The Expression And Regulation Of MiR-185 And Apba-1 In Cerebral Ischemia-reperfusion Injury

Ischemic stroke is the third leading cause of death in developping countries and is a leading cause for human disability as well as in our country. The stroke morbidity rate is increased with the extension of human lifespan. As we all known, the occurrence of stroke is related by multi-factors including genes and environment. However the pathogenesis of stroke are still unknown. Therefore, it is very important to explore the pathogenesis and the protection mechanism of stroke.Micro RNAs(mi RNAs) are small, noncoding RNAs with 22-25 nucleotides. mi RNAs are generated by a two-step processing pathway to yield RNA molecules of approximately 22-25 nucleotides that negatively regulate target gene expression at the post-transcriptional level by Drosha and Dicer. Mature mi RNAs enact m RNA translationalrepression or cleavage via incorporation into a multiprotein complex termed RISC(RNA-induced silencing complex) and subsequent complete or incomplete binding to the 3' untranslated region(UTR) of their respective target m RNAs. Since 1993, mi RNAs were found in growing development of nematode caenorhabditis rod insect larvae, there were more than 1600 mi RNAs recorded in database. Currently the expression profile of ischemic stroke was detected in vitro and vivo. Furthermore, mi RNA micro array detection technology as a kind of screening was used to confirm the expression changes of mi R-185 in ischemic stroke. The result showed that the expression of mi R-185 may be upregulated in stroke. Nevertheless, the Beta-Amyloid precursor protein 1(Apba-1) was extensively researched in Alzheimers disease. Its expression changes in stroke were proved by using isobaric tags for relative and absolute quantitation(i TRAQ). But the changes and the relationship of mi R-185 and Apba-1 in stroke were still unclear. Our studies are to search the changes and the relationship of mi R-185 and Apba-1 in stroke by oxygen glucose deprivation(OGD) and middle cerebral artery occlusion(MCAO).Part I Prediction of regulation pathways of mi R-185 in stroke based on bioinformatics analysisObjectives: To predict the regulation pathways of mi R-185 in stroke based on the methods of bioinformaticsMethods: The pubmed was used to find the micro RNA regulated with ischemic stroke. The UCSC genome browser, the human mi RNA disease database(HMDD), the transcription factor-mi RNA regulation database(Transmi R), Target Scan 6.2 were employed to study the upstream transcription factor, downstream target genes of mi R-185.Results: The study shows that mi R-185 is regulated with many diseased, and one of these is pathophysiologic of stroke such as inflammatory response, ion transport and oxidative stress. The UCSC genome browser shows that mi R-185 is demonstrated with high conservatism in several species. Mi R-185 is regulated by the transcription factor EGR1, and at the same time, it could regulate Apba-1 as a target gene.Conclusions: The bioinformatics analysis predicted the relationships between mi R-185 and stroke, and at the same time, predicted its downstream target genes for further research.Part II The expressions of mi R-185 and Apba-1 in OGD-induced neurons injuryObjectives: To explore the expressions of mi R-185 and Apba-1 in OGD-induced neurons injuryMethods: Rat cortical neurons were originally cultured for 8-10 days in vitro before OGD was carried out. Then MAP2 was used to identify the cortical neurons. After1 h and 2h OGD, 0h, 12 h, 24 h reoxygenation was followed. CCK-8 was used to evaluate cell viability. QRT-PCR was used to detect the expressions of mi R-185 and Apba-1 on m RNA level.Results: The result of MAP2 showed that cortical neurons were well-grown. CCK-8 assays showed that about 20% cells death occurred after 1h OGD.and 50%-55% cells death occurred after 2h OGD. The result demonstrated that OGD was successful and 2h OGD was fit for experiment. The expression of mi R-185 was higher in OGD cell than that in normal cell and with the increasing of the reoxygenation time, the expression of mi R-185 was increased. Oppositely, the expression of Apba-1 was significantly decreased in OGD cell compared with normal cell and with the increasing of the reoxygenation time, the expression of Apba-1 was decreased.Conclusions: Our results revealed that the expression of mi R-185 and Apba-1 varied markedly.Part III The expressions of mi R-185 and Apba-1 on MCAOObjectives: To explore the expressions of mi R-185 and Apba-1 on MCAOMethods: Ten of sixty adult male SD rats were randomly chosen for sham group.the others were subjected to transient middle cerebral artery occlusion(MCAO), and then divided into three groups: 1 day post-ischemia group, 3 day post-ischemia group,and 7 day post-ischemia group and each group has ten rats. Real time PCR was used todetermine the expression of mi R-185 and Apba-1 on m RNA level. The Apba-1 protein level was detected by Western blot.Results: Mi R-185 expression was higher in ischemic cortex than that in normal and with the increasing of the reperfusion time, the expression of mi R-185 is increased. Oppositely, the expression of Apba-1 significantly decreased in ischemic cortex compared with sham group and with the increasing of the reperfusion time, the expression of Apba-1 is decreased.Conclusions: There is a different expression of mi R-185 and Apba-1 after cerebral ischemia.Part IV The relationship between mi R-185 and Apba-1 in ischemic strokeObjectives: To determine the relationship between mi R-185 and Apba-1 in ischemic strokeMethods: Cloning Rno-mi R-185 and Rno-Apba1 sequence, and connected them to the construction of expression vector report gene plasmid. The interaction between the mi R-185 and Apba-1 was determined by using the dual luciferase reporter gene assay.Results:Mi R-185 can regulate Apba-1 during the cerebral ischemia condition.Conclusions:Mi R-185 can reduce the expression of Apba-1 in cerebral ischemia.