The Role Of Circular RNA TLK1 In Neuronal Injury After Cerebral Ischemic Stroke

BackgroundIschemic stroke is a common neurological disorder and a major cause of permanent disability worldwide.Clinical treatments,such as thrombolysis,are often restricted by the narrow therapeutic time window and insufficient long-term effects.The extent of neuronal injury after brain ischemia is a primary factor determining outcomes.Hence,therapeutic options that rescue damaged neurons are important;however,to date,only a few therapeutic agents have been reported to relieve neurological deficits after stroke.Therefore,novel therapeutic approaches that decrease neuronal injury are urgently needed.Circular RNAs(circRNAs),the newly identified endogenous noncoding RNAs(ncRNAs),are characterized by back splicing resulting from covalently closed continuous loops.Many circRNAs are abundantly expressed in the brain and are involved in various central nervous system diseases.As shown in our previous studies,circHECTD1 aggravates ischemic damage by regulating astrocyte activation,and circDLGAP4 overexpression improves the blood-brain barrier(BBB)integrity in mice after cerebral ischemia.However,the potential role of circRNAs in ischemic stroke-associated neuronal injury remains largely unknown.In the present study,circTLK1 was upregulated during the acute period after focal ischemia(GEO accession number: GSE115697),but its function remains unclear.However,researchers have not determined whether the circTLK1 is involved in cerebral ischemic injury,particularly neuronal injury.Therefore,this study first investigated the regulation of circTLK1 on neural injury after transient middle cerebral artery occlusion(tMCAO)reperfusion in adult mice.Secondly,this study explored the molecular mechanism of circTLK1 in regulation of ischemic neuronal injury.Thirdly,the study analyzed the expression of circTLK1 in patients with acute ischemic stroke(AIS)and explored the potential of circTLK1 as a biomarker of neuronal injury after cerebral ischemia.Finally,above all,this study is excepted to provide new theoretical support for elucidating the function of circRNA in cerebral ischemia injury,and contribute to the in-depth understanding in pathophysiological mechanism of ischemic stroke,thus laying a foundation for circRNA-based biomarkers and therapeutic targets of ischemic stroke.Part 1 Effect of circTLK1 on the neurological injury after ischemic stroke Objective: To investigate the effect of circTLK1 on neurological defect and neuronal injury after cerebral ischemic stroke.Methods:(1)Lentivirus and neuron-specific adeno-associated virus were used to interfer circTLK1 expression in mouse brain.Modified neurological severity score,cylinder test and adhesive-removal test were used to evaluate the neurological function of mice after cerebral ischemia.Brain infarction and atrophy volume were determined by 2,3,5-triphenyltetrazolium staining,magnetic resonance imaging and immunohistochemical staining.The dendritic morphology and the number of dendritic spines of neurons in the peri-infarct zone were observed by Golgi staining.Western blotting was used to detect the expression of apoptosisrelated proteins or synapse-related proteins in ischemic cortex.Cell localization of circTLK1 was observed by in situ hybridization.(2)In vitro,primary cortical neurons were used to suffered an oxygen-glucose deprivation/reperfusion(OGD/R)model,which was combined with lentivirus,cell counting kit-8,immunofluorescence staining and other methods to evaluate neuronal injury,morphology of neuronal dendrites,dendritic spines and axon growth.Western blotting was used to measure the expression of apoptosis-related proteins or synapse-related proteins in primary neurons.Results:(1)circTLK1 was increased in the ischemic cortex and plasma of tMCAO mice.(2)Knockdown of circTLK1 in the brain reduced the cerebral infarct volume after tMCAO.(3)Knockdown of circTLK1 in the brain improved the neurological deficits and long-term functional prognosis of tMCAO mice.(4)Post-stroke knockdown of circTLK1 in the brain significantly improved the neurological deficits and somatosesthetic dysfunction in tMCAO mice.(5)Neuron-specific knockdown of circTLK1 could improve neurological deficits and reduce brain atrophy in tMCAO mice.(6)Knockdown of circTLK1 improved OGD/R-induced decreased neuronal cell viability and reduced apoptotic protein expression in vitro.(7)Knockdown of circTLK1 increased the expression of synaptic related proteins reduced by OGD/R,and increased the total dendrite length and dendrite complexity,as well as the axon length and dendrite spine density after OGD/R.Conclusion: circTLK1 is involved in neuronal injury after cerebral ischemia in mice and is involved in the regulation of neuronal damage induced by oxygen-glucose deprivation.Part 2 Mechanism of circ TLK1 in regulating neuronal injury in ischemic strokeObjective: To investigate the molecular mechanism of circTLK1 in regulating neuronal injury in ischemic stroke.Methods:(1)Affinity analysis and luciferase assays were used to verify the interaction between mi R-335-3p and circ TLK1,as well as the mutual binding between mi R-335-3p and its target gene 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD)-inducible poly(adp-ribose)polymerase(TIPARP).(2)Neuron-specific adeno-associated virus was used to knockdown the expression of TIPARP in mouse brain.Modified neurological severity score,cylinder test and adhesiveremoval test were used to evaluate the neurological function of mice after cerebral ischemia.The cerebral infarction volume and atrophy volume were measured by magnetic resonance imaging.Western blotting was used to analyze the expression of apoptosis-related proteins or synapse-related proteins in ischemic tissues.(3)High throughput RNA sequencing was used to identify TIPARP regulated signaling pathways in regulating neuronal injury after ischemic stroke.Results:(1)circ TLK1 binded to mi R-335-3p.(2)circ TLK1/mi R-335-3p axis aggravated neuronal injury by increasing TIPARP.(3)Neuron-specific TIPARP knockdown improved neurological defect and somatosensory dysfunction in t MCAO mice.Neuron-specific TIPARP knockdown reduced brain atrophy volume and apoptotic protein expression,and improved the reduction of synapse related protein expression induced by cerebral ischemia.(4)Transcriptome sequencing analysis showed that 516 differentially expressed genes(DEGs) were identified between t MCAO + AAV-SYN-sh RNA-TIPARP group and t MCAO + AAVSYN-sh RNA-Con group at 24 hours after t MCAO.Through Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,it was found that these DEGs were enriched in the pathways involved in cell processes,signal transduction and nervous system.(5)The results of transcriptome sequencing showed that there were 192 significantly DEGs between t MCAO + AAV-SYN-sh RNA-TIPARP group and t MCAO + AAV-SYN-sh RNA-Con group at 28 days after t MCAO.Through KEGG analysis,these DEGs were found to be enriched in biological processes involved in the regulation of neurology,signal transduction pathways and immunology.Conclusion:(1)Circ TLK1/mi R-335-3p/TIPARP axis is involved in ischemic brain injury,especially neuronal injury.(2)The upregulated circ TLK1 binds to mi R-335-3p,inhibiting the activity of mi R-335-3p,and increases the expression of TIPARP,resulting in neuronal damage and neurological dysfunction.Part 3 Circ TLK1 was up-regulated in plasma of patients with acute ischemic stroke Objective: To explore the expression of circ TLK1 in plasma of patients with acute ischemic stroke.Methods:(1)Seventy-one patients(59 males,12 females)with AIS and 71 healthy controls matched by gender and age were involved in this study.Quantitative reverse transcription-PCR was performed to examine the expression of circ TLK1 in plasma.(2)According to the classification criteria of the etiology of ischemic stroke,patients were divided into three subtypes: large artery arteriosclerosis(LA),small-artery occlusion(SA)and cardioembolism.(3)According to imaging data,AIS patients were divided into three types of infarction sites: cortical infarction,subcortical infarction and subtentorial infarction.(4)Receiver operating characteristic(ROC)curve and area under ROC curve(AUC)were assessed to evaluate the diagnostic value of plasma circ TLK1 level in AIS.Pearson correlation test was employed to evaluate the correlation between relative plasma circ TLK1 level and cerebral infarction volume in LA patients.Results:(1)circ TLK1 was up-regulated in plasma of patients with acute ischemic stroke.(2)Plasma circ TLK1 in LA and SA patients were significantly increased.(3)Plasma circ TLK1 in AIS patients with cortical infarction and subcortical infarction were significantly increased,but there was no significant change in patients with subtentorial cerebral infarction.(4)ROC curve analysis showed that the area under the circ TLK1 curve was 0.868,the sensitivity was 0.789,and the specificity was 0.915.(5)The regression analysis showed that the larger the size of cerebral infarction in AIS patients,the higher the expression of circ TLK1 in plasma(r = 0.7197,p = 0.0017).Conclusion: circ TLK1 is up-regulated in the plasma of patients with acute ischemic stroke,and the plasma circ TLK1 level is correlated with the cerebral infarction volume in LA patients.