Hsa_circ₀060180/HECTD1 Mediate Endothelial Cell Dysfunction Induced By Ischemia/Reperfusion Injury

Objective: Ischemia/reperfusion（I/R）injury,a common aspect of cardiovascular diseases that is characterized by the over-production of inflammatory factors,such as cytokines and chemokines.Recent studies have shown that the microvascular system which closely related to vascular endothelial cell dysfunction plays a crucial role in I/R injury.As a new class of non-coding RNAs,circular RNA（circ RNA）has gradually been clarified in its biological functions,and some circular RNAs have been confirmed to be participated in the evolution of diverse diseases,such as cardiovascular diseases.Previous research by our group showed that HECTD1,a member of the E3 ubiquitin ligase family,can regulate endothelial mesenchymal transition,and cell migration.However,it has not been reported in the damage of microvascular system induced by I/R.Therefore,current study mainly focused on the role of hsa_circ₀060180 / HECTD1 in endothelial cell dysfunction induced by I/R injury,which opens a new perspective and strategy for the treatment of I/R injury.Methods: Human umbilical vein endothelial cells（HUVECs）were applied to establish the in vitro model of I/R stimulation.Western blot and immunocytochemistry were combined to detect the level of target protein HECTD1 in HUVECs stimulated by I/R.The detection of HUVECs viability was confirmed by CCK-8.Hoechst staining,apoptosis-related biomarkers were combined with Brd U assay to identify HUVECs apoptosis and proliferation.The migration of HUVECs were evaluated by both 2D scratch and 3D nested migration model.The mechanism of apoptosis and migration of HUVECs was investigated by using CRISPR/Cas9 to regulate HECTD1 in combination with pharmacological methods.q RT-PCR and fluorescence in situ hybridization（FISH）assay were used to detect the variational expression of hsa_circ₀060180 and mi R-143.Lentivirus transfection which regulated hsa_circ₀060180 binding with the application of mimics and antagonists of mi R-143 to explore the molecular regulation on HECTD1 and the effects on downstream function of ECs.In vivo,FISH combined with immunohistochemistry were used to detect the expression and distribution of mmu_circ₀001098 which is highly homologous to hsa_circ₀060180 and HECTD1 in cardiac vascular tissue of mice which stimulated by acute myocardial I/R.Results: 1)I/R induced a significant time-dependent decrease in HECTD1 associated with an approximate 50% decrease in cell viability and upregulation in both cell apoptosis and migration.2)Overexpression of HECTD1 reversed these I/R-induced effects.3)I/R-induced upregulation of ERS-related proteins were attenuated by overexpression of HECTD1,suggesting that HECTD1 regulates endothelial cell function through ERS.4)The ubiquitination blocker MG-132 can abolish the inhibition of HECTD1 on ERS in a certain extent.5)I/R induced a significant decrease in hsa_circ₀060180 and a 2.5 fold increase in mi R-143.6)Overexpression of hsa_circ₀060180 can up-regulate the expression of HECTD1,meanwhile the antagonists and mimics of mi R-143 cause increase and decrease of HECTD1 expression respectively.7)hsa_circ₀060180 can abolish the inhibition of mi R-143 on HECTD1 by competitive endogenous RNA（ce RNA）mechanism.Conclusion: Our results suggest a critical role for hsa_circ₀060180 and HECTD1 in endothelial cell dysfunction induced by I/R,providing a new perspective and potential therapeutic targets for clinical treatment of I/R injury and cardiovascular diseases.