays a regulatory role in many pathophysiological processes including pyroptosis.In a model of acute pancreatitis,upregulated miR-135a promoted cell death and increased the expression of inflammatory cytokines(tumor necrosis factor-?,IL-6,and IL-1?).Moreover,It was found that miR-135a promoted the inflammatory responses of vascular smooth muscle cells by down-regulating FOXO1.These researches suggest that miR-135a is involved in the regulation of inflammation and cell survival,but whether miR-135a participates in the pyroptosis of endothelial cell has not been reported.Bioinformatics database showed that abasic sites of miR-135 and CDR1as are highly complementary.Therefore,we hypothesized that CDR1as may be involved in the process of endothelial cell pyroptosis through miR-135a.Meanwhile,bioinformatics database showed that miR-135a and SIRT1 had a good base complementary pairing site.SIRT1 is an NAD+-dependent histone deacetylase.Previous studies have shown that SIRT1 plays a protective role in regulating pyroptosis.In addition,SIRT1 plays a protective role in vascular diseases by regulating key metabolic processes through the deacetylation of a variety of substrates.Researches showed the expression level of SIRT1 were reduced in HUVEC treated by ox-LDL.Low expression level of SIRT1 gene is closely related to ROS production.Therefore,SIRT1 may exert protective effects in pyroptosis of endothelial cells.To sum up,we hypothesized that CDR1as affected pyroptosis of HCAECs by inhibiting miR-135a,which further targeted on SIRT1.HCAECs treated by H₂O₂ was used to build a model of pyroptosis.Then we explore the mechanism of CDR1as regulating SIRT1 to impact the pyroptosis of HCAECs through miR-135a.ObjectsTo define the expression levels of CDR1as and miR-135a in H₂O₂ treated HCAECs,explore whether CDR1as and miR-135a are involved in the pyroptosis.Furthermore,we explore the mechanism of CDR1as regulating SIRT1 on the pyproptosis of HCAECs through miR-135a.This provides a new concept and a new basis for the early prevention and treatment of AS.Methods1.We constructed the pyroptosis model of HCAECs by H₂O₂.HCAECs were stimulated by 600?mol/L H₂O₂ for different time(0,1h,2h,4h,8h).Then the pyrolysis of HCAECs was assessed by detecting lactic dehydrogenase(LDH)level and Hoechst33342/PI fluorescence double staining kit.2',7'-Dichloro Fluorescent Yellow Diacetate(DCFH-DA)probe was used to detect intracellular Reactive oxygen species(ROS)levels.In addition,the expression of NLR family Pyrin domain protein3(NLRP3),cysteinyl aspartate specific proteinase(caspase)-1,apoptosis-associated speck-like protein containing a CARD(ASC)was detected by Western blot,and IL-1?and IL-18 was detected by Enzyme-linked immunosorbent assay(ELISA)kit.2.The expression level of CDR1as,miR-135a and SIRT1 in H₂O₂treated HCAECs was detected by q RT-PCR.3.The high and low expression model of CDR1as were constructed by transfecting CDR1as overexpression and sh-CDR1as lentivirus to explore the effect of CDR1as on the pyroptosis of HCAECs induced by H₂O₂.LDH detection kit and Hoechst 33342/PI fluorescence double staining kit were used to evaluate pyroptosis in different groups.NLRP3,caspase-1,ASC was detected by western blot,and NLRP3was observed by immunofluorescence staining.In addition,IL-1?and IL-18 was detected by ELISA kit.4.The level of miR-135a were detected in HCAECs with high and low expression of CDR1as by q RT-PCR.Double luciferase assay was used to verify the binding of CDR1as to miR-135a.The level of SIRT1 was detected by q RT-PCR and Western blot in HCAECs with high and low expression of CDR1as.5.MiR-135a mimics and miR-135a inhibitor were transfected to construct high and low expression models of miR-135a in HCAECs,exploring the effect of CDR1as on the pyroptosis of HCAECs induced by H₂O₂.Pyrolysis was evaluated by detecting the level of LDH and Hoechst33342/PI fluorescence double staining in different groups.NLRP3,caspase-1 and ASC was detected by Western blot,and NLRP3 was observed by immunofluorescence staining.In addition,IL-1?and IL-18 was detected by ELISA kit.6.The level of SIRT1 was detected by q RT-PCR and western blot in HCAECs with high and low expression of miR-135a.Double luciferase assay was used to verify the binding of SIRT1 to miR-135a.The Sh-SIRT1 plasmid was used to construct cell model with the low-expression SIRT1.Pyroptosis was evaluated by detecting the level of LDH and Hoechst33342/PI fluorescence double staining in different groups.NLRP3,caspase-1,ASC was detected by Western blot,and NLRP3was observed by immunofluorescence staining.The expression of IL-1?and IL-18was detected by ELISA kit.Results1.After HCAECs being stimulated by 600?mol/L H₂O₂ for different time(0,1 h,2h,4h,8h),the release of LDH,intracellular ROS level and PI+cell number were increased,and Western blot shown that the level of NLRP3,caspase-1,ASC were also increased.The level of IL-1?and IL-18 was also significantly increased by ELISA kit.It is suggested that the pyroptosis model of HCAECs induced by H₂O₂ was successfully constructed.2.The expressions of CDR1as and SIRT1 were decreased,while the expression of miR-135a was increased with a time-dependent manner in H₂O₂-induced pyroptosis of HCAECs.3.In the pyroptotic model of HCAECs induced by H₂O₂,compared with the empty plasmid transfected group,LDH release,PI+cell number,expression of NLRP3,caspase-1,ASC,point aggregation of NLRP3 and the release of IL-1?and IL-18 were reduced in the overexpressed CDR1as group.However,The opposite results were found in the low expression group of CDR1as,suggesting that CDR1as can reduce the pyproptosis of HCAECs induced by H₂O₂.4.MiR-135a was down-regulated in the overexpression group of CDR1as,while miR-135a was up-regulated in the low expression group of CDR1as.Dual luciferase assay shown the fluorescence activity of co-transfected wt-CDR1as and miR-135a mimics was significantly decreased,suggesting that CDR1as inhibits the expression of miR-135a by binding with it.SIRT1 was up-regulated in overexpression group of CDR1as,while down-regulated in low-expression group CDR1as,suggesting that CDR1as promotes the expression of SIRT1.5.In the pyroptotic model of HCAECs induced by H₂O₂,compared with the miR-NC group,LDH release,PI+cell number,expression of NLRP3,caspase-1,ASC,point aggregation of NLRP3 and the release of IL-1?and IL-18 were reduced in the miR-135a inhibitor group.However,the opposite results were found in the miR-135a mimics group.These results suggest that miR-135a can promote H₂O₂-induced pyroptosis.6.SIRT1 was up-regulated in miR-135a inhibitor group,while down-regulated in miR-135a mimics group.Dual luciferase assay shown the fluorescence activity of co-transfected wt-SIRT1 and miR-135amimics was decreased compared with NC group.LDH release level and PI+cell number were increased in Sh-SIRT1 group.The expression of NLRP3,caspase-1 and ASC was increased by Western blot,and the fluorescence intensity of NLRP3 was increased by immunofluorescence staining.IL-1?and IL-18 showed increased by ELISA kit.These results suggest miR-135a promoted H₂O₂-induced pyroptosis of HCAECs by targeting SIRT1.Conclusions1.We find that H₂O₂ can pyroptosis in HCAECs through the detection of LDH,PI staining,pyroptosis related proteins and inflammatory factors,.2.CDR1as inhibites H₂O₂-induced pyroptosis of HCAECs,suggesting that CDR1as is related to H₂O₂-induced pyroptosis of HCAECs..3.CDR1as inhibits the expression of miR-135a,while miR-135a promotes pyroptosis of HCAECs,which suggest that the inhibition of H₂O₂-induced pyroptosis by CDR1as may be related to miR-135a in HCAECs.4.CDR1as and miR-135a may regulate H₂O₂-induced pyroptosis of HCAECs through SIRT1.CDR1as inhibits pyroptosis of HCAECs,while miR-135a promotes pyroptosis of HCAECs.In conclusion,it may provide a new approach for the treatment of cardiovascular diseases such AS by exploring the mechanism of CDR1as in inhibiting oxidative stress-induced pyroptosis.Innovation and significanceThis study reveales that CDR1as has the effect of anti-pyroptosis in HCAECs,and further elucidates that CDR1as alleviates pyroptosis of HCAECs through inhibiting miR-135a targeting SIRT1 which provides a new theoretical and scientific basis for the early prevention and treatment of AS.