The Relationship Between MiRNA-98 And Ischemic Stroke

Stroke is one of the most serious diseases that threaten human health.It is the third most deadly disease in the world.Epidemiology shows that the morbidity of stroke is younger and increasing year by year.According to the different causes of cerebral infarction,Ischemic stroke occupied 87%groups.The main pathological mechanism research reveals the impact of stroke occurrence and development of neurons including energy metabolism disorder,excessive depolarization,excitotoxicity,ion homeostasis,inflammation and immune disorders,apoptosis and autophagy.To control the mortality and improve the quality of life is a difficult problem to be solved.FDA is currently approved for the treatment of stroke only tissue plasminogen activator(tPA).However,the strict treatment time window and easy to induce cerebral hemorrhage and other complications greatly limits its clinical application,the drug research and development of stroke is still a long way to go.Platelet activating factor(platelet activating,factor,PAF)is a kind of endogenous active phosphoric acid medium closely related to the metabolism and four arachidonic acid,by a variety of organizations involved in allergy and inflammatory reaction and cell(such as macrophages,endothelial cells,platelets and polymorphonuclear cells),widely involved in the pathophysiological process in the body.Its specific receptor is PAFR(platelet activating factor receptor,PAFR).The process of ischemic stroke,abnormal release of PAF binding to its receptor PAFR after brain injury caused by the following mechanism:(1)it increased nerve cell calcium concentration,disrupt the function of cell membrane,causing direct damage to neurons;(2)promote platelet aggregation and activation,release of vasoactive substances,causing vascular embolism so,aggravate brain edema;(3)chemotaxis and activation of inflammatory cells,promote the release of inflammatory factors,initiation and development of inflammatory injury etc.A large number of animal experiments and clinical trials showed that PAF content in brain tissue of rats with acute cerebral infarction was significantly increased.But its activation is not clear.MicroRNA(miRNA,a tiny RNA),a class of non protein coding RNA family,can regulate gene expression by binding to specific mRNA or regulating the translation of specific mRNA proteins.At present,more than one thousand miRNAs have been identified in the human genome,which may be involved in the regulation of the expression of the human gene encoding protein 30%.MiRNA plays an important role in regulating the expression of target genes in cells,and is closely related to the development,growth,proliferation and differentiation of cells.In the human genome sequence,miRNA-98 is a MicroRNA sequence of 5 '-UGAGGUAGUAAG UUGUAUUGUU-3'(GeneBank:GeneID:407054).MiRNA-98 is highly conserved in evolution and has homology among different species.Has a similar effect on animals or humans?In conclusion,this study suggests that miRNA can regulate the expression of platelet activating factor receptor and influence the downstream signaling pathway of platelet activating factor,and then participate in the process of ischemic stroke.In order to investigate the effect and related mechanism,we do the following research.The experiment is divided into two parts.Part I:The study of miRNA-98 and PAFR correlationObjective:To study the expression of miRNA-98 and platelet activating factor receptor PAFR.Methods:A plasmid was constructed to transfect the PAFR gene expression vector and the reporter gene plasmid into HEK293T,and to study the correlation between miRNA-98 and DNA.To collect the serum of patients with stroke,and to extract the same sex and age matched with the case group as the healthy control group.Blood samples were collected from two groups of subjects,and the expression of miRNA-98 was detected by real-time PCR.The platelet activating factor was detected by Elisa method.Results:The effects of hsa-miR-98-5p on the expression of PTAFR WT and Mut were observed.Since hsa-miR-98-5p mimics and 3'UTR containing PTAFR gene fragment of dual luciferase reporter gene co transfection experiments compared with the negative control group(mimic control and 3'UTR containing PTAFR gene fragment of dual luciferase reporter gene transfection test)had significant difference(P=0.001),the ratio of fluorescence as negative control 65.8%.Hsa-miR-98-5p mimics had an inhibitory effect on the gene expression level of the predicted sites with PTAFR-3'UTR,but the inhibition effect was significantly decreased after the predicted site mutation.Therefore,hsa-miR-98-5p is likely to play a role through PAFR.The PAF505.7±93.6pg/ml,in serum of patients with stroke,while the control group was 163±36.6pg/ml,which was higher than that of the control group,and the serum PAF in patients with cerebral apoplexy was increased by 3.4 times compared with the control group.The content of miRNA-98 in the serum of patients with stroke was 0.23±0.07,while that of the healthy control group was about 0.92±0.18.Compared with the healthy control group,the serum miRNA-98 levels were significantly lower in patients with stroke.Conclusion:Studies in vitro showed that miRNA-98 could bind to the promoter region of PAFR.MiRNA-98 can increase the expression level of PAFR.Clinical study showed that the serum PAF levels in patients with ischemic stroke were significantly increased,miRNA-98 content was significantly lower.Part II:The relationship between miRNA-98 and PAFR related signal pathwayObjective:To study the expression and correlation of miRNA-98 and PAFR in penumbra region and its relationship with downstream NF-kB,MAPK family and apoptosis.Methods:The model of focal cerebral ischemia reperfusion injury was prepared.Detect the expression of miRNA-98 in serum and brain tissue penumbra by real-time PCR.The expression of PAFR in the penumbra of serum and brain tissue was measured by Western blot.The expression of NF-kB,MAPK family and apoptosis signal pathway of PAFR in serum and brain tissues were measured by Western blotting.Results:Compared with sham operation group,the expression of miRNA-98 in serum and penumbra of ischemic stroke rats was significantly lower.The expression of PAFR protein in the penumbra region of the model group was 1.72±0.06,while that of the sham operated group was about 1.00±0.14.Compared with sham operation group,the expression of PAFR in ischemic penumbra of the ischemic stroke increased,and there was significant difference.Compared with sham operation group,the expression of pIKK in model group was significantly increased,and the level of P65 activation was significantly increased.The phosphorylation level of JNK and P38 increased in ischemic penumbra.The expression of Bcl-2 protein was down regulated and the expression of Bax was significantly increased in the downstream apoptotic signal pathway of PAFR.The expression of Caspase-3 was significantly increased in the penumbra region.Conclusion:Studies in vivo showed that miRNA-98 could bind to the promoter region of PAFR.The expression level of miRNA-98 was decreased and the expression level of PAFR was increased in the penumbra of cerebral ischemia injury.In vivo study of the downstream signal pathway of PAFR,the phosphorylation level of IKK,P65,JNK and P38 in model group increased.The expression of Bcl-2 protein was significantly down-regulated,and the expression of Bax and caspase-3 were significantly increased.