The Role Of CircHECTD1 In Astrocyte Activation And Its Regulatory Mechanisms After Cerebral Ischemic Stroke

Aims: Stroke,which has a high incidence,is a common neurological disorder and one of the major causes of permanent disability worldwide.Due to a narrow time window and the side effects of recombinant tissue plasminogen activator,the efficacy of ischemic stroke thrombolytic therapy is limited.Therefore,novel treatment approaches that enable clinicians and researchers to overcome the above side effects and to extend the therapeutic time windows of existing agents are urgently needed.Circular RNAs(circ RNAs)are highly expressed in the central nervous system and are involved in the regulation of physiological and pathophysiological processes.However,the potential role of circ RNAs in stroke remains largely unknown.We sought to explore the role of circ HECTD1 in the pathogenesis of stroke,and then examined the effective therapeutic targets for stroke.Methods: The characteristics of circ RNA and m RNA expression in cerebral infarction and peripheral areas of transient middle cerebral artery occlusion(t MCAO)model mice were studied by using high-throughput microarry technology.Real-time PCR was used to detect the level of circ HECTD1 in cerebral infarction and peripheral areas of t MCAO mice and plasma of acute ischemic stroke(AIS)patients.Magnetic resonance imaging(MRI)and TTC staining were used to detect the volume of cerebral infarction of t MCAO model mice.The relationships among circ HECTD1,mi R-142(micro RNA 142)and TIPARP(TCDD inducible poly[ADP-ribose] polymerase)were predicted by using bioinformatics analysis software such as RNAhybrid and Target Scan.The expression of GFAP,TIPARP,LC3B-?,p62,Beclin-1 were detected by western blot.The correlation between circ HECTD1 and mi R-142 was verified by pull-down and in situ hybridization.The binding between mi R-142 and TIPARP was verified by luciferase assay.The effect of circ HECTD1/mi R-142/TIPARP on ischemia-reperfusion induced astrocyte activation was verified in vivo and in vitro by microinjection and lentivirus transfection,combined with immunofluorescence staining and western blot.Results: By using a circ RNA microarray,we showed that circ HECTD1 levels were significantly increased in ischemic brain tissues in t MCAO mouse stroke models and further validated this finding in plasma samples from acute ischemic stroke patients.Genome-wide bioinformatic analysis revealed that circ HECTD1,which is derived from exons 23 and 24 of the HECTD1 gene,acts as a sponge for mi R-142.We also found that TIPARP,which is predicted to be a target of mi R-142,is upregulated in ischemic tissues of the t MCAO mouse model using a gene expression microarray.Knockdown of circ HECTD1 expression significantly decreased infarct areas,attenuated neuronal deficits,and ameliorated astrocyte activation in t MCAO mice.Mechanistically,circ HECTD1 functions as an endogenous mi R-142 sponge to inhibit mi R-142 activity,knockdown the expression of circ HECTD1 result in the inhibition of TIPARP expression with subsequent inhibition of astrocyte activation via autophagy in oxygen glucose deprivation-reperfusion(ODG-R)condition.Conclusion: Our study revealed previously unappreciated regulatory mechanisms of circ HECTD1 that regulate astrocyte activation through targeting of the MIR142-TIPARP pathway via the regulation of autophagy.Specific blockage of circ HECTD1 is predicted to be a potential therapeutic target for the inhibition of astrocyte activation in stroke patients.