Study On The Mechanism Of Long Non-Coding RNA TUG1 Regulating Ischemic Stroke

Background: Ischemic stroke(IS)is a common nervous system disease with high mortality,disability and recurrence rate,which is caused by insufficient blood flow perfusion of brain tissue caused by cerebrovascular disease or intravascular obstruction,resulting in insufficient oxygen supply and accumulation of metabolic wastes.At present,targeted clinical thrombolytic therapy or intravascular thrombolytic therapy are limited by the narrow time window of treatment,and the long-term effect is not ideal.Only a few therapeutic drugs can save ischemic injured neurons to alleviate the neurological deficit after stroke.Long noncoding RNA(lncRNA)widely exists in the central nervous system and regulates the development and disease progression of the central nervous system at the levels of epigenetic,transcription,post transcription and chromatin remodeling.There are differences in the level and type of lncRNA expression between patients with IS and healthy individuals,and the genetic variation of lncRNA loci is also related to the increased risk of stroke.Some lncRNAs have been reported to be related to the occurrence of IS and involved in complex pathological processes related to stroke,such as neuronal apoptosis and inflammation.Recently,it has been found that lncTUG1 increases the expression of inflammatory factors and the occurrence of apoptosis in neurons by combining with the target micro RNA(miRNA)and using the mechanism of competitive endogenous RNA(ceRNA).However,there is no report on the mechanism of the interaction between lncTUG1 and protein in neuronal ischemic injury,which needs further study.HuR is a member of ELAV protein family,and is the only RNA Binding Protein(RBP)widely expressed in all human tissues.The functional activity of HuR is regulated by dynamic subcellular localization: under normal cellular physiological conditions,HuR is mainly located in the nucleus;However,after exposure to stimulation or injury,HuR transfers to the cytoplasm to stabilize and increase the translation of target mRNA,leading to the activation of inflammatory phenotype,which is the basis of its involvement in the occurrence and development of many diseases.In the ischemic cerebral ischemia model of HuR transgenic mice,the expression of HuR in astrocytes can aggravate vasogenic brain edema and deteriorate the neural function of acute ischemic stroke mice,but the specific mechanism remains to be further studied.Methods: The model of neuronal ischemia in vitro was established by OGD treatment of HT22 cell line,and the subcellular distribution of lncTUG1 was detected by real-time quantitative PCR and FISH staining after the cytoplasmic,nuclear and total RNA were extracted respectively.The computer bioinformatics prediction software was used to predict the binding of lncTUG1 to HuR,and then the formaldehyde RNA immunoprecipitation test(fRIP)was used to verify the binding ability of HuR and lncTUG1,as well as the effect of ischemic stimulation on the binding ability.The subcellular localization and distribution changes of HuR protein were determined by Western Blot experiment by extracting nucleus,cytoplasm,and total protein respectively.Si RNA and ASO were used to inhibit the expression of lncTUG1 in HT22 cytoplasm and nucleus,respectively.Western Blot and brain tissue section immunofluorescence assay were used to verify the distribution changes of HuR in nucleus and cytoplasm in vitro and in vivo,respectively.The differential expression of COX-2 mRNA in OGD model,the binding of HuR to COX-2 mRNA and the effect of ischemic stimulation on the binding ability were verified by real-time quantitative fluorescent PCR and formaldehyde RNA immunoprecipitation assay(f RIP).Finally,CCK8 experiment was used to explore the effect of inhibiting lncTUG1 in HT22 cytoplasm and nucleus on cell apoptosis;CRISPR-Cas9 technique was used to construct lncTUG1 KO rats and establish stroke models.TTC staining,neurologic score,HE staining and TUNEL staining were used to verify the effects of lncTUG1 gene knockout on the infarct area,the proportion of neuronal apoptosis and the damage of neural function in stroke models.Results: q PCR and FISH experiments confirmed that lncTUG1 shuttled from nucleus to cytoplasm after OGD.The bioinformatics analysis results predicted by Tartagliaab Webservices showed that there were binding sites and binding tendencies between lncTUG1 and HuR.Through f RIP,it was verified that lncTUG1 and HuR could form complexes and the binding tendency was enhanced under ischemic stimulation.HuR in the nucleus was transferred to the cytoplasm after OGD,but remained unchanged in the total number of HT22 cells.The inhibition of lncTUG1 in nucleus reverses the translocation of HuR to cytoplasm,on the contrary,the inhibition of lncTUG1 in cytoplasm cannot reverse the translocation of HuR to cytoplasm.The expression of COX-2mRNA increased several times after OGD.It was verified by f RIP that COX-2mRNA could form a complex with HuR and the binding tendency was enhanced under ischemic stimulation.When inhibition of lncTUG1 in nucleus reverses the translocation of HuR to cytoplasm,COX-2 mRNA expression also decreases significantly.The immunofluorescence test after MCAO in SD rats showed that HuR in the nucleus was transferred to the cytoplasm,while the distribution of HuR in lncTUG1 KO rats after MCAO did not change significantly.Compared with SD rats,after MCAO in lncTUG1 KO rats,TTC staining showed that the infarct volume was significantly reduced,the proportion of neuronal apoptosis and loss was significantly reduced,and the m NSS neurological function score was reduced.Conclusion: This study confirmed that lncTUG1 promotes the expression and translation of COX-2 mRNA by promoting the transfer of HuR protein from nucleus to cytoplasm in vivo and in vitro and plays an important regulatory role in promoting neuronal apoptosis.