Role And Mechanism Of CircZKSCAN1 In Regulating Endothelial Cell Function And Endothelial Repair

Background and AimsCoronary artery disease(CAD)is one of the most common causes of death in the world.The introduction of percutaneous vascular reconstruction has significantly improved the therapeutic effect of CAD patients.At present,percutaneous coronary intervention(PCI),including percutaneous transluminal coronary angioplasty(PTCA),stent implantation and drug-coated balloon(DCB),has greatly reduced the mortality associated with CAD,especially those cases related to acute myocardial infarction(AMI).However,at the same time,restenosis(ISR)has gradually become the main problem and challenge of PCI treatment CAD.At present,although there are drugs for restenosis,the delivery of these drugs can not completely eradicate ISR,and there is still a risk of intimal hyperplasia,so the exploration of new therapeutic targets and therapeutic schemes for ISR still needs further study.Circular non-coding nucleic acids(RNAs)have been reported to be closely related to cardiovascular diseases.Among them,circZKSCAN1 is one of the newly discovered Circular RNAs molecules with high expression in vascular intima in recent years,but its relationship with endothelial function and intima injury and its regulatory mechanism have not been reported.MethodsFirstly,we used sanger sequencing,fluorescence semi-quantitative and cycloheximide experiments to verify the circular structure of circZKSCAN1,and detected the content of circZKSCAN1 in ISR clinical samples and animal models of intimal injury.Then,the rat carotid artery balloon injury model was established and treated with sh-circZKSCAN1adeno-associated virus.Carotid artery tissues were collected and the pathological changes were verified by hematoxylin-eosin staining,immunohistochemistry,immunofluorescence and in situ hybridization experiments.Then circZKSCAN1 was overexpressed or knocked down in human umbilical vein endothelial cells(HUVECs)to carry out cell function experiments,including cell proliferation and migration experiments,flow experiments and PI staining experiments,to extract protein and RNA detection signal pathways.Finally,we explored the regulatory mechanism of circZKSCAN1.At first,we predicted that circZKSCAN1 might be IRES or m6 A to regulate translation.We designed and constructed IRES mutant and N6-methyladenosine(m6A)mutant respectively,and then carried out luciferase gene reporting experiments and western blot experiments to verify whether these two mutants encode proteins.Finally,the endothelial cell function experiment was carried out under the condition that the encoded protein was inhibited.ResultsFirst of all,we make it clear that circZKSCAN1 is a stable ring structure.Fluorescence in situ hybridization and immunofluorescence tests in healthy and restenosis femoral arteries after stenting confirmed that circZKSCAN1 was specifically expressed in the intima of blood vessels with significant differences in expression.At the same time,the content of circZKSCAN1 in endothelial cells was significantly higher than that in vascular smooth muscle cells.In the following in vivo treatment test,we first verified the knock-down efficiency of circZKSCAN1 in the rat carotid balloon injury model.HE and Masson staining showed that the injured intima was thickened and collagen was deposited,but the treatment group was significantly relieved.Immunofluorescence results showed that knocking down circZKSCAN1 could significantly inhibit the proliferation of endothelial cells.At the same time,we found for the first time that necroptosis in thickened intima occurred significantly,and the expression of necroptosis markers RIP3,p-RIP3,p-RIP3,p-MLKL decreased significantly after knocking down circZKSCAN1,that is,necroptosis was inhibited.In vitro,overexpression of circZKSCAN1 in HUVECs can promote proliferation,migration and necroptosis,while knocking down circZKSCAN1 has the opposite effect.This is the first time that intimal hyperplasia is related to necroptosis.Next,we explored the molecular mechanism of the function of circZKSCAN1.Through the prediction of biological information website,we found that circZKSCAN1 can encode protein.We designed and constructed an overexpression plasmid with flag sequence.Western blot detection found that circZKSCAN1 can indeed encode protein.We named the encoded protein circZKSCAN1-203 aa.According to the prediction,circZKSCAN1 may encode protein through IRES or m6 A.Therefore,we designed IRES mutant,and the luciferase gene report experiment found that IRES was inactive,that is,circZKSCAN1 did not encode protein through IRES.Next,we constructed mutants for the predicted two m6 A sites.WB detection found that the expression of flag band was significantly inhibited after the mutation at the 325 site of m6 A,that is,circZKSCAN1 translated the protein through the 325 site of m6 A.At the same time,the proliferation,migration and necroptosis of HUVECs were also significantly inhibited after the 325-site mutation of m6 A.Finally,in order to further regulate the enzyme modified by m6 A,we knocked down the translation initiation factor eIF4G2 and the reading protein YTHDF3 of m6 A,and found that the coding protein of circZKSCAN1 was significantly reduced,and the proliferation,migration and necroptosis of HUVECs were also inhibited.This result confirmed that eIF4G2 and YTHDF3 are the main regulatory enzymes that regulate the protein encoded by m6A-mediated circZKSCAN1.ConclusionCircZKSCAN1 can regulate the damage and repair of vascular intima by affecting the proliferation,migration and necroptosis of HUVECs.In mechanism,the function of circZKSCAN1 is mainly realized through the regulation of protein encoding circZKSCAN1 mediated by m6 A modification by eIF4G2 and YTHDF3.